Sample records for y-family dna polymerases

  1. Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

    PubMed Central

    2015-01-01

    The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

  2. A nucleotide binding rectification Brownian ratchet model for translocation of Y-family DNA polymerases

    PubMed Central

    2011-01-01

    Y-family DNA polymerases are characterized by low-fidelity synthesis on undamaged DNA and ability to catalyze translesion synthesis over the damaged DNA. Their translocation along the DNA template is an important event during processive DNA synthesis. In this work we present a Brownian ratchet model for this translocation, where the directed translocation is rectified by the nucleotide binding to the polymerase. Using the model, different features of the available structures for Dpo4, Dbh and polymerase ι in binary and ternary forms can be easily explained. Other dynamic properties of the Y-family polymerases such as the fast translocation event upon dNTP binding for Dpo4 and the considerable variations of the processivity among the polymerases can also be well explained by using the model. In addition, some predicted results of the DNA synthesis rate versus the external force acting on Dpo4 and Dbh polymerases are presented. Moreover, we compare the effect of the external force on the DNA synthesis rate of the Y-family polymerase with that of the replicative DNA polymerase. PMID:21699732

  3. Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

    PubMed Central

    Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2012-01-01

    N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

  4. Kinetic Analysis of the Bypass of a Bulky DNA Lesion Catalyzed by Human Y-family DNA Polymerases

    PubMed Central

    Sherrer, Shanen M.; Sanman, Laura E.; Xia, Cynthia X.; Bolin, Eric R.; Malik, Chanchal K.; Efthimiopoulos, Georgia; Basu, Ashis K.; Suo, Zucai

    2012-01-01

    1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolτ), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dGAP on a synthetic DNA template but hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dGAP sites (t50bypass ) encountered by hPolη, hPolκ and hPolτ was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dGAP (hPolη > hPolκ > hPolτ >> hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dGAP efficiently, replication by both hPolκ and hPolτ was strongly stalled at the lesion site and at a site immediately downstream from dGAP. By employing pre-steady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dGAP in vivo is catalyzed by a human Y-family DNA polymerase, e.g. hPolη, the process is certainly mutagenic. PMID:22324639

  5. Backbone assignment of the little finger domain of a Y-family DNA polymerase.

    PubMed

    Ma, Dejian; Fowler, Jason D; Suo, Zucai

    2011-10-01

    Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

  6. Both High-Fidelity Replicative and Low-Fidelity Y-Family Polymerases Are Involved in DNA Rereplication

    PubMed Central

    Sekimoto, Takayuki; Oda, Tsukasa; Kurashima, Kiminori; Hanaoka, Fumio

    2014-01-01

    DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability. PMID:25487575

  7. Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

    PubMed Central

    Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

    2013-01-01

    Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

  8. Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

    PubMed

    Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

    2013-11-15

    Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

  9. Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

    PubMed Central

    Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2015-01-01

    1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

  10. Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

    PubMed Central

    Jansen, Jacob G.; Temviriyanukul, Piya; Wit, Niek; Delbos, Frédéric; Reynaud, Claude-Agnès; Jacobs, Heinz; de Wind, Niels

    2014-01-01

    Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects. PMID:25170086

  11. (1)H, (13)C, and (15)N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog.

    PubMed

    Moro, Sean L; Cocco, Melanie J

    2015-10-01

    The dinB homolog (Dbh) is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the (15)N, (1)H, and (13)C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains.

  12. Replication of a carcinogenic nitropyrene DNA lesion by human Y-family DNA polymerase

    PubMed Central

    Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

    2013-01-01

    Nitrated polycyclic aromatic hydrocarbons are common environmental pollutants, of which many are mutagenic and carcinogenic. 1-Nitropyrene is the most abundant nitrated polycyclic aromatic hydrocarbon, which causes DNA damage and is carcinogenic in experimental animals. Error-prone translesion synthesis of 1-nitropyrene–derived DNA lesions generates mutations that likely play a role in the etiology of cancer. Here, we report two crystal structures of the human Y-family DNA polymerase iota complexed with the major 1-nitropyrene DNA lesion at the insertion stage, incorporating either dCTP or dATP nucleotide opposite the lesion. Polι maintains the adduct in its active site in two distinct conformations. dCTP forms a Watson–Crick base pair with the adducted guanine and excludes the pyrene ring from the helical DNA, which inhibits replication beyond the lesion. By contrast, the mismatched dATP stacks above the pyrene ring that is intercalated in the helix and achieves a productive conformation for misincorporation. The intra-helical bulky pyrene mimics a base pair in the active site and facilitates adenine misincorporation. By structure-based mutagenesis, we show that the restrictive active site of human polη prevents the intra-helical conformation and A-base misinsertions. This work provides one of the molecular mechanisms for G to T transversions, a signature mutation in human lung cancer. PMID:23268450

  13. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-10-04

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.

  14. Translesion synthesis across the (6-4) photoproduct and its Dewar valence isomer by the Y-family and engineered DNA polymerases.

    PubMed

    Yamamoto, Junpei; Loakes, David; Masutani, Chikahide; Simmyo, Shizu; Urabe, Kumiko; Hanaoka, Fumio; Holliger, Philipp; Iwai, Shigenori

    2008-01-01

    We analyzed the translesion synthesis across the UV-induced lesions, the (6-4) photoproduct and its Dewar valence isomer, by using human DNA polymerases eta and iota in vitro. The primer extension experiments revealed that pol eta tended to incorporate dG opposite the 3' component of both lesions, but the incorporation efficiency for the Dewar isomer was higher than that for the (6-4) photoproduct. On the other hand, pol iota was likely to incorporate dA opposite the 3' components of the (6-4) photoproduct and its Dewar isomer with a similar efficiency. Elongation after the incorporation opposite the UV lesions was not observed for these Y-family polymerases. We further analyzed the bypass ability of an engineered polymerase developed from Thermus DNA polymerase for the amplification of ancient DNA. This polymerase could bypass the Dewar isomer more efficiently than the (6-4) photoproduct.

  15. Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

    PubMed

    Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

    2016-08-01

    Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

  16. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

    PubMed Central

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

  17. Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

    PubMed Central

    Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2014-01-01

    3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

  18. Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase via Raman crystallography.

    PubMed

    Espinoza-Herrera, Shirly J; Gaur, Vineet; Suo, Zucai; Carey, Paul R

    2013-07-23

    Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. (13)C- and (15)N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg(2+) and Mn(2+), nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn(2+) or Mg(2+), the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

  19. Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase.

    PubMed

    Gadkari, Varun V; Tokarsky, E John; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

    2014-09-01

    3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-N-ABA)). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG(C8-N-ABA) is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG(C8-N-ABA) on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG(C8-N-ABA) lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG(C8-N-ABA) lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG(C8-N-ABA) bypass catalyzed by Dpo4. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Conformational Dynamics of a Y-Family DNA Polymerase during Substrate Binding and Catalysis As Revealed by Interdomain Förster Resonance Energy Transfer

    PubMed Central

    2015-01-01

    Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis, with the latter conformational change acting to kinetically limit the correct nucleotide incorporation rate. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA–protein binary state for Y-family DNA polymerases. In previous studies [Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol.7, e1000225], a real-time Förster resonance energy transfer (FRET) method was developed to monitor the global conformational transitions of DNA polymerase IV from Sulfolobus solfataricus (Dpo4), a prototype Y-family enzyme, during nucleotide binding and incorporation by measuring changes in distance between locations on the enzyme and the DNA substrate. To elucidate further details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the real-time FRET technique was used to monitor changes in distance between various pairs of locations in the protein itself. In addition to providing new insight into the conformational changes as revealed in previous studies, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA. PMID:24568554

  1. Human DNA polymerase η accommodates RNA for strand extension.

    PubMed

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2017-11-03

    Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

    PubMed

    Antczak, Nicole M; Packer, Morgan R; Lu, Xueguang; Zhang, Ke; Beuning, Penny J

    2017-11-20

    DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N 6 -furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N 2 -furfuryl-dG or N 6 -furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N 2 -furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

  3. Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

    PubMed

    Makarova, A V; Kulbachinskiy, A V

    2012-06-01

    Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

  4. Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases*

    PubMed Central

    Zhao, Linlin; Pence, Matthew G.; Christov, Plamen P.; Wawrzak, Zdzislaw; Choi, Jeong-Yun; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter

    2012-01-01

    N2,3-Ethenoguanine (N2,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N2,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N2,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N2,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N2,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N2,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N2,3-ϵG. PMID:22910910

  5. Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xing, G.; Kirouac, K.; Shin, Y.J.

    2009-09-16

    DNA polymerases are co-ordinated by sliding clamps (PCNA/{beta}-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 {angstrom} resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with amore » 3{sub 10} helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.« less

  6. Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions

    PubMed Central

    Shanbhag, Vinit; Sachdev, Shrikesh; Flores, Jacqueline A.; Modak, Mukund J.; Singh, Kamalendra

    2018-01-01

    DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 3′-5′ exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes. Here, we review the association of DNA polymerases in cancer from the A and B families, which participate in lesion bypass, and conduct gene replication. We also discuss possible therapeutic interventions that could be used to maneuver the role of these enzymes in tumorigenesis. PMID:29301327

  7. Effect of pH on the Misincorporation Rate of DNA Polymerase η.

    PubMed

    Nishimoto, Naomi; Suzuki, Motoshi; Izuta, Shunji

    2016-01-01

    The many known eukaryotic DNA polymerases are classified into four families; A, B, X, and Y. Among them, DNA polymerase η, a Y family polymerase, is a low fidelity enzyme that contributes to translesional synthesis and somatic hypermutation. Although a high mutation frequency is observed in immunoglobulin genes, translesional synthesis occurs with a high accuracy. We determined whether the misincorporation rate of DNA polymerase η varies with ambient conditions. It has been reported that DNA polymerase η is unable to exclude water molecules from the active site. This finding suggests that some ions affect hydrogen bond formation at the active site. We focused on the effect of pH and evaluated the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite template T by DNA polymerase η at various pH levels with a synthetic template-primer. The misincorporation rate of dGTP by DNA polymerase η drastically increased at pH 8.0-9.0 compared with that at pH 6.5-7.5. Kinetic analysis revealed that the Km value for dGTP on the misincorporation opposite template T was markedly affected by pH. However, this drastic change was not seen with the low fidelity DNA polymerase α.

  8. Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta

    PubMed Central

    Brown, Jessica A.; Zhang, Likui; Sherrer, Shanen M.; Taylor, John-Stephen; Burgers, Peter M. J.; Suo, Zucai

    2010-01-01

    Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides. PMID:20798853

  9. Involvement of Escherichia coli DNA Polymerase IV in Tolerance of Cytotoxic Alkylating DNA Lesions in Vivo

    PubMed Central

    Bjedov, Ivana; Dasgupta, Chitralekha Nag; Slade, Dea; Le Blastier, Sophie; Selva, Marjorie; Matic, Ivan

    2007-01-01

    Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polκ, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment. PMID:17483416

  10. Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

    PubMed

    Tokarsky, E John; Gadkari, Varun V; Zahurancik, Walter J; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

    2016-10-01

    3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG C8- N -ABA ), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dG C8- N -ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dG C8- N -ABA in vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dG C8- N- ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dG C8- N -ABA . In contrast, hPolι and hRev1 were severely stalled by dG C8- N -ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dG C8- N- ABA . Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dG C8- N -ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance*

    PubMed Central

    Schermerhorn, Kelly M.; Gardner, Andrew F.

    2015-01-01

    Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼2.5 s−1) and especially tight nucleotide binding (Kd(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates >40-fold (kexo ≥110 s−1 versus ≥2.5 s−1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families. PMID:26160179

  12. DNA polymerase preference determines PCR priming efficiency.

    PubMed

    Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

    2014-01-30

    Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

  13. Structural and Functional Analysis of Sulfolobus solfataricus Y-Family DNA Polymerase Dpo4-Catalyzed Bypass of the Malondialdehyde−Deoxyguanosine Adduct

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eoff, Robert L.; Stafford, Jennifer B.; Szekely, Jozsef

    2010-01-12

    Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-{alpha}]purin-10(3H)-one (M{sub 1}dG). When paired opposite cytosine in duplex DNA at physiological pH, M{sub 1}dG undergoes ring opening to form N{sup 2}-(3-oxo-1-propenyl)-dG (N{sup 2}-OPdG). Previous work has shown that M{sub 1}dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M{sub 1}dG.more » To probe the mechanism by which translesion polymerases bypass M{sub 1}dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M{sub 1}dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M{sub 1}dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M{sub 1}dG. Two crystal structures were determined, including a 'type II' frameshift deletion complex and another complex with Dpo4 bound to a dC-M{sub 1}dG pair located in the postinsertion context. Importantly, M{sub 1}dG was in the ring-closed state in both structures, and in the structure with dC opposite M{sub 1}dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M{sub 1}dG and illustrate how the lesion may affect replication events.« less

  14. A novel mechanism of sugar selection utilized by a human X-family DNA polymerase.

    PubMed

    Brown, Jessica A; Fiala, Kevin A; Fowler, Jason D; Sherrer, Shanen M; Newmister, Sean A; Duym, Wade W; Suo, Zucai

    2010-01-15

    During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2'-hydroxyl group and the bulky side chain of an active-site residue. In this study, we demonstrated that human DNA polymerase lambda used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2'-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2'-position. Copyright 2009 Elsevier Ltd. All rights reserved.

  15. Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

    PubMed

    Redrejo-Rodríguez, Modesto; Ordóñez, Carlos D; Berjón-Otero, Mónica; Moreno-González, Juan; Aparicio-Maldonado, Cristian; Forterre, Patrick; Salas, Margarita; Krupovic, Mart

    2017-11-07

    Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Transient expression and activity of human DNA polymerase iota in loach embryos.

    PubMed

    Makarova, Irina V; Kazakov, Andrey A; Makarova, Alena V; Khaidarova, Nella V; Kozikova, Larisa V; Nenasheva, Valentina V; Gening, Leonid V; Tarantul, Vyacheslav Z; Andreeva, Ludmila E

    2012-02-01

    Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.

  17. Role of disulfide bridges in archaeal family-B DNA polymerases.

    PubMed

    Killelea, Tom; Connolly, Bernard A

    2011-06-14

    The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Identification of an Unfolding Intermediate for a DNA Lesion Bypass Polymerase

    PubMed Central

    Sherrer, Shanen M.; Maxwell, Brian A.; Pack, Lindsey R.; Fiala, Kevin A.; Fowler, Jason D.; Zhang, Jun; Suo, Zucai

    2012-01-01

    Sulfolobus solfataricusDNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C. PMID:22667759

  19. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana

    DOE PAGES

    Kashiwagi, Sayo; Kuraoka, Isao; Fujiwara, Yoshie; ...

    2010-01-01

    Humore » man DNA polymerase η (HsPol η ) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPol η from the thermophilic worm Alvinella pompejana , which inhabits deep-sea hydrothermal vent chimneys. ApPol η shares sequence homology with HsPol η and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPol η is more thermostable than HsPol η , as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPol η provides a robust, human-like Pol η that is more active after exposure to high temperatures and organic solvents.« less

  20. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana

    PubMed Central

    Kashiwagi, Sayo; Kuraoka, Isao; Fujiwara, Yoshie; Hitomi, Kenichi; Cheng, Quen J.; Fuss, Jill O.; Shin, David S.; Masutani, Chikahide; Tainer, John A.; Hanaoka, Fumio; Iwai, Shigenori

    2010-01-01

    Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents. PMID:20936172

  1. Pre-Steady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogs Catalyzed by Non-Canonical Human DNA Polymerases

    PubMed Central

    Brown, Jessica A.; Pack, Lindsey R.; Fowler, Jason D.; Suo, Zucai

    2011-01-01

    Antiviral nucleoside analogs have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogs can be limited by drug toxicity because the 5′-triphosphates of these nucleoside analogs (nucleotide analogs) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogs are substrates for the recently discovered human X- and Y-family DNA polymerases. Using pre-steady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogs approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogs were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3′-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogs catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity. PMID:22132702

  2. Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η*

    PubMed Central

    Su, Yan; Egli, Martin; Guengerich, F. Peter

    2016-01-01

    Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2′-deoxyguanosine with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion. PMID:26740629

  3. Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.

    PubMed

    Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A

    2004-03-26

    Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

  4. A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

    PubMed Central

    Takami, K; Matsuda, S; Sono, A; Sakaguchi, K

    1994-01-01

    A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state. Images Figure 2 Figure 5 Figure 6 PMID:8172591

  5. Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression*

    PubMed Central

    Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.

    2014-01-01

    Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692

  6. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

    PubMed

    Qi, Yonghe; Gao, Zhenchao; Xu, Guangwei; Peng, Bo; Liu, Chenxuan; Yan, Huan; Yao, Qiyan; Sun, Guoliang; Liu, Yang; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-10-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

  7. Posttranslational Regulation of Human DNA Polymerase ι.

    PubMed

    McIntyre, Justyna; McLenigan, Mary P; Frank, Ekaterina G; Dai, Xiaoxia; Yang, Wei; Wang, Yinsheng; Woodgate, Roger

    2015-11-06

    Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys(11)- and Lys(48)-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys(11) and Lys(48) rather than oxidative damage per se. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  9. Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

    PubMed

    Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

    2011-08-01

    DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

  10. Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η.

    PubMed

    O'Flaherty, D K; Patra, A; Su, Y; Guengerich, F P; Egli, M; Wilds, C J

    2016-08-01

    DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O 4 -Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4- O 4 bond on processing by human DNA polymerase η (hPol η ) was studied for oligonucleotides containing O 4 -methylthymidine, O 4 -ethylthymidine, and analogs restricting the O 4 -methylene group in an anti -orientation. Primer extension assays revealed that the O 4 -alkyl orientation influences hPol η bypass. Crystal structures of hPol η •DNA•dNTP ternary complexes with O 4 -methyl- or O 4 -ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn - O 4 -methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O 4 -methylthymidine with hPol η , inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O 4 MedT and O 4 EtdT when acted upon by hPol η .

  11. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

    PubMed

    Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

    2015-09-01

    Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence

  12. The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

    PubMed Central

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

    2015-01-01

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

  13. The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1*

    PubMed Central

    Boehm, Elizabeth M.; Powers, Kyle T.; Kondratick, Christine M.; Spies, Maria; Houtman, Jon C. D.; Washington, M. Todd

    2016-01-01

    Y-family DNA polymerases, such as polymerase η, polymerase ι, and polymerase κ, catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase η, a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase η mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase η binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase κ and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed. PMID:26903512

  14. Refolding Active Human DNA Polymerase ν from Inclusion Bodies

    PubMed Central

    Arana, Mercedes E.; Powell, Gary K.; Edwards, Lori L.; Kunkel, Thomas A.; Petrovich, Robert M.

    2017-01-01

    Human DNA polymerase ν (Pol ν) is a conserved family A DNA polymerase of uncertain biological function. Physical and biochemical characterization aimed at understanding Pol ν function is hindered by the fact that, when over-expressed in E. coli, Pol ν is largely insoluble, and the small amount of soluble protein is difficult to purify. Here we describe the use of high hydrostatic pressure to refold Pol ν from inclusion bodies, in soluble and active form. The refolded Pol ν has properties comparable to those of the small amount of Pol ν that was purified from the soluble fraction. The approach described here may be applicable to other DNA polymerases that are expressed as insoluble inclusion bodies in E. coli. PMID:19853037

  15. The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.

    PubMed

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F

    2015-05-15

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.

    PubMed

    Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

    2012-06-27

    Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

  17. A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota

    PubMed Central

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L

    2012-01-01

    Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase. PMID:22632140

  18. DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds

    PubMed Central

    Potapova, Olga; Chan, Chikio; DeLucia, Angela M.; Helquist, Sandra A.; Kool, Eric T.; Grindley, Nigel D. F.; Joyce, Catherine M.

    2008-01-01

    We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair in order to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA Polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen bonding ability in the nascent pair, the efficiency (kpol/Kd) of the polymerase reaction is decreased by 30-fold, affecting ground state (Kd) and transition state (kpol) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. By contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions, and more dependent on hydrogen bonding between base-paired partners. PMID:16411765

  19. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  20. The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.

    PubMed

    Donigan, Katherine A; McLenigan, Mary P; Yang, Wei; Goodman, Myron F; Woodgate, Roger

    2014-03-28

    Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A "steric gate" pol ι mutant is considerably more active in the presence of Mn(2+) compared with Mg(2+) and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be "at risk" for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.

  1. The Steric Gate of DNA Polymerase ι Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity*

    PubMed Central

    Donigan, Katherine A.; McLenigan, Mary P.; Yang, Wei; Goodman, Myron F.; Woodgate, Roger

    2014-01-01

    Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A “steric gate” pol ι mutant is considerably more active in the presence of Mn2+ compared with Mg2+ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be “at risk” for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations. PMID:24532793

  2. Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

    PubMed

    Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

    2014-03-07

    The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

  3. DNA Polymerase Eta and Chemotherapeutic Agents

    PubMed Central

    2011-01-01

    Abstract The discovery of human DNA polymerase eta (pol η) has a major impact on the fields of DNA replication/repair fields. Since the discovery of human pol η, a number of new DNA polymerases with the ability to bypass various DNA lesions have been discovered. Among these polymerases, pol η is the most extensively studied lesion bypass polymerase with a defined major biological function, that is, to replicate across the cyclobutane pyrimidine dimers introduced by UV irradiation. Cyclobutane pyrimidine dimer is a major DNA lesion that causes distortion of DNA structure and block the replicative DNA polymerases during DNA replication process. Genetic defects in the pol η gene, Rad30, results in a disease called xeroderma pigmentosum variant. This review focuses on the overall properties of pol η and the mechanism that involved in regulating its activity in cells. In addition, the role of pol η in the action of DNA-targeting anticancer compounds is also discussed. Antioxid. Redox Signal. 14, 2521–2529. PMID:21050139

  4. Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase.

    PubMed

    Ren, Zhong

    2016-09-06

    DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya.

    PubMed

    Wardle, Josephine; Burgers, Peter M J; Cann, Isaac K O; Darley, Kate; Heslop, Pauline; Johansson, Erik; Lin, Li-Jung; McGlynn, Peter; Sanvoisin, Jonathan; Stith, Carrie M; Connolly, Bernard A

    2008-02-01

    Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures approximately 100 degrees C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases delta and epsilon for nuclear DNA and polymerase gamma for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.

  6. Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

    PubMed Central

    Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

    2014-01-01

    The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

  7. NMR Structure and Dynamics of the C-terminal Domain from Human Rev1 and its Complex with Rev1 Interacting Region of DNA Polymerase η

    PubMed Central

    Pozhidaeva, Alexandra; Pustovalova, Yulia; D'Souza, Sanjay; Bezsonova, Irina; Walker, Graham C.; Korzhnev, Dmitry M.

    2013-01-01

    Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases. PMID:22691049

  8. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

    PubMed Central

    Earl, P L; Jones, E V; Moss, B

    1986-01-01

    A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses. Images PMID:3012524

  9. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGES

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error ratemore » measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  10. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  11. How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.

    PubMed

    Sholder, Gabriel; Creech, Amanda; Loechler, Edward L

    2015-11-01

    To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG. To investigate structural differences between Y-Family-classes, regions are swapped between DNAP IV (a κ/IV-class-member) and Dpo4 (a η/V-class-member); the kinetic consequences are evaluated via primer-extension studies with a BP-N(2)-dG-containing template. Four key structural elements are revealed. (1) Y-Family DNAPs have discreet non-covalent contacts between their little finger-domain (LF-Domain) and their catalytic core-domain (CC-Domain), which we call "non-covalent bridges" (NCBs). Arg37 and Arg38 in DNAP IV's CC-Domain near the active site form a non-covalent bridge (AS-NCB) by interacting with Glu251 and Asp252, respectively, in DNAP IV's LF-Domain. Without these interactions dATP/dGTP/dTTP misinsertions increase. DNAP IV's AS-NCB suppresses misinsertions better than Dpo4's equivalent AS-NCB. (2) DNAP IV also suppresses dATP/dGTP/dTTP misinsertions via a second non-covalent bridge, which is ∼8Å from the active site (Distal-NCB). Dpo4 has no Distal-NCB, rendering it inferior at dATP/dGTP/dTTP suppression. (3) dCTP insertion is facilitated by the larger minor groove opening near the active site in DNAP IV versus Dpo4, which is sensible given that Watson/Crick-like [dCTP:BP-N(2)-dG] pairing requires the BP-moiety to be in the minor groove. (4) Compared to Dpo4, DNAP IV has a smaller major groove opening, which suppresses dGTP misinsertion, implying BP-N(2)-dG bulk in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. In summary, DNAP IV has a large minor groove opening to enhance dCTP insertion, a plugged major groove opening to suppress dGTP misinsertion, and two non-covalent bridges (near and distal

  12. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  13. Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

    PubMed Central

    Brown, Jessica A.; Pack, Lindsey R.; Sherrer, Shanen M.; Kshetry, Ajay K.; Newmister, Sean A.; Fowler, Jason D.; Taylor, John-Stephen; Suo, Zucai

    2010-01-01

    DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β (Pol β). Pre-steady state kinetic studies have shown that the Pol λ•DNA complex binds both correct and incorrect nucleotides 130-fold tighter on average than the Pol β•DNA complex, although, the base substitution fidelity of both polymerases is 10−4 to 10−5. To better understand Pol λ’s tight nucleotide binding affinity, we created single- and double-substitution mutants of Pol λ to disrupt interactions between active site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding with each of the common structural moieties in the following order: triphosphate ≫ base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and template base led to a moderate increase in the Kd. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template. PMID:20851705

  14. Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution.

    PubMed

    Gahlon, Hailey L; Romano, Louis J; Rueda, David

    2017-11-20

    Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.

  15. Structure–Function Studies of DNA Polymerase λ

    PubMed Central

    2015-01-01

    DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ. PMID:24716527

  16. Repair of Clustered Damage and DNA Polymerase Iota.

    PubMed

    Belousova, E A; Lavrik, O I

    2015-08-01

    Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

  17. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

    DOE PAGES

    Zahn, Karl E.; Averill, April M.; Aller, Pierre; ...

    2015-03-16

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contactsmore » to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.« less

  18. Eukaryotic DNA polymerase ζ

    PubMed Central

    Makarova, Alena V.; Burgers, Peter M.

    2015-01-01

    This review focuses on eukaryotic DNA polymerase ζ (Pol ζ), the enzyme responsible for the bulk of mutagenesis in eukaryotic cells in response to DNA damage. Pol ζ is also responsible for a large portion of mutagenesis during normal cell growth, in response to spontaneous damage or to certain DNA structures and other blocks that stall DNA replication forks. Novel insights in mutagenesis have been derived from recent advances in the elucidation of the subunit structure of Pol ζ. The lagging strand DNA polymerase δ shares the small Pol31 and Pol32 subunits with the Rev3-Rev7 core assembly giving a four subunit Pol ζ complex that is the active form in mutagenesis. Furthermore, Pol ζ forms essential interactions with the mutasome assembly factor Rev1 and with proliferating cell nuclear antigen (PCNA). These interactions are modulated by posttranslational modifications such as ubiquitination and phosphorylation that enhance translesion synthesis (TLS) and mutagenesis. PMID:25737057

  19. DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

    PubMed Central

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-01-01

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

  20. DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

    PubMed

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-07-14

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

  1. Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro.

    PubMed

    Kazachenko, Konstantin Y; Miropolskaya, Nataliya A; Gening, Leonid V; Tarantul, Vyacheslav Z; Makarova, Alena V

    2017-02-01

    Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg 2+ or Mn 2+ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. DNA polymerase γ and disease: what we have learned from yeast

    PubMed Central

    Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico

    2015-01-01

    Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747

  3. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

    PubMed Central

    Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

    2016-01-01

    DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. PMID:27050298

  4. Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

    PubMed Central

    Niimi, Atsuko; Limsirichaikul, Siripan; Yoshida, Shonen; Iwai, Shigenori; Masutani, Chikahide; Hanaoka, Fumio; Kool, Eric T.; Nishiyama, Yukihiro; Suzuki, Motoshi

    2004-01-01

    We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes. PMID:15024063

  5. PrimPol prevents APOBEC/AID family mediated DNA mutagenesis

    PubMed Central

    Pilzecker, Bas; Buoninfante, Olimpia Alessandra; Pritchard, Colin; Blomberg, Olga S.; Huijbers, Ivo J.; van den Berk, Paul C.M.; Jacobs, Heinz

    2016-01-01

    Abstract PrimPol is a DNA damage tolerant polymerase displaying both translesion synthesis (TLS) and (re)-priming properties. This led us to study the consequences of a PrimPol deficiency in tolerating mutagenic lesions induced by members of the APOBEC/AID family of cytosine deaminases. Interestingly, during somatic hypermutation, PrimPol counteracts the generation of C>G transversions on the leading strand. Independently, mutation analyses in human invasive breast cancer confirmed a pro-mutagenic activity of APOBEC3B and revealed a genome-wide anti-mutagenic activity of PRIMPOL as well as most Y-family TLS polymerases. PRIMPOL especially prevents APOBEC3B targeted cytosine mutations within TpC dinucleotides. As C transversions induced by APOBEC/AID family members depend on the formation of AP-sites, we propose that PrimPol reprimes preferentially downstream of AP-sites on the leading strand, to prohibit error-prone TLS and simultaneously stimulate error-free homology directed repair. These in vivo studies are the first demonstrating a critical anti-mutagenic activity of PrimPol in genome maintenance. PMID:26926109

  6. [DNA-dependent DNA polymerase induced by herpes virus papio (HVP) in producing cells].

    PubMed

    D'iachenko, A G; Beriia, L Ia; Matsenko, L D; Kakubava, V V; Kokosh, L V

    1980-11-01

    A new DNA polymerase was found in the cells of suspension lymphoblastoid cultures, which produce lymphotropic baboon herpes virus (HVP). The enzyme was isolated in a partially purified form. In some properties the enzyme differs from other cellular DNA polymerases. The HVP-induced DNA polymerase has the molecular weight of 1,6 x 10(5) and sedimentation coefficient of about 8S. The enzyme is resistant to high salt concentrations and N-ethylmaleimide, but shows a pronounced sensitivity to phosphonoacetate. The enzyme effectively copies "activated" DNA and synthetic deoxyribohomopolymers. The attempts to detect the DNA polymerase activity in HVP virions were unsuccessful.

  7. DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Insdorf, N.F.; Bogenhagen, D.F.

    1989-12-25

    DNA polymerase gamma has been purified over 10,000-fold from mitochondria of Xenopus laevis ovaries. We have developed a novel technique which specifically photolabels DNA polymerases. This procedure, the DNA polymerase trap, was used to identify a catalytic subunit of 140,000 Da from X. laevis DNA polymerase gamma. Additional catalytically active polypeptides of 100,000 and 55,000 Da were identified in the highly purified enzyme. These appear to be products of degradation of the 140,000-Da subunit. The DNA polymerase trap, which does not require large amounts of enzyme or renaturation from sodium dodecyl sulfate, is an alternative to the classic activity gel.

  8. Bypass of a psoralen DNA interstrand cross-link by DNA polymerases beta, iota, and kappa in vitro

    PubMed Central

    Smith, Leigh A.; Makarova, Alena V.; Samson, Laura; Thiesen, Katherine E.; Dhar, Alok; Bessho, Tadayoshi

    2012-01-01

    Repair of DNA inter-strand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated in vitro TLS activity of psoralen DNA inter-strand cross-link by three DNA repair polymerases, DNA polymerase beta, kappa and iota. DNA polymerase beta is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase beta knockout mouse embryonic fibroblast showed a reduced bypass activity of the psoralen cross-link and purified DNA polymerase beta restored the bypass activity. In addition, DNA polymerase iota mis-incorporated thymine across the psoralen cross-link and DNA polymerase kappa extended these mis-paired primer ends, suggesting that DNA polymerase iota may serve as an inserter and DNA polymerase kappa may play a role as an extender in the repair of psoralen DNA inter-strand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA inter-strand cross-link repair. PMID:23106263

  9. Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

    PubMed Central

    Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

    2001-01-01

    We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

  10. Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.

    PubMed

    Makarova, Alena V; Ignatov, Artem; Miropolskaya, Nataliya; Kulbachinskiy, Andrey

    2014-10-01

    Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

    PubMed

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-08-31

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  12. Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

    PubMed

    Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

    2014-10-20

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences

  13. Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

    PubMed Central

    2015-01-01

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N2-ethyl(Et)G, O6-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, N2-EtG, O6-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg2+ (but not with Mn2+), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg2+). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg2+ or Mn2+, except for that opposite N2-EtG with Mn2+ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg2+), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn2+ than with Mg2+. The results indicate that the R96G variation severely impairs most of the Mg2+- and Mn2+-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg2+-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and

  14. DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct.

    PubMed

    Seki, Mineaki; Wood, Richard D

    2008-01-01

    DNA polymerase theta (pol theta) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol theta and of Polq-defective mice have suggested that pol theta participates in DNA damage tolerance. For example, pol theta was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol theta extended relatively efficiently from matched termini as well as termini with A:G, A:T and A:C mismatches, with less descrimination than a well-studied A-family DNA polymerase, exonuclease-free pol I from E. coli. Although pol theta was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6-4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol theta was combined with DNA polymerase iota, an enzyme that can insert a base opposite a UV-induced (6-4) photoproduct, complete bypass of a (6-4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol theta is proficient at extension of unpaired termini. These results show the potential of pol theta to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol theta in somatic mutagenesis and genome instability.

  15. Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

    PubMed

    Zhu, Bin; Wang, Longfei; Mitsunobu, Hitoshi; Lu, Xueling; Hernandez, Alfredo J; Yoshida-Takashima, Yukari; Nunoura, Takuro; Tabor, Stanley; Richardson, Charles C

    2017-03-21

    A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

  16. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    PubMed Central

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  17. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

    PubMed

    Tubeleviciute, Agne; Skirgaila, Remigijus

    2010-08-01

    The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

  18. Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

    PubMed

    Mirzakhanyan, Yeva; Gershon, Paul D

    2017-09-01

    The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

  19. Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

    PubMed Central

    Arbel-Eden, Ayelet; Joseph-Strauss, Daphna; Masika, Hagit; Printzental, Oxana; Rachi, Eléanor; Simchen, Giora

    2013-01-01

    Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed. PMID:23550131

  20. Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase.

    PubMed

    Baños, Benito; Lázaro, José M; Villar, Laurentino; Salas, Margarita; de Vega, Miguel

    2008-10-01

    Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.

  1. Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schormann, Norbert; Zhukovskaya, Natalia; Bedwell, Gregory

    We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymaticmore » function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.« less

  2. Lesion bypass activity of DNA polymerase θ (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts.

    PubMed

    Hogg, Matthew; Seki, Mineaki; Wood, Richard D; Doublié, Sylvie; Wallace, Susan S

    2011-01-21

    DNA polymerase θ (POLQ, polθ) is a large, multidomain DNA polymerase encoded in higher eukaryotic genomes. It is important for maintaining genetic stability in cells and helping protect cells from DNA damage caused by ionizing radiation. POLQ contains an N-terminal helicase-like domain, a large central domain of indeterminate function, and a C-terminal polymerase domain with sequence similarity to the A-family of DNA polymerases. The enzyme has several unique properties, including low fidelity and the ability to insert and extend past abasic sites and thymine glycol lesions. It is not known whether the abasic site bypass activity is an intrinsic property of the polymerase domain or whether helicase activity is also required. Three "insertion" sequence elements present in POLQ are not found in any other A-family DNA polymerase, and it has been proposed that they may lend some unique properties to POLQ. Here, we analyzed the activity of the DNA polymerase in the absence of each sequence insertion. We found that the pol domain is capable of highly efficient bypass of abasic sites in the absence of the helicase-like or central domains. Insertion 1 increases the processivity of the polymerase but has little, if any, bearing on the translesion synthesis properties of the enzyme. However, removal of insertions 2 and 3 reduces activity on undamaged DNA and completely abrogates the ability of the enzyme to bypass abasic sites or thymine glycol lesions. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide gamma-phosphate derivative.

    PubMed

    Hansen, Connie J; Wu, Lydia; Fox, Jeffrey D; Arezi, Bahram; Hogrefe, Holly H

    2011-03-01

    Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.

  4. Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis.

    PubMed

    Livneh, Zvi; Ziv, Omer; Shachar, Sigal

    2010-02-15

    The encounter of replication forks with DNA lesions may lead to fork arrest and/or the formation of single-stranded gaps. A major strategy to cope with these replication irregularities is translesion DNA synthesis (TLS), in which specialized error-prone DNA polymerases bypass the blocking lesions. Recent studies suggest that TLS across a particular DNA lesion may involve as many as four different TLS polymerases, acting in two-polymerase reactions in which insertion by a particular polymerase is followed by extension by another polymerase. Insertion determines the accuracy and mutagenic specificity of the TLS reaction, and is carried out by one of several polymerases such as poleta, polkappa or poliota. In contrast, extension is carried out primarily by polzeta. In cells from XPV patients, which are deficient in TLS across cyclobutane pyrimidine dimers (CPD) due to a deficiency in poleta, TLS is carried out by at least two backup reactions each involving two polymerases: One reaction involves polkappa and polzeta, and the other poliota and polzeta. These mechanisms may also assist poleta in normal cells under an excessive amount of UV lesions.

  5. DNA polymerase θ (POLQ) can extend from mismatches and from bases opposite a (6–4) photoproduct

    PubMed Central

    Seki, Mineaki; Wood, Richard D.

    2007-01-01

    DNA polymerase θ (pol θ) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol θ and of Polq-defective mice have suggested that pol θ participates in DNA damage tolerance. For example, pol θ was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol θ extended relatively efficiently from matched termini as well as termini with A:G, A:T, and A:C mismatches, with less descrimination than a well-studied A family DNA polymerase, exonuclease-free pol I from E. coli. Although pol θ was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6–4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol θ was combined with DNA polymerase ι , an enzyme that can insert a base opposite a UV-induced (6–4) photoproduct, complete bypass of a (6–4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol θ is proficient at extension of unpaired termini. These results show the potential of pol θ to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol θ in somatic mutagenesis and genome instability. PMID:17920341

  6. DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli.

    PubMed Central

    Hendler, R W; Pereira, M; Scharff, R

    1975-01-01

    DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented. PMID:1094453

  7. Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

    PubMed Central

    Laquel, P; Litvak, S; Castroviejo, M

    1993-01-01

    Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta. PMID:7906418

  8. DNA Polymerase in Virions of a Reptilian Type C Virus

    PubMed Central

    Twardzik, Daniel R.; Papas, Takis S.; Portugal, Frank H.

    1974-01-01

    A study was made of the DNA polymerase of reptilian type C virus isolated from Russell's viper spleen cells. Simultaneous detection experiments demonstrated the presence of 70S RNA and RNA-dependent DNA polymerase activity in reptilian type C virions. The endogenous activity was dependent on the addition of all four deoxynucleotide triphosphates and demonstrated an absolute requirement for a divalent cation. The reptilian viral DNA polymerase elutes from phosphocellulose at 0.22 M salt. In this respect, it is similar to the avian (avian myeloblastosis virus; AMV) viral enzyme but is different from the mammalian (Rauscher leukemia virus; RLV) viral enzyme which elutes at 0.4 M salt. The molecular weight of the viper DNA polymerase as estimated from glycerol gradient centrifugation is 109,000. It is a smaller enzyme than the AMV DNA polymerase (180,000 daltons) and somewhat larger than the RLV enzyme (70,000 daltons). A comparison of other properties of the type C reptilian DNA polymerase with the enzyme found in other type C oncogenic viruses is made. PMID:4129837

  9. General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

    PubMed

    Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

    2018-02-19

    DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Mapping DNA polymerase errors by single-molecule sequencing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, David F.; Lu, Jenny; Chang, Seungwoo

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replicationmore » product is tagged with a unique nucleotide sequence before amplification. Here, this allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.« less

  11. Mapping DNA polymerase errors by single-molecule sequencing

    DOE PAGES

    Lee, David F.; Lu, Jenny; Chang, Seungwoo; ...

    2016-05-16

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replicationmore » product is tagged with a unique nucleotide sequence before amplification. Here, this allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.« less

  12. Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling.

    PubMed

    Agarwal, Nayan P; Matthies, Michael; Joffroy, Bastian; Schmidt, Thorsten L

    2018-03-27

    The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

  13. Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

    PubMed

    Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

    2006-08-18

    DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.

  14. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  15. Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

    PubMed Central

    Waqar, M A; Evans, M J; Huberman, J A

    1978-01-01

    We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis. PMID:673840

  16. Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase η.

    PubMed

    Patra, Amritraj; Nagy, Leslie D; Zhang, Qianqian; Su, Yan; Müller, Livia; Guengerich, F Peter; Egli, Martin

    2014-06-13

    DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

    PubMed

    Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

    2006-10-15

    The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

  18. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    PubMed

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  19. The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ

    PubMed Central

    Ma, Emilie; Veaute, Xavier; Coïc, Eric

    2017-01-01

    Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage. PMID:29281621

  20. Unlocking the sugar "steric gate" of DNA polymerases.

    PubMed

    Brown, Jessica A; Suo, Zucai

    2011-02-22

    To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the 2'-hydroxyl of an incoming ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most DNA polymerases to selectively insert deoxyribonucleotides. Recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanistic consequences of ribonucleotide misincorporation on downstream events, such as the bypass of a ribonucleotide in a DNA template and the subsequent extension of the DNA lesion bypass product. These important findings are summarized in this review.

  1. Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes.

    PubMed

    Heyduk, T; Niedziela-Majka, A

    Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated. Copyright 2002 Wiley Periodicals, Inc.

  2. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    PubMed Central

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  3. DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

    PubMed Central

    Erdem, Aysen L; Jaszczur, Malgorzata; Bertram, Jeffrey G; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

    2014-01-01

    Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase. DOI: http://dx.doi.org/10.7554/eLife.02384.001 PMID:24843026

  4. Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η.

    PubMed

    Ucisik, Melek N; Hammes-Schiffer, Sharon

    2017-04-20

    Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine-thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation-π, and π-π interactions of the side chains with the dATP and the TTD or thymine-thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

  5. Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η

    PubMed Central

    2016-01-01

    Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine–thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation−π, and π–π interactions of the side chains with the dATP and the TTD or thymine–thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment. PMID:28423907

  6. Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

    PubMed

    Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

    1997-04-01

    A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

  7. The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

    PubMed

    Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

    1999-03-05

    Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

  8. Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

    PubMed Central

    Kirouac, Kevin N.; Ling, Hong

    2011-01-01

    The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι’s biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase. PMID:21300901

  9. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    PubMed Central

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

  10. DNA polymerases in the rat pituitary gland. Effect of oestrogens and sulpiride.

    PubMed

    Jahn, G A; Kalbermann, L E; Machiavelli, G; Szijan, I; Burdman, J A

    1980-06-01

    Changes in the activity of DNA polymerase and [3H]thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats. The activities of DNA polymerases alpha and beta, extracted in Tris--HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively alpha, while in the phosphate soluble fraction it is a mixture of alpha and beta. The administration of oestrogens to male rats increases [3H]thymidine incorporation and enhances the activity of DNA polymerases in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further increment of [3H]thymidine incorporation and of DNA polymerase activity in the Tris-soluble fraction. In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction. Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase alpha. This activity changes in parallel with [3H]thymidine incorporation into DNA which is an indication of cell proliferation in the gland. This is discussed with respect to a negative feedback mechanism between intracellular prolactin concentration and DNA synthesis in the APG.

  11. Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-04-01

    A kinetic theory is developed for exonuclease-deficient DNA polymerases, based on the experimental observation that the rates depend not only on the newly incorporated nucleotide, but also on the previous one, leading to the growth of Markovian DNA sequences from a Bernoullian template. The dependencies on nucleotide concentrations and template sequence are explicitly taken into account. In this framework, the kinetic and thermodynamic properties of DNA replication, in particular, the mean growth velocity, the error probability, and the entropy production are calculated analytically in terms of the rate constants and the concentrations. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  12. A Polymerase With Potential: The Fe-S Cluster in Human DNA Primase.

    PubMed

    Holt, Marilyn E; Salay, Lauren E; Chazin, Walter J

    2017-01-01

    Replication of DNA in eukaryotes is primarily executed by the combined action of processive DNA polymerases δ and ɛ. These enzymes cannot initiate synthesis of new DNA without the presence of a primer on the template ssDNA. The primers on both the leading and lagging strands are generated by DNA polymerase α-primase (pol-prim). DNA primase is a DNA-dependent RNA polymerase that synthesizes the first ~10 nucleotides and then transfers the substrate to polymerase α to complete primer synthesis. The mechanisms governing the coordination and handoff between primase and polymerase α are largely unknown. Isolated DNA primase contains a [4Fe-4S] 2+ cluster that has been shown to serve as a redox switch modulating DNA binding affinity. This discovery suggests a mechanism for modulating the priming activity of primase and handoff to polymerase α. In this chapter, we briefly discuss the current state of knowledge of primase structure and function, including the role of its iron-sulfur cluster. This is followed by providing the methods for expressing, purifying, and biophysically/structurally characterizing primase and its iron-sulfur cluster-containing domain, p58C. © 2017 Elsevier Inc. All rights reserved.

  13. Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases

    PubMed Central

    Pavlov, Andrey R.; Belova, Galina I.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2002-01-01

    Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes. PMID:12368475

  14. Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

    PubMed

    Frank, K B; Cheng, Y C

    1986-02-05

    Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

  15. Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase.

    PubMed

    Takahashi, Shuntaro; Brazier, John A; Sugimoto, Naoki

    2017-09-05

    Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

  16. Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

    PubMed Central

    Takahashi, Shuntaro; Brazier, John A.; Sugimoto, Naoki

    2017-01-01

    Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases. PMID:28827350

  17. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

    PubMed Central

    Bratic, Ana; Kauppila, Timo E. S.; Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B.; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

    2015-01-01

    Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

  18. A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

    PubMed

    Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

    1986-09-01

    The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

  19. Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η*♦

    PubMed Central

    Patra, Amritraj; Nagy, Leslie D.; Zhang, Qianqian; Su, Yan; Müller, Livia; Guengerich, F. Peter; Egli, Martin

    2014-01-01

    DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion. PMID:24759104

  20. Isolation and characterization of high affinity aptamers against DNA polymerase iota.

    PubMed

    Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

    2012-02-01

    Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

  1. Replicative DNA polymerase mutations in cancer☆

    PubMed Central

    Heitzer, Ellen; Tomlinson, Ian

    2014-01-01

    Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

  2. Editing of misaligned 3′-termini by an intrinsic 3′–5′ exonuclease activity residing in the PHP domain of a family X DNA polymerase

    PubMed Central

    Baños, Benito; Lázaro, José M.; Villar, Laurentino; de Vega, Miguel

    2008-01-01

    Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3′–5′ exonuclease activity specialized in resecting unannealed 3′-termini in a gapped DNA substrate. Biochemical analysis of a PolXBs deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3′–5′ exonuclease activity of PolXBs resides in its PHP domain. Furthermore, site-directed mutagenesis of PolXBs His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3′-termini resection by the 3′–5′ exonuclease activity of PolXBs in the DNA repair context are discussed. PMID:18776221

  3. Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

    PubMed

    Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

    2011-01-21

    In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

    PubMed

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2009-04-01

    The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

  5. Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.

    PubMed

    Lee, M Y; Whyte, W A

    1984-05-01

    The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

  6. PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

    PubMed

    Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

    2009-07-01

    Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

  7. Slow Joining of Newly Replicated DNA Chains in DNA Polymerase I-Deficient Escherichia coli Mutants*

    PubMed Central

    Okazaki, Reiji; Arisawa, Mikio; Sugino, Akio

    1971-01-01

    In Escherichia coli mutants deficient in DNA polymerase I, newly replicated short DNA is joined at about 10% of the rate in the wild-type strains. It is postulated that DNA polymerase I normally functions in filling gaps between the nascent short segments synthesized by the replication complex. Possible implications of the finding are discussed in relation to other abnormal properties of these mutants. PMID:4943548

  8. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    PubMed Central

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  9. Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2[W

    PubMed Central

    Amoroso, Alessandra; Concia, Lorenzo; Maggio, Caterina; Raynaud, Cécile; Bergounioux, Catherine; Crespan, Emmanuele; Cella, Rino; Maga, Giovanni

    2011-01-01

    The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes. PMID:21325140

  10. Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

    PubMed Central

    Yu, Chuanhe; Gan, Haiyun

    2017-01-01

    ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal. PMID:28784720

  11. Molecular Dynamics Study of the Opening Mechanism for DNA Polymerase I

    PubMed Central

    Miller, Bill R.; Parish, Carol A.; Wu, Eugene Y.

    2014-01-01

    During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme∶DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics. PMID:25474643

  12. Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

    PubMed Central

    Suo, Zucai

    2014-01-01

    Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been done to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer (FRET) to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol. PMID:24931550

  13. Structure and mechanism of human DNA polymerase [eta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji

    2010-11-03

    The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assistmore » translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.« less

  14. Getting it Right: How DNA Polymerases Select the Right Nucleotide.

    PubMed

    Ludmann, Samra; Marx, Andreas

    2016-01-01

    All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases.

  15. DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members.

    PubMed Central

    d'Ambrosio, E; Furano, A V

    1987-01-01

    An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members. We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase. The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues. Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues. Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins. The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules. By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde. The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand. Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis. Images PMID:2436148

  16. Techniques used to study the DNA polymerase reaction pathway

    PubMed Central

    Joyce, Catherine M.

    2009-01-01

    Summary A minimal reaction pathway for DNA polymerases was established over 20 years ago using chemical quench methods. Since that time there has been considerable interest in noncovalent steps in the reaction pathway, conformational changes involving the polymerase or its DNA substrate that may play a role in substrate specificity. Fluorescence-based assays have been devised in order to study these conformational transitions and the results obtained have added new detail to the reaction pathway. PMID:19665596

  17. Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-04-01

    Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering of the error probability under physiological conditions. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  18. A Novel Mechanism of Sugar Selection Utilized by a Human X-family DNA Polymerase†

    PubMed Central

    Brown, Jessica A.; Fiala, Kevin A.; Fowler, Jason D.; Sherrer, Shanen M.; Newmister, Sean A.; Dyum, Wade W.; Suo, Zucai

    2009-01-01

    During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2′-hydroxyl group and the bulky side chain of an active site residue. Here, we demonstrated that human DNA polymerase λ used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2′-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such a steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2′ position. PMID:19900463

  19. Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

    PubMed

    Kumar, J K; Tabor, S; Richardson, C C

    2001-09-14

    The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

  20. High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies.

    PubMed

    Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

    2018-03-01

    One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).

    PubMed Central

    Laquel, P.; Litvak, S.; Castroviejo, M.

    1994-01-01

    DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants. PMID:12232187

  2. Replication of N[superscript 2],3-Ethenoguanine by DNA Polymerases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Linlin; Christov, Plamen P.; Kozekov, Ivan D.

    2014-10-02

    The unstable DNA adduct N2,3-ethenoguanine, a product of both exposure to the carcinogen vinyl chloride and of oxidative stress, was built into an oligonucleotide, using an isostere strategy to stabilize the glycosidic bond. This modification was then used to examine the cause of mutations by DNA polymerases, in terms of both the biochemistry of the lesion and a structure of the lesion within a polymerase.

  3. Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

    PubMed Central

    Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

    2010-01-01

    The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

  4. C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

    PubMed Central

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

    2009-01-01

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

  5. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

    PubMed

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

    2009-10-30

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

  6. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    PubMed

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  7. Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

    DOE PAGES

    Wallen, Jamie R.; Zhang, Hao; Weis, Caroline; ...

    2017-01-03

    The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

  8. Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wallen, Jamie R.; Zhang, Hao; Weis, Caroline

    The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

  9. Structural basis for the D-stereoselectivity of human DNA polymerase β

    PubMed Central

    Vyas, Rajan; Reed, Andrew J.; Raper, Austin T.; Zahurancik, Walter J.; Wallenmeyer, Petra C.

    2017-01-01

    Abstract Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolβ, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolβ follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolβ discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolβ to complete thumb domain closure. PMID:28402499

  10. Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae.

    PubMed

    Müller, M; Schnitzler, P; Koonin, E V; Darai, G

    1995-05-01

    Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

  11. Regulation and Modulation of Human DNA Polymerase δ Activity and Function

    PubMed Central

    Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y. C.

    2017-01-01

    This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4). PMID:28737709

  12. How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

    PubMed

    Wu, Wen-Jin; Su, Mei-I; Wu, Jian-Li; Kumar, Sandeep; Lim, Liang-Hin; Wang, Chun-Wei Eric; Nelissen, Frank H T; Chen, Ming-Chuan Chad; Doreleijers, Jurgen F; Wijmenga, Sybren S; Tsai, Ming-Daw

    2014-04-02

    A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

  13. Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

    PubMed Central

    Shock, David D.; Freudenthal, Bret D.; Beard, William A.; Wilson, Samuel H.

    2017-01-01

    DNA polymerases catalyze efficient and high fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, removing the DNA primer terminus and generating deoxynucleoside triphosphates. Since pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that it was limited by a non-chemical step. Utilizing a pyrophosphate analog, where the bridging oxygen is replaced with an imido-group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium that favored the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium. PMID:28759020

  14. A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

    PubMed Central

    Keith, Brian J.; Jozwiakowski, Stanislaw K.; Connolly, Bernard A.

    2013-01-01

    A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated–naphthoylated DEAE–cellulose, resulting in a low background mutation frequency (∼1 × 10−4). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system. PMID:23098700

  15. Conformational transitions in DNA polymerase I revealed by single-molecule FRET

    PubMed Central

    Santoso, Yusdi; Joyce, Catherine M.; Potapova, Olga; Le Reste, Ludovic; Hohlbein, Johannes; Torella, Joseph P.; Grindley, Nigel D. F.; Kapanidis, Achillefs N.

    2010-01-01

    The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates. PMID:20080740

  16. A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aller, Pierre; Duclos, Stéphanie; Wallace, Susan S.

    2012-06-27

    Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. and Doublie S. (2007). A structural rationale for stalling of a replicative DNA polymerase atmore » the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors - including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg - influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.« less

  17. Inhibition of RNA-Dependent DNA Polymerase of Avian Myeloblastosis Virus by Pyran Copolymer

    PubMed Central

    Papas, Takis S.; Pry, Thomas W.; Chirigos, Michael A.

    1974-01-01

    Pyran copolymer, a known immunostimulator, was found to be a potent inhibitor of purified DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) isolated from avian myeloblastosis virus. Unlike other inhibitors, pyran showed unique features of inhibition. It interacts with the polymerase at a region other than the template site. The inhibitory effect was overcome only by excess enzyme and not affected by excess template. The degree of inhibition was not template specific for the templates tested: 70S RNA from avian myeloblastosis virus, synthetic hybrid poly(rA)·oligo(dT)10, synthetic copolymer poly(dA-dT), and activated calf-thymus DNA. The observed rate of inhibition by pyran was shown to vary with the different polymerases tested. Inhibition was shown with all oncornaviral polymerases and, to a lesser extent, with mammalian polymerases. However, two of the three bacterial polymerases, by contrast, showed a marked activation. PMID:4131275

  18. Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

    PubMed

    Hicks, Matt N; Gunasekara, Sanjiva; Serate, Jose; Park, Jin; Mosharaf, Pegah; Zhou, Yue; Lee, Jin-Won; Youn, Hwan

    2017-10-01

    The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

  19. Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone.

    PubMed

    DeMott, Michael S; Beyret, Ergin; Wong, Donny; Bales, Brian C; Hwang, Jae-Taeg; Greenberg, Marc M; Demple, Bruce

    2002-03-08

    Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1' carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase beta is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase beta with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "short-patch" base excision repair but instead function as suicide substrates in the formation of protein-DNA cross-links that may require alternative modes of repair.

  20. Strand displacement synthesis by yeast DNA polymerase ε.

    PubMed

    Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

    2016-09-30

    DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Strand displacement synthesis by yeast DNA polymerase ε

    PubMed Central

    Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

    2016-01-01

    DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

  2. Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerases.

    PubMed

    Belguise-Valladier, P; Maki, H; Sekiguchi, M; Fuchs, R P

    1994-02-11

    In the present work, we have studied in vitro replication of N-2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II (cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the alpha subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G1G2CG3CC-) have been used in primer extension assays. Site-specifically platinated 5'd(ApG) or 5'd(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the NarI site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3'-->5' proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf (exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.

  3. Inhibition of DNA polymerase λ and associated inflammatory activities of extracts from steamed germinated soybeans.

    PubMed

    Mizushina, Yoshiyuki; Kuriyama, Isoko; Yoshida, Hiromi

    2014-04-01

    During the screening of selective DNA polymerase (pol) inhibitors from more than 50 plant food materials, we found that the extract from steamed germinated soybeans (Glycine max L.) inhibited human pol λ activity. Among the three processed soybean samples tested (boiled soybeans, steamed soybeans, and steamed germinated soybeans), both the hot water extract and organic solvent extract from the steamed germinated soybeans had the strongest pol λ inhibition. We previously isolated two glucosyl compounds, a cerebroside (glucosyl ceramide, AS-1-4, compound ) and a steroidal glycoside (eleutheroside A, compound ), from dried soybean, and these compounds were prevalent in the extracts of the steamed germinated soybeans as pol inhibitors. The hot water and organic solvent extracts of the steamed germinated soybeans and compounds and selectively inhibited the activity of eukaryotic pol λ in vitro but did not influence the activities of other eukaryotic pols, including those from the A-family (pol γ), B-family (pols α, δ, and ε), and Y-family (pols η, ι, and κ), and also showed no effect on the activity of pol β, which is of the same family (X) as pol λ. The tendency for in vitro pol λ inhibition by these extracts and compounds showed a positive correlation with the in vivo suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in mouse ear. These results suggest that steamed germinated soybeans, especially the glucosyl compound components, may be useful for their anti-inflammatory properties.

  4. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

    PubMed

    Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

    2018-06-01

    The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

  5. Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi.

    PubMed

    Rojas, Diego A; Urbina, Fabiola; Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo; Maldonado, Edio

    2018-02-01

    Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

  6. Mitochondrial Disorders of DNA Polymerase γ Dysfunction

    PubMed Central

    Zhang, Linsheng; Chan, Sherine S. L.; Wolff, Daynna J.

    2011-01-01

    Context Primary mitochondrial dysfunction is one of the most common causes of inherited disorders predominantly involving the neuromuscular system. Advances in the molecular study of mitochondrial DNA have changed our vision and our approach to primary mitochondrial disorders. Many of the mitochondrial disorders are caused by mutations in nuclear genes and are inherited in an autosomal recessive pattern. Among the autosomal inherited mitochondrial disorders, those related to DNA polymerase γ dysfunction are the most common and the best studied. Understanding the molecular mechanisms and being familiar with the recent advances in laboratory diagnosis of this group of mitochondrial disorders are essential for pathologists to interpret abnormal histopathology and laboratory results and to suggest further studies for a definitive diagnosis. Objectives To help pathologists better understand the common clinical syndromes originating from mutations in DNA polymerase γ and its associated proteins and use the stepwise approach of clinical, laboratory, and pathologic diagnosis of these syndromes. Data Sources Review of pertinent published literature and relevant Internet databases. Conclusions Mitochondrial disorders are now better recognized with the development of molecular tests for clinical diagnosis. A cooperative effort among primary physicians, diagnostic pathologists, geneticists, and molecular biologists with expertise in mitochondrial disorders is required to reach a definitive diagnosis. PMID:21732785

  7. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    NASA Technical Reports Server (NTRS)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

  8. Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2′-deoxyguanosine by Human DNA Polymerase η*

    PubMed Central

    Patra, Amitraj; Zhang, Qianqian; Guengerich, F. Peter; Egli, Martin

    2016-01-01

    O6-Methyl-2′-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the “translesion” Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. PMID:27694439

  9. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

    PubMed Central

    Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

    2018-01-01

    Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

  10. Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine*

    PubMed Central

    Su, Yan; Patra, Amritraj; Harp, Joel M.; Egli, Martin; Guengerich, F. Peter

    2015-01-01

    Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. PMID:25947374

  11. Family-wide analysis of poly(ADP-ribose) polymerase activity

    PubMed Central

    Uchima, Lilen; Rood, Jenny; Zaja, Roko; Hay, Ronald T.; Ahel, Ivan; Chang, Paul

    2014-01-01

    The poly(ADP-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as substrate. Based on the composition of three NAD+ coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino acid targets. In addition, we identify cysteine as a novel amino acid target for ADP-ribosylation on PARPs. PMID:25043379

  12. A Transient Kinetic Approach to Investigate Nucleoside Inhibitors of Mitochondrial DNA polymerase γ

    PubMed Central

    Anderson, Karen S.

    2010-01-01

    Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (kpol), the dissociation constant for the ground state binding (Kd), and the incorporation efficiency (kpol/Kd) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity. PMID:20573564

  13. Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

    PubMed

    Montgomery, Jesse L; Wittwer, Carl T

    2014-02-01

    Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

  14. Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta.

    PubMed

    Yamtich, Jen; Starcevic, Daniela; Lauper, Julia; Smith, Elenoe; Shi, Idina; Rangarajan, Sneha; Jaeger, Joachim; Sweasy, Joann B

    2010-03-23

    DNA polymerase beta (pol beta) is the key gap-filling polymerase in base excision repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol beta is also widely used as a model polymerase for structure and function studies, and several structural regions have been identified as being critical for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains. Previous work by our lab has shown that hinge residues Y265, I260, and F272 are critical for polymerase fidelity by functioning in discrimination of the correct from incorrect dNTP during ground state binding. Our work aimed to elucidate the role of hinge residue I174 in polymerase fidelity. To study this residue, we conducted a genetic screen to identify mutants with a substitution at residue I174 that resulted in a mutator polymerase. We then chose the mutator mutant I174S for further study and found that it follows the same general kinetic pathway as and has an overall protein folding similar to that of wild-type (WT) pol beta. Using single-turnover kinetic analysis, we found that I174S exhibits decreased fidelity when inserting a nucleotide opposite a template base G, and this loss of fidelity is due primarily to a loss of discrimination during ground state dNTP binding. Molecular dynamics simulations show that mutation of residue I174 to serine results in an overall tightening of the hinge region, resulting in aberrant protein dynamics and fidelity. These results point to the hinge region as being critical in the maintenance of the proper geometry of the dNTP binding pocket.

  15. DNA Polymerase III Star Requires ATP to Start Synthesis on a Primed DNA†

    PubMed Central

    Wickner, William; Kornberg, Arthur

    1973-01-01

    DNA polymerase III star replicates a ϕX174 single-stranded, circular DNA primed with a fragment of RNA. This reaction proceeds in two stages. In stage I, a complex is formed requiring DNA polymerase III star, ATP, spermidine, copolymerase III*, and RNA-primed ϕX174 single-stranded, circular DNA. The complex, isolated by gel filtration, contains ADP and inorganic phosphate (the products of a specific ATP cleavage) as well as spermidine, polymerase III star, and copolymerase III star. In stage II, the chain grows upon addition of deoxynucleoside triphosphates; ADP and inorganic phosphate are discharged and chain elongation is resistant to antibody to copolymerase III star. Thus ATP and copolymerase III star are required to initiate chain growth but not to sustain it. Images PMID:4519657

  16. A new building block for DNA network formation by self-assembly and polymerase chain reaction.

    PubMed

    Bußkamp, Holger; Keller, Sascha; Robotta, Marta; Drescher, Malte; Marx, Andreas

    2014-01-01

    The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

  17. Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

    PubMed Central

    Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo

    2018-01-01

    Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control. PMID:29432450

  18. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

    2013-06-25

    A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

  19. Probing Conformational Changes of Human DNA Polymerase λ Using Mass Spectrometry-Based Protein Footprinting

    PubMed Central

    Fowler, Jason D.; Brown, Jessica A.; Kvaratskhelia, Mamuka; Suo, Zucai

    2009-01-01

    SUMMARY Crystallographic studies of the C-terminal, DNA polymerase β-like domain of human DNA polymerase lambda (fPolλ) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase β. To examine solution-phase protein conformation changes in fPolλ, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a Proline-rich domain at its N-terminus, we used a mass spectrometry - based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPolλ versus the binary complex of enzyme•gapped DNA and the ternary complex of enzyme•gapped DNA•dNTP. These experiments suggested that fPolλ does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide dNTP. Site-directed mutagenesis and pre-steady state kinetic studies confirmed the importance of R386 for the enzyme activity, and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases. PMID:19467241

  20. DNA polymerase ɛ and δ exonuclease domain mutations in endometrial cancer

    PubMed Central

    Church, David N.; Briggs, Sarah E.W.; Palles, Claire; Domingo, Enric; Kearsey, Stephen J.; Grimes, Jonathon M.; Gorman, Maggie; Martin, Lynn; Howarth, Kimberley M.; Hodgson, Shirley V.; Kaur, Kulvinder; Taylor, Jenny; Tomlinson, Ian P.M.

    2013-01-01

    Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ɛ, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5–10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis. PMID:23528559

  1. Electron Microscopic Analysis of the Products of DNA Synthesis by DNA Polymerases from Calf Thymus and Herpes Simplex Virus Type I

    DTIC Science & Technology

    1988-10-03

    DNA replication showed an average of 2.5 primers per M13 DNA circle. The measurement of the double stranded length from individual replicative intermediates by electron microscopy was within the accuracy of 10% standard deviation. The product length distribution obtained from the HSV-1 DNA polymerase catalyzed replication of M13 DNA primed with a specific pentadecamer and in the presence of E. Coli SSB protein showed a near Poisson distribution. Replication of the same primer-template system or DNA primase primed M13 DNA template by calf thymus DNA polymerase a showed a

  2. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  3. Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ

    PubMed Central

    Zahurancik, Walter J.; Klein, Seth J.; Suo, Zucai

    2014-01-01

    Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation. PMID:25414327

  4. DNA Polymerase λ Inactivation by Oxidized Abasic Sites&

    PubMed Central

    Stevens, Adam J.; Guan, Lirui; Bebenek, Katarzyna; Kunkel, Thomas A.; Greenberg, Marc M.

    2013-01-01

    Base excision repair plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ is believed to play a backup role to DNA polymerase β in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA damaging agents, including several antitumor antibiotics, the C4′-oxidized abasic site following Ape1 incision (pC4-AP) and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single turnover kinetic experiments show that Pol λ excises DOB almost 13-times more slowly than a 5′-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β at the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β and the difference is attributable to a higher KI (33 ± 7 nM). Inactivation of Pol λ’s lyase activity by DOB also prevents the enzyme from carrying out polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. Protein modification involves reaction with released but-2-ene-1,4-dial. When acting on pC4-AP, Pol λ achieves approximately 4 turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA damaging agents that

  5. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D

    PubMed Central

    Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3’-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotide and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologs of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both, in an M. smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5’-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. PMID:23198659

  6. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

    PubMed

    Ramsay, Nicola; Jemth, Ann-Sofie; Brown, Anthony; Crampton, Neal; Dear, Paul; Holliger, Philipp

    2010-04-14

    DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

  7. Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2'-deoxyguanosine.

    PubMed

    Su, Yan; Patra, Amritraj; Harp, Joel M; Egli, Martin; Guengerich, F Peter

    2015-06-26

    Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I.

    PubMed Central

    Odelberg, S J; Weiss, R B; Hata, A; White, R

    1995-01-01

    Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching. Images PMID:7596836

  9. Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

    PubMed

    Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

    2017-01-01

    Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016

  10. The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.

    PubMed

    Castrec, Benoît; Laurent, Sébastien; Henneke, Ghislaine; Flament, Didier; Raffin, Jean-Paul

    2010-03-05

    A glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D. (c) 2010 Elsevier Ltd. All rights reserved.

  11. Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

    PubMed

    Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

    2011-02-04

    La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

  12. DNA Damage Repair and the Emerging Role of Poly(ADP-ribose) Polymerase Inhibition in Cancer Therapeutics.

    PubMed

    Rabenau, Karen; Hofstatter, Erin

    2016-07-01

    As a result of improved understanding of DNA repair mechanisms, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasingly recognized to play an important therapeutic role in the treatment of cancer. The aim of this article is to provide a review of PARPi function in DNA damage repair and synthetic lethality and to demonstrate how these mechanisms can be exploited to provide new PARPi-based therapies to patients with solid tumors. Literature from a range of sources, including PubMed and MEDLINE, were searched to identify recent reports regarding DNA damage repair and PARPi. DNA damage repair is central to cellular viability. The family of poly(ADP-ribose) polymerase proteins play multiple intracellular roles in DNA repair, but function primarily in the resolution of repair of single-strand DNA breaks. Insights through the discovery of germline BRCA1/2 mutations led to the understanding of synthetic lethality and the potential therapeutic role of PARPi in the treatment of cancer. Further understanding of DNA damage repair and the concept of BRCA-like tumors have catalyzed PARPi clinical investigation in multiple oncologic settings. PARPi hold great promise in the treatment of solid tumors, both as monotherapy and in combination with other cancer therapeutics. Multiple PARPi clinical trials are currently underway. Further understanding of aberrant DNA repair mechanisms in the germline and in the tumor genome will allow clinicians and researchers to apply PARPi most strategically in the era of personalized medicine. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

  13. Fixing the model for transcription: the DNA moves, not the polymerase.

    PubMed

    Papantonis, Argyris; Cook, Peter R

    2011-01-01

    The traditional model for transcription sees active polymerases tracking along their templates. An alternative (controversial) model has active enzymes immobilized in "factories." Recent evidence supports the idea that the DNA moves, not the polymerase, and points to alternative explanations of how regulatory motifs like enhancers and silencers work.

  14. Mechanism of Microhomology-Mediated End-Joining Promoted by Human DNA Polymerase Theta

    PubMed Central

    Kent, Tatiana; Chandramouly, Gurushankar; McDevitt, Shane Michael; Ozdemir, Ahmet Y.; Pomerantz, Richard T.

    2014-01-01

    Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break repair pathway that utilizes sequence microhomology to recombine broken DNA. Although MMEJ is implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3’ single-strand DNA overhangs with two or more base-pairs of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing then utilizes the opposing overhang as a template in trans which stabilizes the DNA synapse. Polθ exhibits a preference for DNA containing a 5’-terminal phosphate, similar to polymerases involved in non-homologous end-joining. Lastly, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ which likely promote DNA synapse formation. PMID:25643323

  15. CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase

    PubMed Central

    2010-01-01

    DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting “CyDNA” displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties. PMID:20235594

  16. Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues.

    PubMed

    Killelea, Tom; Ghosh, Samantak; Tan, Samuel S; Heslop, Pauline; Firbank, Susan J; Kool, Eric T; Connolly, Bernard A

    2010-07-13

    Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

  17. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    PubMed

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. DNA-polymerase induced by Herpesvirus papio (HVP) in cells of lymphoblastoid cultures derived from lymphomatous baboons. Report V.

    PubMed

    Djachenko, A G; Lapin, B A

    1981-01-01

    A new DNA-polymerase was found in the cells of suspension lymphoblastoid cultures which produce lymphotropic baboon herpesvirus (HVP). This enzyme was isolated in a partially purified form. Some of its properties vary from those of other cellular DNA-polymerases. HVP-induced DNA-polymerase has a molecule weight of 160,000 and sedimentation coefficient of about 8 S. The enzyme is resistant to high salt concentration and N-ethylmaleimide, but it is very sensitive to phosphonoacetate. It effectively copies "activated" DNA and synthetic deoxyribohomopolymers. Attempts to reveal the DNA-polymerase activity in HVP virions were unsuccessful.

  19. Engineering of a DNA Polymerase for Direct m6 A Sequencing.

    PubMed

    Aschenbrenner, Joos; Werner, Stephan; Marchand, Virginie; Adam, Martina; Motorin, Yuri; Helm, Mark; Marx, Andreas

    2018-01-08

    Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N 6 -methyladenosine (m 6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m 6 A-containing RNA prior to sequencing, since m 6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m 6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N 6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m 6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m 6 A sites directly from the sequencing data of untreated RNA samples. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

    PubMed

    Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

    1998-08-28

    The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

  1. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase iota: Hoogsteen or Watson-Crick base pairing?

    PubMed

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-13

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase iota (poliota) is a bypass polymerase of the Y family. Crystal structures of poliota suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that poliota is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in poliota for bypass of dG-AAF. In poliota with Hoogsteen-paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick-paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that poliota would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for poliota in lesion bypass.

  2. Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases

    PubMed Central

    Su, Yan; Guengerich, F. Peter

    2016-01-01

    Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. PMID:27248785

  3. Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hogg, Matthew; Rudnicki, Jean; Midkiff, John

    2010-04-12

    The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of amore » lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.« less

  4. Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η.

    PubMed

    Patra, Amitraj; Zhang, Qianqian; Guengerich, F Peter; Egli, Martin

    2016-11-11

    O 6 -Methyl-2'-deoxyguanosine (O 6 -MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O 6 -MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O 6 -MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O 6 - MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O 6 -MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O 6 -MeG:dC or O 6 -MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. © 2016 by

  5. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  6. E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

    PubMed Central

    Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

    2001-01-01

    The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

  7. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome.

    PubMed

    Singh, Bhupendra; Li, Xiurong; Owens, Kjerstin M; Vanniarajan, Ayyasamy; Liang, Ping; Singh, Keshav K

    2015-01-01

    To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.

  8. MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

    PubMed Central

    Stumpf, Jeffrey D.; Copeland, William C.

    2014-01-01

    Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but

  9. MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

    PubMed

    Stumpf, Jeffrey D; Copeland, William C

    2014-10-01

    Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but

  10. Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

    PubMed Central

    Koc, Katrina N.; Stodola, Joseph L.; Burgers, Peter M.; Galletto, Roberto

    2015-01-01

    The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo− to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities. PMID:25813050

  11. Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

    PubMed

    Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

    2018-05-05

    Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Archaeal RNA polymerase arrests transcription at DNA lesions.

    PubMed

    Gehring, Alexandra M; Santangelo, Thomas J

    2017-01-01

    Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

  13. Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues†

    PubMed Central

    Killelea, Tom; Ghosh, Samantak; Tan, Samuel S.; Heslop, Pauline; Firbank, Susan; Kool, Eric T.; Connolly, Bernard A.

    2010-01-01

    Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describes uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at the +2 position explains the stimulation of the polymerase 3′-5′ proof reading exonuclease, observed with deaminated bases at this location. A β hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double stranded DNA. This denatures the two complementary primer bases and directs the resulting 3′ single-stranded extension towards the exonuclease active site. Finally the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using non-polar isosteres. Affinity for both 2,4 difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4 difluorobenzene is seen, confirming a role for shape recognition. PMID:20527806

  14. [Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

    PubMed

    Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

    1997-02-01

    The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

  15. Role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dasgupta, S.; Mitra, S.

    The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an E. coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.

  16. DNA and RNA polymerase activity in a Moniliophthora perniciosa mitochondrial plasmid and self-defense against oxidative stress.

    PubMed

    Andrade, B S; Villela-Dias, C; Gomes, D S; Micheli, F; Góes-Neto, A

    2013-06-13

    Moniliophthora perniciosa (Stahel) Aime and Phillips-Mora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack.

  17. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  18. Each Monomer of the Dimeric Accessory Protein for Human Mitochondrial DNA Polymerase Has a Distinct Role in Conferring Processivity*

    PubMed Central

    Lee, Young-Sam; Lee, Sujin; Demeler, Borries; Molineux, Ian J.; Johnson, Kenneth A.; Yin, Y. Whitney

    2010-01-01

    The accessory protein polymerase (pol) γB of the human mitochondrial DNA polymerase stimulates the synthetic activity of the catalytic subunit. pol γB functions by both accelerating the polymerization rate and enhancing polymerase-DNA interaction, thereby distinguishing itself from the accessory subunits of other DNA polymerases. The molecular basis for the unique functions of human pol γB lies in its dimeric structure, where the pol γB monomer proximal to pol γA in the holoenzyme strengthens the interaction with DNA, and the distal pol γB monomer accelerates the reaction rate. We further show that human pol γB exhibits a catalytic subunit- and substrate DNA-dependent dimerization. By duplicating the monomeric pol γB of lower eukaryotes, the dimeric mammalian proteins confer additional processivity to the holoenzyme polymerase. PMID:19858216

  19. Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ.

    PubMed

    Klvaňa, Martin; Bren, Urban; Florián, Jan

    2016-12-29

    Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3'-OH group of the primer DNA strand, and the subsequent formation and cleavage of P-O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol -1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P-O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pK a of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

  20. Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

    PubMed Central

    2016-01-01

    Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3′-OH group of the primer DNA strand, and the subsequent formation and cleavage of P–O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol–1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P–O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3′-OH group as a predictor of the catalytic rate of X-family DNA polymerases. PMID:27992186

  1. In Vitro Lesion Bypass Studies of O(4)-Alkylthymidines with Human DNA Polymerase η.

    PubMed

    Williams, Nicole L; Wang, Pengcheng; Wu, Jiabin; Wang, Yinsheng

    2016-04-18

    Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O(4)-alkylthymidine (O(4)-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O(4)-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O(4)-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O(4)-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O(4)-iPrdT, O(4)-iBudT, and O(4)-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O(4)-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

  2. Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang, K.H.; /Ohio State U.; Niebuhr, M.

    2009-04-30

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDamore » 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.« less

  3. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Y.; Dunn, J.; Gao, S.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing asmore » little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.« less

  4. Effects of intermediates between vitamins K(2) and K(3) on mammalian DNA polymerase inhibition and anti-inflammatory activity.

    PubMed

    Mizushina, Yoshiyuki; Maeda, Jun; Irino, Yasuhiro; Nishida, Masayuki; Nishiumi, Shin; Kondo, Yasuyuki; Nishio, Kazuyuki; Kuramochi, Kouji; Tsubaki, Kazunori; Kuriyama, Isoko; Azuma, Takeshi; Yoshida, Hiromi; Yoshida, Masaru

    2011-02-10

    Previously, we reported that vitamin K(3) (VK(3)), but not VK(1) or VK(2) (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK(2) and VK(3), namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK(3) was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC(50) value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK(2) and VK(3) intermediates, such as MK-2, that are promising anti-inflammatory candidates.

  5. The actin multigene family and livestock speciation using the polymerase chain reaction.

    PubMed

    Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

    1998-01-01

    Actins constitute a family of highly-conserved multifunctional intracellular proteins, best known as myofibrillar components in striated muscle fibres. Most vertebrate genomes contain numerous actin genes with high sequence homology in protein coding regions but considerable variability in intron number and sizes. This genetic diversity can be utilised for livestock speciation purposes. The high sequence conservation has enabled a single pair of oligonucleotides to be used to prime the polymerase chain reaction (PCR) with DNA extracted from all animals so far studied. Multiple amplification products were obtained which on gel electrophoresis constituted characteristic species-specific 'fingerprints'. The patterns were reproducible, did not vary between individuals of the same breed or between different breeds within a species, and could be generated even from heat-processed muscle held at 120 degrees C for one hour. Given the capacity of PCR to amplify relatively short sequences in highly-degraded DNA, this approach may be suitable for authentication of processed meat products.

  6. Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

    E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

  7. Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η.

    PubMed

    Genna, Vito; Gaspari, Roberto; Dal Peraro, Matteo; De Vivo, Marco

    2016-04-07

    Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Functional Architecture of T7 RNA Polymerase Transcription Complexes

    PubMed Central

    Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

    2007-01-01

    Summary T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. PMID:17580086

  9. Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

    PubMed Central

    Kuhl, Alexander; Svenstrup, Niels; Ladel, Christoph; Otteneder, Michael; Binas, Annegret; Schiffer, Guido; Brands, Michael; Lampe, Thomas; Ziegelbauer, Karl; Rübsamen-Waigmann, Helga; Haebich, Dieter; Ehlert, Kerstin

    2005-01-01

    Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 μg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired. PMID:15728893

  10. Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3'-to-5' Direction and Is Dependent on the 3'-to-5' Exonuclease Active Site.

    PubMed

    Lawler, Jessica L; Mukherjee, Purba; Coen, Donald M

    2018-03-01

    The catalytic subunit (Pol) of herpes simplex virus 1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA-DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3'-to-5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3'-to-5' exonuclease-deficient mutant, D368A Pol, for DNA polymerase activity, 3'-to-5' exonuclease activity, and RNase H activity in vitro Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity but was drastically impaired for 3'-to-5' exonuclease activity, with no activity detected even at high enzyme-to-DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3' or 5' terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA-DNA hairpin template, but only detectably with a 3' RNA terminus in a 3'-to-5' direction and at a rate lower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3'-to-5' exonuclease activity and that this activity prefers DNA degradation over degradation of RNA from RNA-DNA hybrids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a member of the Herpesviridae family of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5'-to-3' RNase H activity intrinsic to this enzyme

  11. Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    PubMed Central

    Sun, Fei; Huang, Li

    2013-01-01

    Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

  12. Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

    PubMed

    Gregory, Mark T; Park, Ga Young; Johnstone, Timothy C; Lee, Young-Sam; Yang, Wei; Lippard, Stephen J

    2014-06-24

    Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

  13. Light-dependent, plastome-wide association of the plastid-encoded RNA polymerase with chloroplast DNA.

    PubMed

    Finster, Sabrina; Eggert, Erik; Zoschke, Reimo; Weihe, Andreas; Schmitz-Linneweber, Christian

    2013-12-01

    Plastid genes are transcribed by two types of RNA polymerases: a plastid-encoded eubacterial-type RNA polymerase (PEP) and nuclear-encoded phage-type RNA polymerases (NEPs). To investigate the spatio-temporal expression of PEP, we tagged its α-subunit with a hemagglutinin epitope (HA). Transplastomic tobacco plants were generated and analyzed for the distribution of the tagged polymerase in plastid sub-fractions, and associated genes were identified under various light conditions. RpoA:HA was detected as early as the 3rd day after imbibition, and was constitutively expressed in green tissue over 60 days of plant development. We found that the tagged polymerase subunit preferentially associated with the plastid membranes, and was less abundant in the soluble stroma fraction. Attachment of RpoA:HA to the membrane fraction during early seedling development was independent of DNA, but at later stages of development, DNA appears to facilitate attachment of the polymerase to membranes. To survey PEP-dependent transcription units, we probed for nucleic acids enriched in RpoA:HA precipitates using a tobacco chloroplast whole-genome tiling array. The most strongly co-enriched DNA fragments represent photosynthesis genes (e.g. psbA, psbC, psbD and rbcL), whose expression is known to be driven by PEP promoters, while NEP-dependent genes were less abundant in RpoA:HA precipitates. Additionally, we demonstrate that the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that plastome-wide PEP-DNA association is a light-dependent process. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  14. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

    PubMed Central

    Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

    2014-01-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

  15. Pyrovanadolysis: a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate MN2plus and DNA Polymerase of Bacteriophage T7

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    B Akabayov; A Kulczyk; S Akabayov

    2011-12-31

    DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP{sub i}). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP{sub i}, a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-Vmore » distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP{sub i} complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn{sup 2+}, larger than Mg{sup 2+}, fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.« less

  16. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  17. Target-induced Catalytic Assembly of Y-Shaped DNA and Its Application for In Situ Imaging of MicroRNAs.

    PubMed

    Xue, Chang; Zhang, Shu-Xin; Ouyang, Chang-He; Chang, Dingran; Salena, Bruno J; Li, Yingfu; Wu, Zai-Sheng

    2018-06-14

    DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase ι: Hoogsteen or Watson-Crick base pairing?†

    PubMed Central

    Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

    2009-01-01

    Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase ι (polι) is a bypass polymerase of the Y family. Crystal structures of polι suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that polι is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in polι for bypass of dG-AAF. In polι with Hoogsteen paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that polι would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for polι in lesion bypass. PMID:19072536

  19. Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

    PubMed Central

    Eissenberg, J C; Ayyagari, R; Gomes, X V; Burgers, P M

    1997-01-01

    The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks. PMID:9343398

  20. Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast

    PubMed Central

    Guo, Xiaoge; Jinks-Robertson, Sue

    2013-01-01

    Transformation-based gap-repair assays have long been used to model the repair of mitotic double-strand breaks (DSBs) by homologous recombination in yeast. In the current study, we examine genetic requirements of two key processes involved in DSB repair: (1) the processive 5′-end resection that is required to efficiently engage a repair template and (2) the filling of resected ends by DNA polymerases. The specific gap-repair assay used allows repair events resolved as crossover versus noncrossover products to be distinguished, as well as the extent of heteroduplex DNA formed during recombination to be measured. To examine end resection, the efficiency and outcome of gap repair were monitored in the absence of the Exo1 exonuclease and the Sgs1 helicase. We found that either Exo1 or Sgs1 presence is sufficient to inhibit gap-repair efficiency over 10-fold, consistent with resection-mediated destruction of the introduced plasmid. In terms of DNA polymerase requirements for gap repair, we focused specifically on potential roles of the Pol ζ and Pol η translesion synthesis DNA polymerases. We found that both Pol ζ and Pol η are necessary for efficient gap repair and that each functions independently of the other. These polymerases may be either in the initiation of DNA synthesis from the an invading end, or in a gap-filling process that is required to complete recombination. PMID:24210827

  1. Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli.

    PubMed

    Corzett, Christopher H; Goodman, Myron F; Finkel, Steven E

    2013-06-01

    Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to <20% during long-term stationary phase. Pol IV transcription dominates as cells transition out of exponential phase into stationary phase and a burst of Pol V transcription is observed as cells transition from death phase to long-term stationary phase. These changes in alternative DNA polymerase transcription occur in the absence of SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

  2. Structural basis for the suppression of skin cancers by DNA polymerase [eta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Silverstein, Timothy D.; Johnson, Robert E.; Jain, Rinku

    2010-09-13

    DNA polymerase {eta} (Pol{eta}) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Pol{eta} (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Pol{eta} (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Pol{eta} to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in anmore » active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln55, Arg73 and Met74. Together, these features define the basis for Pol{eta}'s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.« less

  3. [Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I].

    PubMed

    Shematorova, E K; Shpakovskiĭ, G V

    2002-01-01

    In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

  4. Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays.

    PubMed Central

    deWit, D; Wootton, M; Allan, B; Steyn, L

    1993-01-01

    A simple method for the production of internal control DNA for two well-established Mycobacterium tuberculosis polymerase chain reaction assays is described. The internal controls were produced from Mycobacterium kansasii DNA with the same primers but at a lower annealing temperature than that used in the standard assays. In both assays, therefore, the internal control DNA has the same primer-binding sequences at the target DNA. One-microgram quantities of internal control DNA which was not contaminated with target DNA could easily be produced by this method. The inclusion of the internal control in the reaction mixture did not affect the efficiency of amplification of the target DNA. The method is simple and rapid and should be adaptable to most M. tuberculosis polymerase chain reaction assays. Images PMID:8370752

  5. Estimating HPV DNA Deposition Between Sexual Partners Using HPV Concordance, Y Chromosome DNA Detection, and Self-reported Sexual Behaviors.

    PubMed

    Malagón, Talía; Burchell, Ann N; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L

    2017-12-05

    Detection of human papillomavirus (HPV) DNA in genital samples may not always represent true infections but may be depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker (Y chromosome DNA) were associated with genital HPV partner concordance and estimated the fraction of HPV detections potentially attributable to partner deposition. The HITCH study enrolled young women attending a university or college in Montréal, Canada, and their male partners, from 2005 to 2010. We tested baseline genital samples for Y chromosome DNA and HPV DNA using polymerase chain reaction. Type-specific HPV concordance was 42.4% in partnerships where at least one partner was HPV DNA positive. Y chromosome DNA predicted type-specific HPV concordance in univariate analyses, but in multivariable models the independent predictors of concordance were days since last vaginal sex (26.5% higher concordance 0-1 vs 8-14 days after last vaginal sex) and condom use (22.6% higher concordance in never vs always users). We estimated that 14.1% (95% confidence interval [CI], 6.3-21.9%) of HPV DNA detections in genital samples were attributable to vaginal sex in the past week. A substantial proportion of HPV DNA detections may be depositions due to recent unprotected vaginal sex. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  6. DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

    PubMed

    Barnes, Ryan P; Hile, Suzanne E; Lee, Marietta Y; Eckert, Kristin A

    2017-09-01

    Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

    PubMed Central

    Kelly, Richard D. W.; Mahmud, Arsalan; McKenzie, Matthew; Trounce, Ian A.; St John, Justin C.

    2012-01-01

    DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization. PMID:22941637

  8. Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA

    NASA Astrophysics Data System (ADS)

    Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia; Adhikary, Ramkrishna; Tsuen, Shujian S.; Romesberg, Floyd E.

    2016-06-01

    The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2ʹ positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2ʹ-OMe-modified oligonucleotides and their DNA counterparts via ‘transcription’ and ‘reverse transcription’ or, more importantly, that PCR-amplify partially C2ʹ-OMe- or C2ʹ-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.

  9. Elucidation of Kinetic Mechanisms of Human Translesion DNA Polymerase κ Using Tryptophan Mutants

    PubMed Central

    Zhao, Linlin; Pence, Matthew G.; Eoff, Robert L.; Yuan, Shuai; Fercu, Catinca A.; Guengerich, F. Peter

    2014-01-01

    In order to investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), as judged by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3′-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat, suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair. PMID:25065501

  10. Archaeal replicative primases can perform translesion DNA synthesis.

    PubMed

    Jozwiakowski, Stanislaw K; Borazjani Gholami, Farimah; Doherty, Aidan J

    2015-02-17

    DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

  11. Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor.

    PubMed

    Roux-Michollet, Dad D; Schimel, Joshua P; Holden, Patricia A

    2010-12-01

    Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.

    PubMed

    Plachta, Michal; Halas, Agnieszka; McIntyre, Justyna; Sledziewska-Gojska, Ewa

    2015-05-01

    Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

    NASA Astrophysics Data System (ADS)

    Fujita, Keisuke; Iwaki, Mitsuhiro; Yanagida, Toshio

    2016-12-01

    Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

  14. Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι

    PubMed Central

    McIntyre, Justyna; Vidal, Antonio E.; McLenigan, Mary P.; Bomar, Martha G.; Curti, Elena; McDonald, John P.; Plosky, Brian S.; Ohashi, Eiji; Woodgate, Roger

    2013-01-01

    Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in ‘replication factories’ in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS. PMID:23248005

  15. A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

    PubMed Central

    Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

    2013-01-01

    Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

  16. The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

    PubMed Central

    Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-01-01

    Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system

  17. Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database.

    PubMed

    Nurminen, Anssi; Farnum, Gregory A; Kaguni, Laurie S

    2017-06-01

    DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO). We have collected all publicly available POLG-related patient data and analyzed it using our pathogenic clustering model to provide a new research and clinical tool in the form of an online server. The server evaluates the pathogenicity of both previously reported and novel mutations. There are currently 176 unique point mutations reported and found in mitochondrial patients in the gene encoding the catalytic subunit of POLG, POLG . The mutations are distributed nearly uniformly along the length of the primary amino acid sequence of the gene. Our analysis shows that most of the mutations are recessive, and that the reported dominant mutations cluster within the polymerase active site in the tertiary structure of the POLG enzyme. The POLG Pathogenicity Prediction Server (http://polg.bmb.msu.edu) is targeted at clinicians and scientists studying POLG disorders, and aims to provide the most current available information regarding the pathogenicity of POLG mutations.

  18. Cid1, a Fission Yeast Protein Required for S-M Checkpoint Control when DNA Polymerase δ or ɛ Is Inactivated

    PubMed Central

    Wang, Shao-Win; Toda, Takashi; MacCallum, Robert; Harris, Adrian L.; Norbury, Chris

    2000-01-01

    The S-M checkpoint is an intracellular signaling pathway that ensures that mitosis is not initiated in cells undergoing DNA replication. We identified cid1, a novel fission yeast gene, through its ability when overexpressed to confer specific resistance to a combination of hydroxyurea, which inhibits DNA replication, and caffeine, which overrides the S-M checkpoint. Cid1 overexpression also partially suppressed the hydroxyurea sensitivity characteristic of DNA polymerase δ mutants and mutants defective in the “checkpoint Rad” pathway. Cid1 is a member of a family of putative nucleotidyltransferases including budding yeast Trf4 and Trf5, and mutation of amino acid residues predicted to be essential for this activity resulted in loss of Cid1 function in vivo. Two additional Cid1-like proteins play similar but nonredundant checkpoint-signaling roles in fission yeast. Cells lacking Cid1 were found to be viable but specifically sensitive to the combination of hydroxyurea and caffeine and to be S-M checkpoint defective in the absence of Cds1. Genetic data suggest that Cid1 acts in association with Crb2/Rhp9 and through the checkpoint-signaling kinase Chk1 to inhibit unscheduled mitosis specifically when DNA polymerase δ or ɛ is inhibited. PMID:10757807

  19. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein.more » Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.« less

  20. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    PubMed Central

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  1. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

    PubMed Central

    Dean, Frank B.; Nelson, John R.; Giesler, Theresa L.; Lasken, Roger S.

    2001-01-01

    We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications. PMID:11381035

  2. RNA Polymerase Collision versus DNA Structural Distortion: Twists and Turns Can Cause Break Failure

    PubMed Central

    Pannunzio, Nicholas R.; Lieber, Michael R.

    2016-01-01

    Summary The twisting of DNA due to the movement of RNA polymerases is the basis of numerous classic experiments in molecular biology. Recent mouse genetic models indicate that chromosomal breakage is common at sites of transcriptional turbulence. Two key studies on this point mapped breakpoints to sites of either convergent or divergent transcription, but arrived at different conclusions as to which is more detrimental and why. The issue turns on whether DNA strand separation is the basis for the chromosomal instability or collision of RNA polymerases? PMID:27153532

  3. Coordinating DNA polymerase traffic during high and low fidelity synthesis.

    PubMed

    Sutton, Mark D

    2010-05-01

    With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell's genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  4. RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

    PubMed

    KIreeva, Maria; Trang, Cyndi; Matevosyan, Gayane; Turek-Herman, Joshua; Chasov, Vitaly; Lubkowska, Lucyna; Kashlev, Mikhail

    2018-06-20

    Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA-DNA base pair at the upstream edge of the transcription bubble. DNA-DNA base pairing immediately upstream from the RNA-DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA-DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

  5. A novel variant of DNA polymerase ζ, Rev3ΔC, highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

    PubMed Central

    Siebler, Hollie M.; Lada, Artem G.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Pavlov, Youri I.

    2014-01-01

    Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches. PMID:24819597

  6. Laser crosslinking of E. coli RNA polymerase and T7 DNA.

    PubMed Central

    Harrison, C A; Turner, D H; Hinkle, D C

    1982-01-01

    The first photochemical crosslinking of a protein to a nucleic acid using laser excitation is reported. A single, 120 mJ, 20 ns pulse at 248 nm crosslinks about 10% of bound E. coli RNA polymerase to T7 DNA under the conditions studied. The crosslinking yield depends on mercaptoethanol concentration, and is a linear function of laser intensity. The protein subunits crosslinked to DNA are beta, beta' and sigma. PMID:7045809

  7. Revealing the role of the product metal in DNA polymerase β catalysis

    PubMed Central

    Freudenthal, Bret D.; Beard, William A.; Pedersen, Lee G.; Wilson, Samuel H.

    2017-01-01

    Abstract DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction. PMID:28108654

  8. Plasimids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, Sanford A.; Martinez, Susana; Lopez, Paloma; Espinosa, Manuel

    1991-01-01

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme.

  9. An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis.

    PubMed

    Li, Jing; Chen, Ran; Yang, Ye; Zhang, Zhemin; Fang, Guang-Chen; Xie, Wei; Cao, Weiguo

    2017-12-01

    The uracil DNA glycosylase superfamily consists of at least six families with a diverse specificity toward DNA base damage. Family 1 uracil N-glycosylase (UNG) exhibits exclusive specificity on uracil-containing DNA. Here, we report a family 1 UNG homolog from Nitratifractor salsuginis with distinct biochemical features that differentiate it from conventional family 1 UNGs. Globally, the crystal structure of N. salsuginisUNG shows a few additional secondary structural elements. Biochemical and enzyme kinetic analysis, coupled with structural determination, molecular modeling, and molecular dynamics simulations, shows that N. salsuginisUNG contains a salt bridge network that plays an important role in DNA backbone interactions. Disruption of the amino acid residues involved in the salt bridges greatly impedes the enzymatic activity. A tyrosine residue in motif 1 (GQDPY) is one of the distinct sequence features setting family 1 UNG apart from other families. The crystal structure of Y81G mutant indicates that several subtle changes may account for its inactivity. Unlike the conventional family 1 UNG enzymes, N. salsuginisUNG is not inhibited by Ugi, a potent inhibitor specific for family 1 UNG. This study underscores the diversity of paths that a uracil DNA glycosylase may take to acquire its unique structural and biochemical properties during evolution. Structure data are available in the PDB under accession numbers 5X3G and 5X3H. © 2017 Federation of European Biochemical Societies.

  10. Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight.

    PubMed

    Kozmin, Stanislav G; Pavlov, Youri I; Kunkel, Thomas A; Sage, Evelyne

    2003-08-01

    Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase eta (Poleta) and polymerase zeta (Polzeta), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310-1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Delta, rev3Delta and rev3Delta rad30Delta strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6-4) photoproducts derived from studies with UVC. They further suggest that Poleta participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polzeta is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polzeta, Poleta contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.

  11. DNA polymerase η mutational signatures are found in a variety of different types of cancer.

    PubMed

    Rogozin, Igor B; Goncearenco, Alexander; Lada, Artem G; De, Subhajyoti; Yurchenko, Vyacheslav; Nudelman, German; Panchenko, Anna R; Cooper, David N; Pavlov, Youri I

    2018-01-01

    DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.

  12. A structural role for the PHP domain in E. coli DNA polymerase III

    PubMed Central

    2013-01-01

    Background In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Results Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. Conclusions While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase. PMID:23672456

  13. A structural role for the PHP domain in E. coli DNA polymerase III.

    PubMed

    Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O'Donnell, Mike; Kuriyan, John; Lamers, Meindert H

    2013-05-14

    In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

  14. A RecA Protein Surface Required for Activation of DNA Polymerase V

    PubMed Central

    Gruber, Angela J.; Erdem, Aysen L.; Sabat, Grzegorz; Karata, Kiyonobu; Jaszczur, Malgorzata M.; Vo, Dan D.; Olsen, Tayla M.; Woodgate, Roger; Goodman, Myron F.; Cox, Michael M.

    2015-01-01

    DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. PMID:25811184

  15. Effect of human cell malignancy on activity of DNA polymerase iota.

    PubMed

    Kazakov, A A; Grishina, E E; Tarantul, V Z; Gening, L V

    2010-07-01

    An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by

  16. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  17. Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

    PubMed Central

    Skinner, Gary M.; Kalafut, Bennett S.; Visscher, Koen

    2011-01-01

    Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the “scrunching” model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

  18. Localized Cerebral Energy Failure in DNA Polymerase Gamma-Associated Encephalopathy Syndromes

    ERIC Educational Resources Information Center

    Tzoulis, Charalampos; Neckelmann, Gesche; Mork, Sverre J.; Engelsen, Bernt E.; Viscomi, Carlo; Moen, Gunnar; Ersland, Lars; Zeviani, Massimo; Bindoff, Laurence A.

    2010-01-01

    Mutations in the catalytic subunit of the mitochondrial DNA-polymerase gamma cause a wide spectrum of clinical disease ranging from infantile hepato-encephalopathy to juvenile/adult-onset spinocerebellar ataxia and late onset progressive external ophthalmoplegia. Several of these syndromes are associated with an encephalopathy that…

  19. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1991-03-26

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 figure.

  20. DNA extraction from coral reef sediment bacteria for the polymerase chain reaction.

    PubMed

    Guthrie, J N; Moriarty, D J; Blackall, L L

    2000-12-15

    A rapid and effective method for the direct extraction of high molecular weight amplifiable DNA from two coral reef sediments was developed. DNA was amplified by the polymerase chain reaction (PCR) using 16S rDNA specific primers. The amplicons were digested with HaeIII, HinP1I and MspI and separated using polyacrylamide gel electrophoresis and silver staining. The resulting amplified ribosomal DNA restriction analysis (ARDRA) patterns were used as a fingerprint to discern differences between the coral reef sediment samples. Results indicated that ARDRA is an effective method for determining differences within the bacterial community amongst different environmental samples.

  1. DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

    PubMed Central

    Masuda, Keiji; Ouchida, Rika; Takeuchi, Arata; Saito, Takashi; Koseki, Haruhiko; Kawamura, Kiyoko; Tagawa, Masatoshi; Tokuhisa, Takeshi; Azuma, Takachika; O-Wang, Jiyang

    2005-01-01

    Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase η (Polη) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Polθ) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Polθ) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the JH4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Polθ activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Polθ efficiently catalyzes the bypass of abasic sites, lead us to propose that Polθ introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase. PMID:16172387

  2. Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

    PubMed

    He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

    2013-11-01

    Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

  3. Inhibitory effects of vitamin K3 on DNA polymerase and angiogenesis.

    PubMed

    Matsubara, Kiminori; Kayashima, Tomoko; Mori, Masaharu; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2008-09-01

    Vitamins play essential roles in cellular reactions and maintain human health. Recent studies have revealed that some vitamins including D3, B6 and K2 and their derivatives have an anti-cancer effect. As a mechanism, their inhibitory effect on cancer-related angiogenesis has been demonstrated. Vitamin K2 (menaquinones) has an anti-cancer effect in particular for hepatic cancer and inhibits angiogenesis. In the current study, we demonstrated that sole vitamin K3 (menadione) selectively inhibits the in vitro activity of eukaryotic DNA polymerase gamma, which is a mitochondrial DNA polymerase, and suppresses angiogenesis in a rat aortic ring model. The anti-angiogenic effect of vitamin K3 has been shown in angiogenesis models using human umbilical vein endothelial cells (HUVECs) with regard to HUVEC growth, tube formation on reconstituted basement membrane and chemotaxis. These results suggest that vitamin K3 may be a potential anti-cancer agent like vitamin K2.

  4. Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

    PubMed Central

    Shcherbakov, V. P.; Plugina, L. A.; Kudryashova, E. A.

    1995-01-01

    Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates. PMID:7635281

  5. Nested polymerase chain reaction study of 53 cases with Turner`s syndrome: Is cytogenetically undetected Y mosaicism common?

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Binder, G.; Koch, A.; Ranke, M.B.

    1995-12-01

    Turner`s syndrome patients with Y mosaicism face a high risk of developing gonadoblastoma. Cytogenetic analysis can fail to detect rare cells bearing a normal or structurally abnormal Y chromosome (low level Y mosaicism). We screened 53 individuals with Turner`s syndrome for presence of sex-determining region Y (SRY), the testis-specific protein, Y encoded, gene, and the Y centromeric DYZ3 repeat using nested polymerase chain reaction (PCR). Thirty girls (57%) had the 45,X karyotype, determined through standard analysis of blood lymphocytes. The remaining 23 girls (43%) were mosaics and/or had structural abnormalities in 1 X-chromosome. Genomic DNA from blood leukocytes was amplifiedmore » using 2 rounds of PCR. This method was sensitive enough to detect 0.0001% male DNA on a female background. None of 53 Turner`s syndrome cases was positive for Y-specific loci after the first round of PCR. After the second round, 2 of 53 Turner`s syndrome cases were positive for SRY mapping to the distal short arm of chromosome Y. In 1 SRY-positive subject, the karyotype was 45,X, and in the other, it was 46,Xi(Xq). None of 53 Turner`s syndrome individuals, including the 2 SRY-positive subjects, were positive for the testis-specific protein, Y encoded, gene on the proximal short arm of chromosome Y or the centromeric DYZ3 repeat. These data exclude low level Y mosaicism in almost all Turner`s syndrome cases tested. 35 refs., 3 figs., 1 tab.« less

  6. Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries.

    PubMed

    Randrianjatovo-Gbalou, Irina; Rosario, Sandrine; Sismeiro, Odile; Varet, Hugo; Legendre, Rachel; Coppée, Jean-Yves; Huteau, Valérie; Pochet, Sylvie; Delarue, Marc

    2018-05-21

    Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.

  7. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    PubMed

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  8. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1987-08-28

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of /und Streptococcus/ /und pneumoniae/. Plasmid pSM22, the vector containing the pneumococcal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 fig., 1 tab.

  9. Domain structure, localization, and function of DNA polymerase η, defective in xeroderma pigmentosum variant cells

    PubMed Central

    Kannouche, Patricia; Broughton, Bernard C.; Volker, Marcel; Hanaoka, Fumio; Mullenders, Leon H.F.; Lehmann, Alan R.

    2001-01-01

    DNA polymerase η carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that polη is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of polη is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Polη truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains. PMID:11157773

  10. DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens.

    PubMed

    Gowda, A S Prakasha; Spratt, Thomas E

    2016-03-21

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O(2)-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP ≫ dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O(2)-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O(2)-POB-dT, and then, pol ζ extends past the adduct.

  11. Evidence for Moonlighting Functions of the θ Subunit of Escherichia coli DNA Polymerase III

    PubMed Central

    Dietrich, M.; Pedró, L.; García, J.; Pons, M.; Hüttener, M.; Paytubi, S.; Madrid, C.

    2014-01-01

    The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no detectable homology to other ORFs in a database). It encodes the θ subunit of the DNA polymerase III core complex. The precise function of the θ subunit within this complex is not well established, and loss of holE does not result in a noticeable phenotype. Paralogs of holE are also present on many conjugative plasmids and on phage P1 (hot gene). In this study, we provide evidence indicating that θ (HolE) exhibits structural and functional similarities to a family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins that are also encoded by enterobacterial ORFan genes. Microarray studies comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT mutants revealed highly similar expression patterns for strains harboring holE and ydgT alleles. Among the genes differentially regulated in both mutants were genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE and YdgT downregulate expression of the tna operon by possibly increasing the level of Rho-dependent transcription termination at the tna operon's leader region. Thus, for the first time, a regulatory function can be attributed to HolE, in addition to its role as structural component of the DNA polymerase III complex. PMID:24375106

  12. Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ: Novel Mechanisms of Function and Pathogenesis.

    PubMed

    Euro, Liliya; Haapanen, Outi; Róg, Tomasz; Vattulainen, Ilpo; Suomalainen, Anu; Sharma, Vivek

    2017-03-07

    DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory β-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol γ-associated neurodegenerative disorders.

  13. Cloning and characterization of a DNA polymerase beta gene from Trypanosoma cruzi.

    PubMed

    Venegas, Juan A; Aslund, Lena; Solari, Aldo

    2009-06-01

    A gene coding for a DNA polymerase beta from the Trypanosoma cruzi Miranda clone, belonging to the TcI lineage, was cloned (Miranda Tcpol beta), using the information from eight peptides of the T. cruzi beta-like DNA polymerase purified previously. The gene encodes for a protein of 403 amino acids which is very similar to the two T. cruzi CL Brener (TcIIe lineage) sequences published, but has three different residues in highly conserved segments. At the amino acid level, the identity of TcI-pol beta with mitochondrial pol beta and pol beta-PAK from other trypanosomatids was between 68-80% and 22-30%, respectively. Miranda Tc-pol beta protein has an N-terminal sequence similar to that described in the mitochondrial Crithidia fasciculata pol beta, which suggests that the TcI-pol beta plays a role in the organelle. Northern and Western analyses showed that this T. cruzi gene is highly expressed both in proliferative and non-proliferative developmental forms. These results suggest that, in addition to replication of kDNA in proliferative cells, this enzyme may have another function in non-proliferative cells, such as DNA repair role similar to that which has extensively been described in a vast spectrum of eukaryotic cells.

  14. Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions

    NASA Astrophysics Data System (ADS)

    Kemp, David J.; Smith, Donald B.; Foote, Simon J.; Samaras, N.; Peterson, M. Gregory

    1989-04-01

    The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.

  15. Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richardson, C.C.

    1993-12-31

    This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less

  16. Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

    PubMed

    Heyduk, E; Baichoo, N; Heyduk, T

    2001-11-30

    The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

  17. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    PubMed Central

    Chénard, Caroline; Wirth, Jennifer F.

    2016-01-01

    ABSTRACT   Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. PMID:27302758

  18. The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

    PubMed

    Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek; Langel, Ülo

    2017-04-01

    The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

  19. Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

    PubMed Central

    Stranges, P. Benjamin; Palla, Mirkó; Kalachikov, Sergey; Nivala, Jeff; Dorwart, Michael; Trans, Andrew; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Tao, Chuanjuan; Morozova, Irina; Li, Zengmin; Shi, Shundi; Aberra, Aman; Arnold, Cleoma; Yang, Alexander; Aguirre, Anne; Harada, Eric T.; Korenblum, Daniel; Pollard, James; Bhat, Ashwini; Gremyachinskiy, Dmitriy; Bibillo, Arek; Chen, Roger; Davis, Randy; Russo, James J.; Fuller, Carl W.; Roever, Stefan; Ju, Jingyue; Church, George M.

    2016-01-01

    Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform. PMID:27729524

  20. RNA Polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes.

    PubMed

    Gruber, Andreas R

    2014-07-10

    RNA Polymerase III is a highly specialized enzyme complex responsible for the transcription of a very distinct set of housekeeping noncoding RNAs including tRNAs, 7SK snRNA, Y RNAs, U6 snRNA, and the RNA components of RNaseP and RNaseMRP. In this work we have utilized the conserved promoter structure of known RNA Polymerase III transcripts consisting of characteristic sequence elements termed proximal sequence elements (PSE) A and B and a TATA-box to uncover a novel RNA Polymerase III-transcribed, noncoding RNA family found to be conserved in Caenorhabditis as well as other clade V nematode species. Homology search in combination with detailed sequence and secondary structure analysis revealed that members of this novel ncRNA family evolve rapidly, and only maintain a potentially functional small stem structure that links the 5' end to the very 3' end of the transcript and a small hairpin structure at the 3' end. This is most likely required for efficient transcription termination. In addition, our study revealed evidence that canonical C/D box snoRNAs are also transcribed from a PSE A-PSE B-TATA-box promoter in Caenorhabditis elegans. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

    PubMed Central

    Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C.M.; Jansen, Jacob G.; Hogenbirk, Marc A.; de Wind, Niels; Jacobs, Heinz

    2015-01-01

    Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. PMID:25505145

  2. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    PubMed Central

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  3. Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins.

    PubMed

    Bowen, Nikki; Kolodner, Richard D

    2017-04-04

    Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using S. cerevisiae proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.

  4. DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

    NASA Astrophysics Data System (ADS)

    Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

    2013-11-01

    Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

  5. Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

    PubMed

    Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-12-01

    Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

  6. Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

    PubMed

    Yuan, Quan; McHenry, Charles S

    2009-11-13

    In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

  7. Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis.

    PubMed

    Pedroza-Garcia, José Antonio; Domenichini, Séverine; Mazubert, Christelle; Bourge, Mickael; White, Charles; Hudik, Elodie; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Del Olmo, Ivan; Piñeiro, Manuel; Jarillo, Jose Antonio; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2016-09-06

    Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Role of Human DNA Polymerase and Its Accessory Proteins in Breast Cancer

    DTIC Science & Technology

    2000-09-01

    10, 13, 15, and 19 are abnormal and indicate mutants in POLD1 gene . Determination of NIRCA detected mutations by DNA sequencing NIRCA detected...CAGCAA; GnGln) in codon 461. Table III. Summary of mutation identified in the Exo motif of POLD1 Gene from breast cancer. Patient/Cell line Nucleotide...the gene for human DNA polymerase 8 catalytic p125 (POLDI) and p50 ( POLD2 ) subunits (Chang et al., 1995, Perez et al., 2000).. Normal and breast

  9. Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

    PubMed

    Lin, H J; Wu, P C; Lai, C L; Chak, W

    1984-04-01

    A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

  10. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

    PubMed

    Jamsen, Joonas A; Beard, William A; Pedersen, Lars C; Shock, David D; Moon, Andrea F; Krahn, Juno M; Bebenek, Katarzyna; Kunkel, Thomas A; Wilson, Samuel H

    2017-08-15

    DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP i . The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

  11. Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

    PubMed

    Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

    2017-11-20

    DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

  12. Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

    PubMed

    Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

    2017-10-24

    Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

  13. RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction

    PubMed Central

    Leis, Jonathan P.; Hurwitz, Jerard

    1972-01-01

    The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg2+, and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qβ; and synthetic homopolymers such as polyadenylate·polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3′ hydroxyl ends of primer strands. The product is an RNA·DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S. PMID:4333539

  14. Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

    PubMed

    Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C M; Jansen, Jacob G; Hogenbirk, Marc A; de Wind, Niels; Jacobs, Heinz

    2015-01-01

    Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

    PubMed

    Juhas, Mario; Ajioka, James W

    2017-11-01

    The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (η) in pathogenic yeast Candida albicans

    NASA Astrophysics Data System (ADS)

    Satpati, Suresh; Manohar, Kodavati; Acharya, Narottam; Dixit, Anshuman

    2017-01-01

    Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

  17. Roles of Saccharomyces cerevisiae DNA polymerases Polη and Polζ in response to irradiation by simulated sunlight

    PubMed Central

    Kozmin, Stanislav G.; Pavlov, Youri I.; Kunkel, Thomas A.; Sage, Evelyne

    2003-01-01

    Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase η (Polη) and polymerase ζ (Polζ), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310–1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Δ, rev3Δ and rev3Δ rad30Δ strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6–4) photoproducts derived from studies with UVC. They further suggest that Polη participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polζ is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polζ, Polη contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine. PMID:12888515

  18. Phylogenetic analysis of DNA and RNA polymerases from a Moniliophthora perniciosa mitochondrial plasmid reveals probable lateral gene transfer.

    PubMed

    Andrade, B S; Góes-Neto, A

    2015-10-30

    The filamentous fungus Moniliophthora perniciosa is a hemibiotrophic basidiomycete that causes witches' broom disease of cacao (Theobroma cacao L.). Many fungal mitochondrial plasmids are DNA and RNA polymerase-encoding invertrons with terminal inverted repeats and 5'-linked proteins. The aim of this study was to carry out comparative and phylogenetic analyses of DNA and RNA polymerases for all known linear mitochondrial plasmids in fungi. We performed these analyses at both gene and protein levels and assessed differences between fungal and viral polymerases in order to test the lateral gene transfer (LGT) hypothesis. We analyzed all mitochondrial plasmids of the invertron type within the fungal clade, including five from Ascomycota, seven from Basidiomycota, and one from Chytridiomycota. All phylogenetic analyses generated similar tree topologies regardless of the methods and datasets used. It is likely that DNA and RNA polymerase genes were inserted into the mitochondrial genomes of the 13 fungal species examined in our study as a result of different LGT events. These findings are important for a better understanding of the evolutionary relationships between fungal mitochondrial plasmids.

  19. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

    DOE PAGES

    Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; ...

    2015-01-20

    In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5´-templating base, indicating that it derives from nontemplated synthesismore » according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. The translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.« less

  20. Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

    PubMed

    Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

    2013-03-01

    How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.

  1. Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

    PubMed Central

    Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

    2013-01-01

    How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair. PMID:23388460

  2. Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.

    PubMed

    Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina; Lee, Young-Sam; Zhang, Qianqian; Egli, Martin; Guengerich, F Peter

    2016-09-30

    DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn 2+ than Mg 2+ The human germline R96G variant impairs both Mn 2+ -dependent and Mg 2+ -dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg 2+ -dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn 2+ (0.15 mm) instead of Mg 2+ (2 mm) caused a 770-fold increase in efficiency (k pol /K d ,dCTP ) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K d ,dCTP The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k pol /K d ,dCTP for dCTP insertion opposite G with Mg 2+ when compared with wild type, substantially attenuated by substitution with Mn 2+ Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg 2+ or Mn 2+ , revealed that Mn 2+ achieves more optimal octahedral coordination geometry than Mg 2+ , with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι*

    PubMed Central

    Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina; Lee, Young-Sam; Zhang, Qianqian; Egli, Martin; Guengerich, F. Peter

    2016-01-01

    DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn2+ than Mg2+. The human germline R96G variant impairs both Mn2+-dependent and Mg2+-dependent activities of pol ι, whereas the Δ1–25 variant selectively enhances its Mg2+-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1–445) proteins. The presence of Mn2+ (0.15 mm) instead of Mg2+ (2 mm) caused a 770-fold increase in efficiency (kpol/Kd,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in Kd,dCTP. The R96G and Δ1–25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in kpol/Kd,dCTP for dCTP insertion opposite G with Mg2+ when compared with wild type, substantially attenuated by substitution with Mn2+. Crystal structures of pol ι ternary complexes, including the primer terminus 3′-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg2+ or Mn2+, revealed that Mn2+ achieves more optimal octahedral coordination geometry than Mg2+, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation. PMID:27555320

  4. [Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

    PubMed

    Wang, Ling; Yang, Peng; Li, Shuang-qing; Xu, Shu-hui; Cao, Gui-qun; Zhang, Fa-qiang; Zhang, Mei-xia; Chen, Qing-ying; Xia, Qing-jie; Liu, Kai; Tang, Fang; Zhang, Yuan-zheng

    2004-11-01

    To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.

  5. DNA polymerase ι: The long and the short of it!

    PubMed

    Frank, Ekaterina G; McLenigan, Mary P; McDonald, John P; Huston, Donald; Mead, Samantha; Woodgate, Roger

    2017-10-01

    The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform. Published by Elsevier B.V.

  6. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

    Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

  7. The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

    PubMed

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-11-01

    Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

    PubMed Central

    Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

    2015-01-01

    Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

  9. A patient with polymerase E1 deficiency (POLE1): clinical features and overlap with DNA breakage/instability syndromes.

    PubMed

    Thiffault, Isabelle; Saunders, Carol; Jenkins, Janda; Raje, Nikita; Canty, Kristi; Sharma, Mukta; Grote, Lauren; Welsh, Holly I; Farrow, Emily; Twist, Greyson; Miller, Neil; Zwick, David; Zellmer, Lee; Kingsmore, Stephen F; Safina, Nicole P

    2015-05-07

    Chromosome instability syndromes are a group of inherited conditions associated with chromosomal instability and breakage, often leading to immunodeficiency, growth retardation and increased risk of malignancy. We performed exome sequencing on a girl with a suspected chromosome instability syndrome that manifested as growth retardation, microcephaly, developmental delay, dysmorphic features, poikiloderma, immune deficiency with pancytopenia, and myelodysplasia. She was homozygous for a previously reported splice variant, c.4444 + 3A > G in the POLE1 gene, which encodes the catalytic subunit of DNA polymerase E. This is the second family with POLE1-deficency, with the affected individual demonstrating a more severe phenotype than previously described.

  10. DNA stabilization at the Bacillus subtilis PolX core—a binding model to coordinate polymerase, AP-endonuclease and 3′-5′ exonuclease activities

    PubMed Central

    Baños, Benito; Villar, Laurentino; Salas, Margarita; de Vega, Miguel

    2012-01-01

    Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs. PMID:22844091

  11. The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

    PubMed Central

    Bogani, Federica; Boehmer, Paul E.

    2008-01-01

    Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5′-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3′ side of abasic sites and 5′-dRP residues that remain after cleavage by 5′-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency. PMID:18695225

  12. Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jain, Rinku; Choudhury, Jayati Roy; Buku, Angeliki

    N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the synmore » conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.« less

  13. Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

    PubMed Central

    Williams, Lindsey N.; Herr, Alan J.; Preston, Bradley D.

    2013-01-01

    DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity. PMID:23307893

  14. Primers for polymerase chain reaction to detect genomic DNA of Toxocara canis and T. cati.

    PubMed

    Wu, Z; Nagano, I; Xu, D; Takahashi, Y

    1997-03-01

    Primers for polymerase chain reaction to amplify genomic DNA of both Toxocara canis and T. cati were constructed by adapting cloning and sequencing random amplified polymorphic DNA. The primers are expected to detect eggs and/or larvae of T. canis and T. cati, both of which are known to cause toxocariasis in humans.

  15. Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

    PubMed

    Pedroza-García, José-Antonio; Mazubert, Christelle; Del Olmo, Ivan; Bourge, Mickael; Domenichini, Séverine; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Piñeiro, Manuel; Jarillo, José A; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2017-03-01

    Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis ( Arabidopsis thaliana ). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase β's activity.

    PubMed

    Homouz, Dirar; Joyce-Tan, Kwee Hong; Shahir Shamsir, Mohd; Moustafa, Ibrahim M; Idriss, Haitham

    2018-01-01

    DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity. Copyright © 2017. Published by Elsevier Inc.

  17. Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae

    PubMed Central

    Donigan, Katherine A.; Cerritelli, Susana M.; McDonald, John P.; Vaisman, Alexandra; Crouch, Robert J.; Woodgate, Roger

    2015-01-01

    DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of S. cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related polι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S. cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNaseH2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. PMID:26340535

  18. Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.

    PubMed

    Donigan, Katherine A; Cerritelli, Susana M; McDonald, John P; Vaisman, Alexandra; Crouch, Robert J; Woodgate, Roger

    2015-11-01

    DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved "steric gate" residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. Published by Elsevier B.V.

  19. Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

    PubMed Central

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

    2012-01-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

  20. Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

    PubMed Central

    Waisertreiger, Irina S.-R.; Liston, Victoria G.; Menezes, Miriam R.; Kim, Hyun-Min; Lobachev, Kirill S.; Stepchenkova, Elena I.; Tahirov, Tahir H.; Rogozin, Igor B.; Pavlov, Youri. I.

    2014-01-01

    The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair. PMID:23055184

  1. Interaction between the Rev1 C-terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis

    PubMed Central

    Pustovalova, Yulia; Magalhães, Mariana T. Q.; D’Souza, Sanjay; Rizzo, Alessandro A.; Korza, George; Walker, Graham C.; Korzhnev, Dmitry M.

    2016-01-01

    Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι or Polκ, inserts a nucleotide across DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of 'inserter' to 'extender' DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the 'inserter' Polη, Polι or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit 'extender' Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits. PMID:26982350

  2. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

    PubMed Central

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2016-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

  3. The difference in the stimulation by putrescine of DNA synthesis using DNA polymerase extracts of normal rat liver or of tumour tissue or host liver from tumour-bearing rats.

    PubMed

    Taguchi, Takahiko; Kurata, Sumiko; Ohashi, Mochihiko

    2002-09-01

    Putrescine biosynthesis is elevated before DNA replication, and a stimulation of DNA synthesis by 20 mM putrescine has been found using an in vitro DNA synthesizing system. Furthermore, this stimulation of DNA synthesis by putrescine involves a particular factor (factor PA). This factor PA stimulates DNA polymerases alpha, beta, and gamma, and is present in nuclei and mitochondria but not in cytoplasm. Factor PA loses about 80% of its activity by heating at 45 degrees C for 15 min or by hydrolysis with 100 mg ml(-1) Enzygel trypsin. These properties indicate that factor PA is a protein. Its size is estimated to be about 2.1 S. DNA synthesis in nuclear and mitochondrial DNA polymerase extracts from tumour tissues and host livers of tumour-bearing rats are not stimulated by 20 mM putrescine. However, the addition of excess factor PA to DNA synthesizing systems using DNA polymerase extracts from proliferative tissues again results in a stimulation of DNA synthesis by exogenous putrescine. These findings indicate that the stimulatory effect of DNA synthesis in vitro by exogenous putrescine is controlled by the ratio between factor PA and endogenously synthesized putrescine in proliferative tissues or that sent by the bloodstream from proliferative tissues. These results suggest that a non-stimulatory effect of putrescine on DNA synthesis may be diagnostic in tumour-bearing patients. Copyright 2002 John Wiley & Sons, Ltd.

  4. Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.

    PubMed Central

    Jeruzalmi, D; Steitz, T A

    1998-01-01

    The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold. PMID:9670025

  5. Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

    PubMed Central

    Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.

    2015-01-01

    DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is “balanced,” as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability. PMID:26351676

  6. Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase*

    PubMed Central

    Jin, Zhinan; Johnson, Kenneth A.

    2011-01-01

    A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. PMID:20978284

  7. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

  8. Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niyogi, S.K.; Mitra, S.

    Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

  9. DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

    PubMed

    Focke, Felix; Haase, Ilka; Fischer, Markus

    2011-01-26

    Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.

  10. RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

    PubMed Central

    Spiering, Michelle M.; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J.

    2017-01-01

    The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes. PMID:28507156

  11. RNA primer-primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication.

    PubMed

    Spiering, Michelle M; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J

    2017-05-30

    The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior-that is, the signaling mechanism-have not been definitively identified. We examined the role of RNA primer-primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer-primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer-primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer-primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.

  12. A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E. coli O157:H7 DNA detection.

    PubMed

    Wang, Lijiang; Liu, Qingjun; Hu, Zhaoying; Zhang, Yuanfan; Wu, Chunsheng; Yang, Mo; Wang, Ping

    2009-05-15

    A novel biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for Escherichia coli O157:H7 DNA detection. An original and dynamic polymerase-extending (PE) DNA hybridization procedure is proposed, where hybridization happens in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strand would be extended as long as the target DNA strand, then the capability to block the ionic flow in the pores has been prominently enhanced by the double strand complex. We have investigated the variation of ionic conductivity during the fabrication of the film and the hybridization using cyclic voltammetry and impedance spectroscopy. The present approach provides low detection limit for DNA (a few hundreds of pmol), rapid label-free and easy-to-use bacteria detection, which holds the potential for future use in various ss-DNA analyses by integrated into a self-contained biochip.

  13. Study of Pure Proteins, Nucleic Acids and their Complexes from Extreme Halobacteria of the Dead Sea: RNA Polymerase-DNA Interaction

    DTIC Science & Technology

    1988-10-10

    identify by block number) FIELD GROUP S OUP - Archaebacteria , Halobacteria, Proteins Nucleic Acids, 08 RNA Polymerase-DNA Interactionsi R soimal operons...objectives of our program are to isolate and characterize a fully active DNA dependent RNA polymerase from the extremely halophilic archaebacteria from...Woese and his colleagues to suggest that all living organisms can be classified into three phylogenetic kingdoms : the eukaryotes, the eubacterla and

  14. Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

    PubMed

    Kaminiwa, Junko; Honda, Katsuya; Sugano, Yukiko; Yano, Shizue; Nishi, Takeki; Sekine, Yuko

    2013-05-01

    Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology. Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  15. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    PubMed

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  16. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  17. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  18. Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

    PubMed Central

    Weisshart, Klaus; Chow, Connie S.; Coen, Donald M.

    1999-01-01

    Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ∼15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ∼2-fold faster than did Pol and dissociated ∼10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ∼30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented. PMID:9847307

  19. The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa

    PubMed Central

    Maddukuri, Leena; Ketkar, Amit; Eddy, Sarah; Zafar, Maroof K.; Eoff, Robert L.

    2014-01-01

    Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells. PMID:25294835

  20. Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences.

    PubMed Central

    Bjourson, A J; Stone, C E; Cooper, J E

    1992-01-01

    A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells. Images PMID:1637166

  1. Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding.

    PubMed

    Basu, A; Williams, K R; Modak, M J

    1987-07-15

    Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.

  2. Localized cerebral energy failure in DNA polymerase gamma-associated encephalopathy syndromes.

    PubMed

    Tzoulis, Charalampos; Neckelmann, Gesche; Mørk, Sverre J; Engelsen, Bernt E; Viscomi, Carlo; Moen, Gunnar; Ersland, Lars; Zeviani, Massimo; Bindoff, Laurence A

    2010-05-01

    Mutations in the catalytic subunit of the mitochondrial DNA-polymerase gamma cause a wide spectrum of clinical disease ranging from infantile hepato-encephalopathy to juvenile/adult-onset spinocerebellar ataxia and late onset progressive external ophthalmoplegia. Several of these syndromes are associated with an encephalopathy that characteristically shows episodes of rapid neurological deterioration and the development of acute cerebral lesions. The purpose of this study was to investigate the nature, distribution and natural evolution of central nervous system lesions in polymerase gamma associated encephalopathy focusing particularly on lesions identified by magnetic resonance imaging. We compared radiological, electrophysiological and pathological findings where available to study potential mechanisms underlying the episodes of exacerbation and acute cerebral lesions. We studied a total of 112 magnetic resonance tomographies and 11 computed tomographies in 32 patients with polymerase gamma-encephalopathy, including multiple serial examinations performed during both the chronic and acute phases of the disease and, in several cases, magnetic resonance spectroscopy and serial diffusion weighted studies. Data from imaging, electroencephalography and post-mortem examination were compared in order to study the underlying disease process. Our findings show that magnetic resonance imaging in polymerase gamma-related encephalopathies has high sensitivity and can identify patterns that are specific for individual syndromes. One form of chronic polymerase gamma-encephalopathy, that is associated with the c.1399G > A and c.2243G > C mutations, is characterized by progressive cerebral and cerebellar atrophy and focal lesions of the thalamus, deep cerebellar structures and medulla oblongata. Acute encephalopathies, both infantile and later onset, show similar pictures with cortical stroke-like lesions occurring during episodes of exacerbation. These lesions can occur both

  3. Biochemical analysis of active site mutations of human polymerase η.

    PubMed

    Suarez, Samuel C; Beardslee, Renee A; Toffton, Shannon M; McCulloch, Scott D

    2013-01-01

    DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis-syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called 'damage tolerance' to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine-thymine CPD (T-T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T-T CPD. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

    PubMed

    Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

    1985-12-05

    The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

  5. Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions.

    PubMed

    Tsunoda, Hirosuke; Kudo, Tomomi; Masaki, Yoshiaki; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2011-04-01

    To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.

  6. Characterization of an Avipoxvirus From a Bald Eagle ( Haliaeetus leucocephalus ) Using Novel Consensus PCR Protocols for the rpo147 and DNA-Dependent DNA Polymerase Genes.

    PubMed

    Stephen, Alexa A; Leone, Angelique M; Toplon, David E; Archer, Linda L; Wellehan, James F X

    2016-12-01

    A juvenile female bald eagle ( Haliaeetus leucocephalus ) was presented with emaciation and proliferative periocular lesions. The eagle did not respond to supportive therapy and was euthanatized. Histopathologic examination of the skin lesions revealed plaques of marked epidermal hyperplasia parakeratosis, marked acanthosis and spongiosis, and eosinophilic intracytoplasmic inclusion bodies. Novel polymerase chain reaction (PCR) assays were done to amplify and sequence DNA polymerase and rpo147 genes. The 4b gene was also analyzed by a previously developed assay. Bayesian and maximum likelihood phylogenetic analyses of the obtained sequences found it to be poxvirus of the genus Avipoxvirus and clustered with other raptor isolates. Better phylogenetic resolution was found in rpo147 rather than the commonly used DNA polymerase. The novel consensus rpo147 PCR assay will create more accurate phylogenic trees and allow better insight into poxvirus history.

  7. Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.

    PubMed

    Sihi, Sayantani; Bakshi, Sankar; Sengupta, Dibyendu Narayan

    2015-02-01

    DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

  8. Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

    PubMed

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-09-01

    Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights

  9. Unequal rates of Y chromosome gene divergence during speciation of the family Ursidae.

    PubMed

    Nakagome, Shigeki; Pecon-Slattery, Jill; Masuda, Ryuichi

    2008-07-01

    Evolution of the bear family Ursidae is well investigated in terms of morphological, paleontological, and genetic features. However, several phylogenetic ambiguities occur within the subfamily Ursinae (the family Ursidae excluding the giant panda and spectacled bear), which may correlate with behavioral traits of female philopatry and male-biased dispersal which form the basis of the observed matriarchal population structure in these species. In the process of bear evolution, we investigate the premise that such behavioral traits may be reflected in patterns of variation among genes with different modes of inheritance: matrilineal mitochondrial DNA (mtDNA), patrilineal Y chromosome, biparentally inherited autosomes, and the X chromosome. In the present study, we sequenced 3 Y-linked genes (3,453 bp) and 4 X-linked genes (4,960 bp) and reanalyzed previously published sequences from autosome genes (2,347 bp) in ursid species to investigate differences in evolutionary rates associated with patterns of inheritance. The results describe topological incongruence between sex-linked genes and autosome genes and between nuclear DNA and mtDNA. In more ancestral branches within the bear phylogeny, Y-linked genes evolved faster than autosome and X-linked genes, consistent with expectations based on male-driven evolution. However, this pattern changes among branches leading to each species within the lineage of Ursinae whereby the evolutionary rates of Y-linked genes have fewer than expected substitutions. This inconsistency between more recent nodes of the bear phylogeny with more ancestral nodes may reflect the influences of sex-biased dispersal as well as molecular evolutionary characteristics of the Y chromosome, and stochastic events in species natural history, and phylogeography unique to ursine bears.

  10. Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

    PubMed Central

    Yao, Chengguo; Müller, Ulrich F.

    2011-01-01

    The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3′-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world. PMID:21622900

  11. In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

    PubMed Central

    Hirota, Kouji; Tsuda, Masataka; Mohiuddin; Tsurimoto, Toshiki; Cohen, Isadora S.; Livneh, Zvi; Kobayashi, Kaori; Narita, Takeo; Nishihara, Kana; Murai, Junko; Iwai, Shigenori; Guilbaud, Guillaume; Sale, Julian E.; Takeda, Shunichi

    2016-01-01

    The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass. PMID:27185888

  12. New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

    PubMed Central

    Price, Alan R.; Cook, Sandra J.

    1972-01-01

    The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection. PMID:4623224

  13. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

    PubMed

    Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

    2017-11-01

    5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    PubMed

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  15. Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

    PubMed

    Masumura, Kenichi; Toyoda-Hokaiwado, Naomi; Niimi, Naoko; Grúz, Petr; Wada, Naoko A; Takeiri, Akira; Jishage, Kou-Ichi; Mishima, Masayuki; Nohmi, Takehiko

    2017-12-01

    DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-03

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

  17. Functional analysis of H. sapiens DNA polymerase γ spacer mutation W748S with and without common variant E1143G

    PubMed Central

    Palin, Eino JH; Lesonen, Annamari; Farr, Carol L; Euro, Liliya; Suomalainen, Anu; Kaguni, Laurie S

    2010-01-01

    Mitochondrial DNA polymerase, POLG, is the sole DNA polymerase found in animal mitochondria. In humans, POLGα W748S in cis with an E1143G mutation has been linked to a new type of recessive ataxia, MIRAS, which is the most common inherited ataxia in Finland. We investigated the biochemical phenotypes of the W748S amino acid change, using recombinant human POLG. We measured processive and non-processive DNA polymerase activity, DNA binding affinity, enzyme processivity, and subunit interaction with recombinant POLGβ. In addition, we studied the effects of the W748S and E1143G mutations in primary human cell cultures using retroviral transduction. Here, we examined cell viability, mitochondrial DNA copy number, and products of mitochondrial translation. Our results indicate that the W748S mutant POLGα does not exhibit a clear biochemical phenotype, making it indistinguishable from wild type POLGα and as such, fail to replicate previously published results. Furthermore, results from the cell models were concurrent with the findings from patients, and support our biochemical findings. PMID:20153822

  18. The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

    PubMed Central

    Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

    2016-01-01

    DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

  19. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

    PubMed

    Ririe, K M; Rasmussen, R P; Wittwer, C T

    1997-02-15

    A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

  20. Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

    PubMed Central

    Lucas, Daniel; Escudero, Beatriz; Ligos, José Manuel; Segovia, Jose Carlos; Estrada, Juan Camilo; Terrados, Gloria; Blanco, Luis; Samper, Enrique; Bernad, Antonio

    2009-01-01

    Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues. PMID:19229323

  1. Understanding the role of DNA polymerase λ gene in different growth and developmental stages of Oryza sativa L. indica rice cultivars.

    PubMed

    Sihi, Sayantani; Maiti, Soumitra; Bakshi, Sankar; Nayak, Arup; Chaudhuri, Shubho; Sengupta, Dibyendu Narayan

    2017-11-01

    DNA polymerase λ (Pol λ) is the only member of DNA polymerase family X present in plants. The enzyme is ddNTP sensitive as it contains the conserved C-terminal Pol β domain. The 1.1 kb partial coding sequence isolated spanned the whole 3' regions of the gene containing functionally important domains of the Pol λ gene. Comparative in silico studies from both indica and japonica cultivars involving homology modelling showed that the model for the partial Pol λ gene was stable and acceptable. The alignment of both the protein models showed a RMS value of 0.783. Apart from this, expression of Pol λ and its relative activity is studied during different development stages of three different indica rice cultivars (IR29, Nonabokra and N22). Enhanced accumulation and higher activity of Pol λ during the early seedling stage was detected. Higher expression and activity were observed in the anthers, which was probably necessary for DNA repair during microspore formation. However, during the maturation stage of seed development and plant growth, expression and the activity of Pol λ decreased due to slow metabolic activity and a reduced rate of cell division respectively. Furthermore, the expression and activity of Pol λ were found to be higher in IR29 in comparison to Nonabokra and N22. IR29 is a rice cultivar susceptible to environmental stresses and hence it encounters higher DNA damages. The enhanced presence and activity of the Pol λ enzyme in IR29 with respect to the other two cultivars, which are more tolerant to the environmental stresses during various developmental stages, is therefore explainable. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.

    PubMed

    Koponen, Jonna K; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

    2002-03-01

    Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

  3. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  4. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    PubMed Central

    Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

    2015-01-01

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  5. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    PubMed

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

    PubMed

    Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

    2015-06-16

    A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

  7. Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats*

    PubMed Central

    Delagoutte, Emmanuelle; Goellner, Geoffrey M.; Guo, Jie; Baldacci, Giuseppe; McMurray, Cynthia T.

    2008-01-01

    Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5′-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5′-CAG and 5′-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5′-CAG is the template. We propose a “template-push” model in which the slow speed of DNA polymerase across the 5′-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over. PMID:18263578

  8. A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

    PubMed Central

    Wu, Jiqin; Liu, Weichi; Gong, Peng

    2015-01-01

    RNA-dependent RNA polymerases (RdRPs) from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions. PMID:26062131

  9. Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

    PubMed Central

    Tsunoda, Hirosuke; Kudo, Tomomi; Masaki, Yoshiaki; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2011-01-01

    To clarify the biochemical behavior of 2′-deoxyribonucleoside 5′-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNA–DNA duplexes incorporating 2′-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo−) and Vent (exo−) suggested that Co and Ao selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo−) toward Ao on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator. PMID:21300642

  10. Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa.

    PubMed

    Yasui, Manabu; Laxmi, Y R Santosh; Ananthoju, Sreenivasa R; Suzuki, Naomi; Kim, Sung Yeon; Shibutani, Shinya

    2006-05-16

    Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C --> G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the

  11. The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an “Ajar” Intermediate Conformation in the Nucleotide Selection Mechanism*

    PubMed Central

    Wu, Eugene Y.; Beese, Lorena S.

    2011-01-01

    To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established “open” and “closed” states. In this “ajar” conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation. PMID:21454515

  12. Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

    PubMed Central

    Loregian, Arianna; Rigatti, Roberto; Murphy, Mary; Schievano, Elisabetta; Palu, Giorgio; Marsden, Howard S.

    2003-01-01

    In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially α-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction. PMID:12857903

  13. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai

    2009-12-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predictedmore » to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.« less

  14. Promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Henderson, S.L.; Ryan, K.; Sollner-Webb, B.

    1989-02-01

    We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promotermore » in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.« less

  15. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

    PubMed Central

    Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

    2011-01-01

    DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

  16. Specific inhibitors of mammalian DNA polymerase species.

    PubMed

    Mizushina, Yoshiyuki

    2009-06-01

    In screening of selective inhibitors of eukaryotic DNA polymerases (pols) for 15 years, more than 100 inhibitors have been discovered from natural and chemical sources. Some compounds selectively inhibit the activities of mammalian pols, and in particular, dehydroaltenusin and curcumin derivatives, such as monoacetyl-curcumin, were found to be specific inhibitors of pol alpha and pol lambda, respectively. Dehydroaltenusin was isolated from a fungus (Alternaria tennuis), and this compound inhibited cell proliferation of human cancer cell lines by arresting the cells at the S-phase, and was effective in suppressing the growth on nude mice of solid tumors of human cervical cancer cell line HeLa. Curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol lambda inhibitory activity. These compounds might be useful not only as "molecular probes" for pol research, but also as biomedical and chemotherapeutic drugs for anti-cancer or anti-inflammation.

  17. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  18. Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity.

    PubMed

    Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G; Petruska, John; Kool, Eric T; Goodman, Myron F

    2016-04-19

    What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 10(3)- to 10(4)-fold, corresponding to free energy differences of ΔΔGinc∼ 5.5-7 kcal/mol? Standard ΔΔG° values (∼0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper [Olson et al. (2013)J Am Chem Soc135:1205-1208] used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent ΔG° = -RTlnKeq, indicating ΔΔG° of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore,[Formula: see text]values obtained from such steady-state evaluations ofKeqare not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site.

  19. DNA polymerase iota (Pol ι) promotes invasion and metastasis of esophageal squamous cell carcinoma.

    PubMed

    Zou, Shitao; Shang, Zeng-Fu; Liu, Biao; Zhang, Shuyu; Wu, Jinchang; Huang, Min; Ding, Wei-Qun; Zhou, Jundong

    2016-05-31

    DNA polymerase iota (Pol ι) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that contributes to the accumulation of DNA mutations. We recently showed that Pol ι is overexpressed in human esophageal squamous cell cancer (ESCC) tissues which promotes ESCC' progression. The present study was aimed at investigating the molecular mechanisms by which Pol ι enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol ι is significantly higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed an inverse correlation between Pol ι expression and patient prognosis. The expression levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two essential regulators of cells' invasiveness, were positively associated with Pol ι expression in ESCC tissues. Ectopic expression of Pol ι enhanced the motility and invasiveness of ESCC cells as evaluated by wound-healing and transwell assays, respectively. A xenograft nude mouse model showed that Pol ι promotes the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis identified the JNK-AP-1 cascade as a mediator of the Pol ι-induced increase in the expression of MMP-2/9 and enhancement of ESCC progression. These data demonstrate the underlying mechanism by which Pol ι promotes ESCC progression, suggesting that Pol ι is a potential novel prognostic biomarker and therapeutic target for ESCC.

  20. DNA polymerase iota (Pol ι) promotes invasion and metastasis of esophageal squamous cell carcinoma

    PubMed Central

    Liu, Biao; Zhang, Shuyu; Wu, Jinchang; Huang, Min; Ding, Wei-Qun; Zhou, Jundong

    2016-01-01

    DNA polymerase iota (Pol ι) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that contributes to the accumulation of DNA mutations. We recently showed that Pol ι is overexpressed in human esophageal squamous cell cancer (ESCC) tissues which promotes ESCC' progression. The present study was aimed at investigating the molecular mechanisms by which Pol ι enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol ι is significantly higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed an inverse correlation between Pol ι expression and patient prognosis. The expression levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two essential regulators of cells' invasiveness, were positively associated with Pol ι expression in ESCC tissues. Ectopic expression of Pol ι enhanced the motility and invasiveness of ESCC cells as evaluated by wound-healing and transwell assays, respectively. A xenograft nude mouse model showed that Pol ι promotes the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis identified the JNK-AP-1 cascade as a mediator of the Pol ι-induced increase in the expression of MMP-2/9 and enhancement of ESCC progression. These data demonstrate the underlying mechanism by which Pol ι promotes ESCC progression, suggesting that Pol ι is a potential novel prognostic biomarker and therapeutic target for ESCC. PMID:27057634

  1. Absence of giant blood Marseille-like virus DNA detection by polymerase chain reaction in plasma from healthy US blood donors and serum from multiply transfused patients from Cameroon.

    PubMed

    Phan, Tung Gia; Desnues, Christelle; Switzer, William M; Djoko, Cyrille F; Schneider, Bradley S; Deng, Xutao; Delwart, Eric

    2015-06-01

    A new Marseilleviridae virus family member, giant blood Marseille-like (GBM) virus, was recently reported in persons from France in the serum of an infant with adenitis, in the blood of 4% of healthy blood donors, and in 9% of multiply transfused thalassemia patients. These results suggested the presence of a nucleocytoplasmic large DNA virus potentially transmissible by blood product transfusion. To investigate this possibility we tested the plasma from 113 US blood donors and 74 multiply transfused Cameroon patients for GBM viral DNA using highly sensitive polymerase chain reaction (PCR) assays. GBM DNA was not detected by nested PCR in any of these 187 human specimens. Further testing is required to confirm the occurrence of human GBM virus infections. © 2015 AABB.

  2. Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

    PubMed

    Sang, Fuming; Zhang, Zhizhou; Yuan, Lin; Liu, Deli

    2018-02-26

    Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

  3. Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamb, A.; Weir, M.; Rudy, B.

    1989-06-01

    The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this methodmore » to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.« less

  4. Nickel(II) affects poly(ADP-ribose) polymerase-mediated DNA repair in normal and cancer cells.

    PubMed

    Wozniak, Katarzyna; Czechowska, Agnieszka; Blasiak, Janusz

    2006-01-01

    Nickel(II) can be genotoxic, but the mechanism of its genotoxicity is not fully understood and the process of DNA repair may be considered as its potential target. We studied the effect of nickel chloride on the poly(ADP-ribose) polymerase (PARP)-mediated repair of DNA damaged by gamma-radiation and idarubicin with the alkaline comet assay in normal and cancer cells. Our results indicate that nickel chloride at very low, non-cytotoxic concentration of 1 microM can affect PARP-mediated DNA repair of lesions evoked by idarubicin and gamma-radiation. We also suggest that in the quiescent lymphocytes treated with gamma-radiation, nickel(II) could interfere with DNA repair process independent of PARP.

  5. Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1.

    PubMed

    Gu, Shiling; Xiong, Jingyuan; Shi, Ying; You, Jia; Zou, Zhenyu; Liu, Xiaoying; Zhang, Huidong

    2017-09-01

    O 6 -Methylguanine (O 6 -MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O 6 -MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O 6 -MeG by gp90 exo - . O 6 -MeG partially inhibited full-length extension by gp90 exo - . O 6 -MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo - extends beyond T:O 6 -MeG 2-fold more efficiently than C:O 6 -MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O 6 -MeG show fast burst phases. The pre-steady-state incorporation efficiency (k pol /K d,dNTP ) is decreased in the order of dCTP:G>dTTP:O 6 -MeG>dCTP:O 6 -MeG. The presence of O 6 -MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg 2+ . Misincorporation of dTTP opposite O 6 -MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O 6 -MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O 6 -MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

    PubMed Central

    Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

    2006-01-01

    DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

  7. A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

    PubMed

    Litschko, Christa; Oldrini, Davide; Budde, Insa; Berger, Monika; Meens, Jochen; Gerardy-Schahn, Rita; Berti, Francesco; Schubert, Mario; Fiebig, Timm

    2018-05-29

    Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis , Actinobacillus pleuropneumoniae , Haemophilus influenzae , Bibersteinia trehalosi , and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis. IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy

  8. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

    PubMed Central

    Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

    2017-01-01

    Background: Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor. PMID:29279831

  9. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction.

    PubMed

    Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

    2017-01-01

    Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA ® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. The CTAB method showed more positive results at 1:10-1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

  10. Food Fish Identification from DNA Extraction through Sequence Analysis

    ERIC Educational Resources Information Center

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  11. Structural basis for inhibition of DNA replication by aphidicolin

    DOE PAGES

    Baranovskiy, A. G.; Babayeva, N. D.; Suwa, Y.; ...

    2014-11-27

    Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with anmore » RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.« less

  12. Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

    PubMed

    Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

    2009-07-01

    XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

  13. Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II.

    PubMed

    Steurer, Barbara; Marteijn, Jurgen A

    2017-10-27

    The faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The stalling of RNAP2 on a transcription-blocking lesion triggers a series of highly regulated events, including RNAP2 processing to make the lesion accessible for DNA repair, R-loop-mediated DNA damage signaling, and the initiation of transcription-coupled DNA repair. The correct execution and coordination of these processes is vital for resuming transcription following the successful repair of transcription-blocking lesions. Here, we outline recent insights into the molecular consequences of RNAP2 stalling on transcription-blocking DNA lesions and how these lesions are resolved to restore mRNA synthesis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  14. Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

    PubMed

    Miropolskaya, Nataliya; Petushkov, Ivan; Kulbachinskiy, Andrey; Makarova, Alena V

    2017-08-31

    Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ι, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ι, with possible involvement of residues from the finger domain in the dRP group cleavage.

  15. Polymerase Gamma Disease through the Ages

    ERIC Educational Resources Information Center

    Saneto, Russell P.; Naviaux, Robert K.

    2010-01-01

    The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

  16. RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

    PubMed

    NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

    2016-12-27

    Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

  17. Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer

    PubMed Central

    Kinoshita, Kenji; Fujimoto, Kentaro; Yakabe, Toru; Saito, Shin; Hamaguchi, Yuzo; Kikuchi, Takayuki; Nonaka, Ken; Murata, Shigenori; Masuda, Daisuke; Takada, Wataru; Funaoka, Sohei; Arai, Susumu; Nakanishi, Hisao; Yokoyama, Kanehisa; Fujiwara, Kazuhiko; Matsubara, Kenichi

    2007-01-01

    DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3′ end of the immobilized DNA primers on the S-Bio® by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA. PMID:17135189

  18. Novel human CRYGD rare variant in a Brazilian family with congenital cataract

    PubMed Central

    Giordano, Gabriel Gorgone; Tavares, Anderson; da Silva, Márcio José; de Vasconcellos, José Paulo Cabral; Arieta, Carlos Eduardo Leite; de Melo, Mônica Barbosa

    2011-01-01

    Purpose To describe a novel polymorphism in the γD-crystallin (CRYGD) gene in a Brazilian family with congenital cataract. Methods A Brazilian four-generation family was analyzed. The proband had bilateral lamellar cataract and the phenotypes were classified by slit lamp examination. Genomic DNA was extracted from peripheral blood and coding regions and intron/exon boundaries of the αA-crystallin (CRYAA), γC-crystallin (CRYGC), and CRYGD genes were amplified by polymerase chain reaction and directly sequenced. Results Sequencing of the coding regions of CRYGD showed the presence of a heterozygous A→G transversion at c.401 position, which results in the substitution of a tyrosine to a cysteine (Y134C). The polymorphism was identified in three individuals, two affected and one unaffected. Conclusions A novel rare variant in CRYGD (Y134C) was detected in a Brazilian family with congenital cataract. Because there is no segregation between the substitution and the phenotypes in this family, other genetic alterations are likely to be present. PMID:21866214

  19. The Anti-Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase.

    PubMed

    Andrei, Graciela; Gillemot, Sarah; Topalis, Dimitrios; Snoeck, Robert

    2018-02-14

    Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  20. Functional analysis of CedA based on its structure: residues important in binding of DNA and RNA polymerase and in the cell division regulation

    PubMed Central

    Abe, Yoshito; Fujisaki, Naoki; Miyoshi, Takanori; Watanabe, Noriko; Katayama, Tsutomu; Ueda, Tadashi

    2016-01-01

    DnaAcos, a mutant of the initiator DnaA, causes overinitiation of chromosome replication in Escherichia coli, resulting in inhibition of cell division. CedA was found to be a multi-copy suppressor which represses the dnaAcos inhibition of cell division. However, functional mechanism of CedA remains elusive except for previously indicated possibilities in binding to DNA and RNA polymerase. In this study, we searched for the specific sites of CedA in binding of DNA and RNA polymerase and in repression of cell division inhibition. First, DNA sequence to which CedA preferentially binds was determined. Next, the several residues and β4 region in CedA C-terminal domain was suggested to specifically interact with the DNA. Moreover, we found that the flexible N-terminal region was required for tight binding to longer DNA as well as interaction with RNA polymerase. Based on these results, several cedA mutants were examined in ability for repressing dnaAcos cell division inhibition. We found that the N-terminal region was dispensable and that Glu32 in the C-terminal domain was required for the repression. These results suggest that CedA has multiple roles and residues with different functions are positioned in the two regions. PMID:26400504

  1. A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

    PubMed Central

    Al-Khouri, Anna Maria; Paule, Marvin R.

    2002-01-01

    In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

  2. A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

    PubMed

    Al-Khouri, Anna Maria; Paule, Marvin R

    2002-02-01

    In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

  3. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    PubMed

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

  4. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

    PubMed Central

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3′ end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells. PMID:25764216

  5. A Paper and Plastic Device for Performing Recombinase Polymerase Amplification of HIV DNA

    PubMed Central

    Rohrman, Brittany A.; Richards-Kortum, Rebecca R.

    2013-01-01

    Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 minutes. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings. PMID:22733333

  6. A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

    PubMed

    Rohrman, Brittany A; Richards-Kortum, Rebecca R

    2012-09-07

    Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

  7. Problem-Solving Test: Real-Time Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, "HER2" gene, genomic DNA, "in vitro" DNA synthesis, template, primer, Taq polymerase, 5[prime][right arrow]3[prime] elongation activity, 5[prime][right arrow]3[prime] exonuclease activity, deoxyribonucleoside…

  8. Using a Fluorescent Cytosine Analogue tC[superscript o] To Probe the Effect of the Y567 to Ala Substitution on the Preinsertion Steps of dNMP Incorporation by RB69 DNA Polymerase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xia, Shuangluo; Beckman, Jeff; Wang, Jimin

    2012-10-10

    Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC{sup o}), to identify preinsertion step(s) altered by NBP flexibility. When tC{sup o} is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC{sup o} is also quenched in the presence of dATP. We determined the crystalmore » structure of the dATP/tC{sup o}-containing ternary complex of the RB69pol Y567A mutant at 1.9 {angstrom} resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC{sup o}. Stabilization of the dATP/tC{sup o} base pair involved movement of the tC{sup o} backbone sugar into the DNA minor groove and required tilting of the tC{sup o} tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity.« less

  9. Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination

    PubMed Central

    Markkanen, Enni; van Loon, Barbara; Ferrari, Elena; Parsons, Jason L.; Dianov, Grigory L.; Hübscher, Ulrich

    2012-01-01

    It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2′-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ’s affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation. PMID:22203964

  10. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    PubMed

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa.

    PubMed

    Suzuki, Naomi; Yasui, Manabu; Santosh Laxmi, Y R; Ohmori, Haruo; Hanaoka, Fumio; Shibutani, Shinya

    2004-09-07

    Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.

  12. RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Corda, Y.; Job, D.; Anin, M.F.

    1992-02-25

    Two duplexes (20-mers) were constructed containing either a single cis-(Pt(NH{sub 3}){sub 2}(d(GpG))) or cis-(Pt(NH{sub 3}){sub 2}(d(ApG))) intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute blockmore » to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent K{sub m} of enzyme for the platinated polymer is increased by a factor of 4-5. These results are discussed in reaction to the distortions induced in DNA by the two adducts.« less

  13. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

    PubMed Central

    Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1986-01-01

    To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

  14. Oligomerization of the E. coli Core RNA Polymerase: Formation of (α2ββ'ω)2–DNA Complexes and Regulation of the Oligomerization by Auxiliary Subunits

    PubMed Central

    Kansara, Seema G.; Sukhodolets, Maxim V.

    2011-01-01

    In this work, using multiple, dissimilar physico-chemical techniques, we demonstrate that the Escherichia coli RNA polymerase core enzyme obtained through a classic purification procedure forms stable (α2ββ'ω)2 complexes in the presence or absence of short DNA probes. Multiple control experiments indicate that this self-association is unlikely to be mediated by RNA polymerase-associated non-protein molecules. We show that the formation of (α2ββ'ω)2 complexes is subject to regulation by known RNA polymerase interactors, such as the auxiliary SWI/SNF subunit of RNA polymerase RapA, as well as NusA and σ70. We also demonstrate that the separation of the core RNA polymerase and RNA polymerase holoenzyme species during Mono Q chromatography is likely due to oligomerization of the core enzyme. We have analyzed the oligomeric state of the polymerase in the presence or absence of DNA, an aspect that was missing from previous studies. Importantly, our work demonstrates that RNA polymerase oligomerization is compatible with DNA binding. Through in vitro transcription and in vivo experiments (utilizing a RapAR599/Q602 mutant lacking transcription-stimulatory function), we demonstrate that the formation of tandem (α2ββ'ω)2–DNA complexes is likely functionally significant and beneficial for the transcriptional activity of the polymerase. Taken together, our findings suggest a novel structural aspect of the E. coli elongation complex. We hypothesize that transcription by tandem RNA polymerase complexes initiated at hypothetical bidirectional “origins of transcription” may explain recurring switches of the direction of transcription in bacterial genomes. PMID:21533049

  15. Biochemical analysis of DNA polymerase η fidelity in the presence of replication protein A.

    PubMed

    Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

    2014-01-01

    DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

  16. Size-Expanded yDNA bases: An Ab Initio Study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fuentes-Cabrera, Miguel A; Sumpter, Bobby G; Lipkowski, Pawel

    2006-01-01

    xDNA and yDNA are new classes of synthetic nucleic acids characterized by having base-pairs with one of the bases larger than the natural congeners. Here these larger bases are called x- and y-bases. We recently investigated and reported the structural and electronic properties of the x-bases (Fuentes-Cabrera et al. J. Phys. Chem. B 2005, 109, 21135-21139). Here we extend this study by investigating the structure and electronic properties of the y-bases. These studies are framed within our interest that xDNA and yDNA could function as nanowires, for they could have smaller HOMO-LUMO gaps than natural DNA. The limited amount ofmore » experimental structural data in these synthetic duplexes makes it necessary to first understand smaller models and, subsequently, to use that information to build larger models. In this paper, we report the results on the chemical and electronic structure of the y-bases. In particular, we predict that the y-bases have smaller HOMO-LUMO gaps than their natural congeners, which is an encouraging result for it indicates that yDNA could have a smaller HOMO-LUMO gap than natural DNA. Also, we predict that the y-bases are less planar than the natural ones. Particularly interesting are our results corresponding to yG. Our studies show that yG is unstable because it is less aromatic and has a Coulombic repulsion that involves the amino group, as compared with a more stable tautomer. However, yG has a very small HOMO-LUMO gap, the smallest of all the size-expanded bases we have considered. The results of this study provide useful information that may allow the synthesis of an yG-mimic that is stable and has a small HOMO-LUMO gap.« less

  17. A major role of DNA polymerase δ in replication of both the leading and lagging DNA strands

    PubMed Central

    Prakash, Louise; Prakash, Satya

    2015-01-01

    SUMMARY Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. PMID:26145172

  18. Functional analysis of CedA based on its structure: residues important in binding of DNA and RNA polymerase and in the cell division regulation.

    PubMed

    Abe, Yoshito; Fujisaki, Naoki; Miyoshi, Takanori; Watanabe, Noriko; Katayama, Tsutomu; Ueda, Tadashi

    2016-02-01

    DnaAcos, a mutant of the initiator DnaA, causes overinitiation of chromosome replication in Escherichia coli, resulting in inhibition of cell division. CedA was found to be a multi-copy suppressor which represses the dnaAcos inhibition of cell division. However, functional mechanism of CedA remains elusive except for previously indicated possibilities in binding to DNA and RNA polymerase. In this study, we searched for the specific sites of CedA in binding of DNA and RNA polymerase and in repression of cell division inhibition. First, DNA sequence to which CedA preferentially binds was determined. Next, the several residues and β4 region in CedA C-terminal domain was suggested to specifically interact with the DNA. Moreover, we found that the flexible N-terminal region was required for tight binding to longer DNA as well as interaction with RNA polymerase. Based on these results, several cedA mutants were examined in ability for repressing dnaAcos cell division inhibition. We found that the N-terminal region was dispensable and that Glu32 in the C-terminal domain was required for the repression. These results suggest that CedA has multiple roles and residues with different functions are positioned in the two regions. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  19. Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

    PubMed

    Pilger, Beatrice D; Cui, Can; Coen, Donald M

    2004-05-01

    The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

  20. Keeping mammalian mutation load in check: regulation of the activity of error-prone DNA polymerases by p53 and p21.

    PubMed

    Livneh, Zvi

    2006-09-01

    To overcome DNA lesions that block replication the cell employs translesion DNA synthesis (TLS) polymerases, a group of low fidelity DNA polymerases that have the capacity to bypass a wide range of DNA lesions. This TLS process is also termed error-prone repair, due to its inherent mutagenic nature. We have recently shown that the tumor suppressor p53 and the cell cycle inhibitor p21 are global regulators of TLS. When these proteins are missing or nonfunctional, TLS gets out of control: its extent increases to very high levels, and its fidelity decreases, causing an overall increase in mutation load. This may be explained by the loss of selectivity in the bypass of specific DNA lesions by their cognate specialized polymerases, such that lesion bypass continues to a maximum, regardless of the price paid in increased mutations. The p53 and p21 proteins are also required for efficient UV light-induced monoubiquitination of PCNA, which is consistent with a model in which this modification of PCNA is necessary but not sufficient for the normal activity of TLS. This regulation suggests that TLS evolved in mammals as a system that balances gain in survival with a tolerable mutational cost, and that disturbing this balance causes a potentially harmful increase in mutations, which might play a role in carcinogenesis.

  1. Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

    PubMed

    Nilforoushan, Arman; Furrer, Antonia; Wyss, Laura A; van Loon, Barbara; Sturla, Shana J

    2015-04-15

    Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

  2. Clinical and genetic investigation of families with type II Waardenburg syndrome

    PubMed Central

    CHEN, YONG; YANG, FUWEI; ZHENG, HEXIN; ZHOU, JIANDA; ZHU, GANGHUA; HU, PENG; WU, WEIJING

    2016-01-01

    The present study aimed to investigate the molecular pathology of Waardenburg syndrome type II in three families, in order to provide genetic diagnosis and hereditary counseling for family members. Relevant clinical examinations were conducted on the probands of the three pedigrees. Peripheral blood samples of the probands and related family members were collected and genomic DNA was extracted. The coding sequences of paired box 3 (PAX3), microphthalmia-associated transcription factor (MITF), sex-determining region Y-box 10 (SOX10) and snail family zinc finger 2 (SNAI2) were analyzed by polymerase chain reaction and DNA sequencing. The heterozygous mutation, c.649_651delAGA in exon 7 of the MITF gene was detected in the proband and all patients of pedigree 1; however, no pathological mutation of the relevant genes (MITF, SNAI2, SOX10 or PAX3) was detected in pedigrees 2 and 3. The heterozygous mutation c.649_651delAGA in exon 7 of the MITF gene is therefore considered the disease-causing mutation in pedigree 1. However, there are novel disease-causing genes in Waardenburg syndrome type II, which require further research. PMID:26781036

  3. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

    PubMed

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-07-13

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

  4. The role of polymerase III in conjugation between E. coli K12 donor and recipient strains carrying dnaE ts mutation.

    PubMed

    Blinkowa, A

    1976-01-01

    The possible role of DNA polimerase III in conjugation was studied in a series of mutants temperature-sensitive for DNA polymerase III synthesis. The temperature-sensitive DNA mutation called dnaE 486 (ts) prohibits vegetative DNA replication at 41-45 degrees. Transfer of episome and chromosome from temperature-sensitive donor, carrying dnaE mutation to wild-type recipient strains, revertants and dnaE recipients was investigated. In the first two cases the number of Lac+ sexductants being even slightly higher at 43 degrees. Conjugational synthesis accompanying transfer involving the combination of dnaE (ts) thymine dependent and thymine independent donor and recipient strains measured by incorporation of 14C thymine was observed at the restrictive temperature. In the case of conjugation with temperaturesensitive recipient strains a drop of Lac+ sexductants and Pro+ recombinants may be as a result of disturbances in the synthesis of complementary strand in recipient, known to be dependent on pol III. However, the episome investigated by centrifugation in neutral CsC1 gradient after its transfer to the recipient with faulty polymerase III was double stranded (replicated) at the restrictive temperature.

  5. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

    PubMed

    Takahashi, Masateru; Takahashi, Etsuko; Joudeh, Luay I; Marini, Monica; Das, Gobind; Elshenawy, Mohamed M; Akal, Anastassja; Sakashita, Kosuke; Alam, Intikhab; Tehseen, Muhammad; Sobhy, Mohamed A; Stingl, Ulrich; Merzaban, Jasmeen S; Di Fabrizio, Enzo; Hamdan, Samir M

    2018-01-24

    The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of KOD DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

  6. Molecular Cytogenetic Characterization of an inv(Y)(p11.2q11.221∼q11.222) in a Syrian Family.

    PubMed

    Al-Achkar, W; Wafa, A; Al-Ablog, A; Moassass, F; Liehr, T

    2013-12-01

    Constitutional chromosomal abnormalities are an important cause of miscarriage, infertility, congenital anomalies and mental retardation in humans. Pericentric inversions of the human Y-chromosome [inv(Y)] are rather common and show an estimated incidence of 0.6-1:1,000 in males in the general population. Most of the reported cases with inv(Y) are familial. For carriers of pericentric inversions the risk of mental retardation or multiple abortions is not apparently increased and there is no relation with abnormal phenotypic features. Polymerase chain reaction (PCR) analysis to detect microdeletions along the Y-chromosome as well as cytogenetic and fluorescence in situ hybridization (FISH) analysis were done to delineate the characteristics of an inv(Y) in a Syrian family. Thus, we present a detailed molecular-cytogenetic characterization of a father and his two sons having an inv(Y)(p11. 2q11.221∼q11.222) with varying mental retardation features but otherwise normal phenotype.

  7. Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

    PubMed

    Teixeira, M M; Campaner, M; Camargo, E P

    1994-01-01

    To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

  8. Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV.

    PubMed

    Church, Trenton Mel; Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

    2018-03-15

    Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81. IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in

  9. Mispairs with Watson-Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant.

    PubMed

    Xia, Shuangluo; Konigsberg, William H

    2014-04-01

    Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson-Crick geometry in the presence of Mn(2+) indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base-pair could be affected by its microenvironment, we determined 10 structures of an RB69 DNA polymerase quadruple mutant with dG/dT or dT/dG mispairs at position n-1 to n-5 of the Primer/Template duplex. Different shapes of the mispairs, including Watson-Crick geometry, have been observed, strongly suggesting that the local environment of base-pairs plays an important role in their tautomeric or ionization states. © 2014 The Protein Society.

  10. Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota.

    PubMed

    Pence, Matthew G; Choi, Jeong-Yun; Egli, Martin; Guengerich, F Peter

    2010-12-24

    O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

  11. Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal.

    PubMed

    Janowska, Beata; Kurpios-Piec, Dagmara; Prorok, Paulina; Szparecki, Grzegorz; Komisarski, Marek; Kowalczyk, Paweł; Janion, Celina; Tudek, Barbara

    2012-01-03

    One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>C≫A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA(-)Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA(-) bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T→C:G, A:T→G:C, G:C→A:T and G:C→T:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

    PubMed

    Lindow, Janet C; Dohrmann, Paul R; McHenry, Charles S

    2015-07-03

    Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Variations in Nuclear Localization Strategies Among Pol X Family Enzymes.

    PubMed

    Kirby, Thomas W; Pedersen, Lars C; Gabel, Scott A; Gassman, Natalie R; London, Robert E

    2018-06-22

    Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase μ (pol μ), and DNA polymerase λ (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes, and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.

    PubMed

    Konopiński, R; Nowak, R; Siedlecki, J A

    1996-10-17

    Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

  15. Uranyl mediated photofootprinting reveals strong E. coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex.

    PubMed Central

    Jeppesen, C; Nielsen, P E

    1989-01-01

    Employing a newly developed uranyl photofootprinting technique (Nielsen et al. (1988) FEBS Lett. 235, 122), we have analyzed the structure of the E. coli RNA polymerase deoP1 promoter open complex. The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region. These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region. The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded. The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region. Images PMID:2503811

  16. Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

    PubMed

    Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

    2012-03-21

    We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

  17. Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

    PubMed Central

    Vyas, Rajan; Reed, Andrew J.; Tokarsky, E. John; Suo, Zucai

    2015-01-01

    One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion. PMID:25825995

  18. Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

    DOE PAGES

    Vyas, Rajan; Reed, Andrew J.; Tokarsky, E. John; ...

    2015-03-31

    One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here in this article, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg 2+ appeared during the process of phosphodiester bond formation and was located between the reactingmore » α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.« less

  19. First Nuclear DNA C-values for 18 Eudicot Families

    PubMed Central

    HANSON, LYNDA; BOYD, AMY; JOHNSON, MARGARET A. T.; BENNETT, MICHAEL D.

    2005-01-01

    • Background and Aims A key target set at the second Plant Genome Size Workshop, held at the Royal Botanic Gardens, Kew in 2003, was to produce first DNA C-value data for an additional 1 % of angiosperm species, and, within this, to achieve 75 % familial coverage overall (up from approx. 50 %) by 2009. The present study targeted eudicot families for which representation in 2003 (42·5 %) was much lower than monocot (72·8 %) and basal angiosperm (69·0 %) families. • Methods Flow cytometry or Feulgen microdensitometry were used to estimate nuclear DNA C-values, and chromosome counts were obtained where possible. • Key Results First nuclear DNA C-values are reported for 20 angiosperm families, including 18 eudicots. This substantially increases familial representation to 55·2 % for angiosperms and 48·5 % for eudicots. • Conclusions The importance of targeting specific plant families to improve familial nuclear DNA C-value representation is reconfirmed. International collaboration will be increasingly essential to locate and obtain material of unsampled plant families, if the target set by the second Plant Genome Size Workshop is to be met. PMID:16239248

  20. Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

    PubMed Central

    Baril, E; Bonin, P; Burstein, D; Mara, K; Zamecnik, P

    1983-01-01

    A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A. Images PMID:6576366

  1. Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

    PubMed Central

    Flood, Carrie L.; Rodriguez, Gina P.; Bao, Gaobin; Shockley, Arthur H.; Kow, Yoke Wah; Crouse, Gray F.

    2015-01-01

    It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants. PMID:25742645

  2. Nested methylation-specific polymerase chain reaction cancer detection method

    DOEpatents

    Belinsky, Steven A [Albuquerque, NM; Palmisano, William A [Edgewood, NM

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  3. StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

    PubMed

    Boudreau, Beth A; Hron, Daniel R; Qin, Liang; van der Valk, Ramon A; Kotlajich, Matthew V; Dame, Remus T; Landick, Robert

    2018-06-20

    In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

  4. Glucose-nucleobase pairs within DNA: impact of hydrophobicity, alternative linking unit and DNA polymerase nucleotide insertion studies† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04850e

    PubMed Central

    Vengut-Climent, Empar; Peñalver, Pablo; Lucas, Ricardo; Gómez-Pinto, Irene; Aviñó, Anna; Muro-Pastor, Alicia M.; Galbis, Elsa; de Paz, M. Violante; Fonseca Guerra, Célia; Bickelhaupt, F. Matthias; Eritja, Ramón; González, Carlos

    2018-01-01

    Recently, we studied glucose-nucleobase pairs, a binding motif found in aminoglycoside–RNA recognition. DNA duplexes with glucose as a nucleobase were able to hybridize and were selective for purines. They were less stable than natural DNA but still fit well on regular B-DNA. These results opened up the possible use of glucose as a non-aromatic DNA base mimic. Here, we have studied the incorporation and thermal stability of glucose with different types of anchoring units and alternative apolar sugar-nucleobase pairs. When we explored butanetriol instead of glycerol as a wider anchoring unit, we did not gain duplex thermal stability. This result confirmed the necessity of a more conformationally restricted linker to increase the overall duplex stability. Permethylated glucose-nucleobase pairs showed similar stability to glucoside-nucleobase pairs but no selectivity for a specific nucleobase, possibly due to the absence of hydrogen bonds between them. The three-dimensional structure of the duplex solved by NMR located both, the hydrophobic permethylated glucose and the nucleobase, inside the DNA helix as in the case of glucose-nucleobase pairs. Quantum chemical calculations on glucose-nucleobase pairs indicate that the attachment of the sugar to the DNA skeleton through the OH1 or OH4 positions yields the highest binding energies. Moreover, glucose was very selective for guanine when attached through OH1 or OH4 to the DNA. Finally, we examined DNA polymerase insertion of nucleotides in front of the saccharide unit. KF– polymerase from E. coli inserted A and G opposite glc and 6dglc with low efficiency but notable selectivity. It is even capable of extending the new pair although its efficiency depended on the DNA sequence. In contrast, Bst 2.0, SIII and BIOTAQ™ DNA polymerases seem to display a loop-out mechanism possibly due to the flexible glycerol linker used instead of deoxyribose. PMID:29780486

  5. Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta.

    PubMed

    Nicolay, Nils H; Carter, Rebecca; Hatch, Stephanie B; Schultz, Niklas; Prevo, Remko; McKenna, W Gillies; Helleday, Thomas; Sharma, Ricky A

    2012-11-01

    DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

  6. Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain

    PubMed Central

    Contesto-Richefeu, Céline; Tarbouriech, Nicolas; Brazzolotto, Xavier; Betzi, Stéphane; Morelli, Xavier; Burmeister, Wim P.; Iseni, Frédéric

    2014-01-01

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A201–50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A201–50 clearly behaves as a heterodimer. The crystal structure of D4/A201–50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A201–50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A201–50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A201–50 interaction. Finally, we propose a model of D4/A201–50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface. PMID:24603707

  7. DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

    PubMed Central

    Zhong, Xuehua; Hale, Christopher J.; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A.; Henderson, Ian R.; Wohlschlegel, James A.; Jacobsen, Steven E.

    2015-01-01

    DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

  8. Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli.

    PubMed

    Sutton, Mark D; Duzen, Jill M

    2006-03-07

    Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.

  9. Molecular propulsion: chemical sensing and chemotaxis of DNA driven by RNA polymerase.

    PubMed

    Yu, Hua; Jo, Kyubong; Kounovsky, Kristy L; de Pablo, Juan J; Schwartz, David C

    2009-04-29

    Living cells sense extracellular signals and direct their movements in response to stimuli in environment. Such autonomous movement allows these machines to sample chemical change over a distance, leading to chemotaxis. Synthetic catalytic rods have been reported to chemotax toward hydrogen peroxide fuel. Nevertheless individualized autonomous control of movement of a population of biomolecules under physiological conditions has not been demonstrated. Here we show the first experimental evidence that a molecular complex consisting of a DNA template and associating RNA polymerases (RNAPs) displays chemokinetic motion driven by transcription substrates nucleoside triphosphates (NTPs). Furthermore this molecular complex exhibits a biased migration into a concentration gradient of NTPs, resembling chemotaxis. We describe this behavior as "Molecular Propulsion", in which RNAP transcriptional actions deform DNA template conformation engendering measurable enhancement of motility. Our results provide new opportunities for designing and directing nanomachines by imposing external triggers within an experimental system.

  10. Direct non transcriptional role of NF-Y in DNA replication.

    PubMed

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Heltzel, J.; Scouten Ponticelli, S; Sanders, L

    2009-01-01

    Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that themore » poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive

  12. Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta.

    PubMed

    Yang, Juntang; Wang, Rong; Liu, Binyan; Xue, Qizhen; Zhong, Mengyu; Zeng, Hao; Zhang, Huidong

    2015-09-01

    Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼ 50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol η(core) (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP > dCTP and dTTP. Pol η(core) showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol η(core) bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol η(core) relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

    PubMed

    Grover, Abhinav; Agrawal, Vibhuti; Shandilya, Ashutosh; Bisaria, Virendra S; Sundar, Durai

    2011-01-01

    Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies

  14. Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

    PubMed Central

    2011-01-01

    Background Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Results Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. Conclusions We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex

  15. The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

    PubMed Central

    Powers, Kyle T; Washington, M Todd

    2018-01-01

    Abstract Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion. PMID:29385534

  16. Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion.

    PubMed

    Hakonen, Anna H; Isohanni, Pirjo; Paetau, Anders; Herva, Riitta; Suomalainen, Anu; Lönnqvist, Tuula

    2007-11-01

    Twinkle is a mitochondrial replicative helicase, the mutations of which have been associated with autosomal dominant progressive external ophthalmoplegia (adPEO), and recessively inherited infantile onset spinocerebellar ataxia (IOSCA). We report here a new phenotype in two siblings with compound heterozygous Twinkle mutations (A318T and Y508C), characterized by severe early onset encephalopathy and signs of liver involvement. The clinical manifestations included hypotonia, athetosis, sensory neuropathy, ataxia, hearing deficit, ophthalmoplegia, intractable epilepsy and elevation of serum transaminases. The liver showed mtDNA depletion, whereas the muscle mtDNA was only slightly affected. Alpers-Huttenlocher syndrome has previously been associated with mutations of polymerase gamma, a replicative polymerase of mtDNA. We show here that recessive mutations of the close functional partner of the polymerase, the Twinkle helicase, can also manifest as early encephalopathy with liver involvement, a phenotype reminiscent of Alpers syndrome, and are a new genetic cause underlying tissue-specific mtDNA depletion.

  17. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    NASA Astrophysics Data System (ADS)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  18. Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells.

    PubMed

    Lerner, Leticia K; Francisco, Guilherme; Soltys, Daniela T; Rocha, Clarissa R R; Quinet, Annabel; Vessoni, Alexandre T; Castro, Ligia P; David, Taynah I P; Bustos, Silvina O; Strauss, Bryan E; Gottifredi, Vanesa; Stary, Anne; Sarasin, Alain; Chammas, Roger; Menck, Carlos F M

    2017-02-17

    Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

    PubMed Central

    Usdin, K; Furano, A V

    1988-01-01

    The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

  20. Detection of cashew nut DNA in spiked baked goods using a real-time polymerase chain reaction method.

    PubMed

    Brzezinski, Jennifer L

    2006-01-01

    The detection of potentially allergenic foods, such as tree nuts, in food products is a major concern for the food processing industry. A real-time polymerase chain reaction (PCR) method was designed to determine the presence of cashew DNA in food products. The PCR amplifies a 67 bp fragment of the cashew 2S albumin gene, which is detected with a cashew-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other tree nut species, such as almond, Brazil nut, hazelnut, and walnut, as well as 4 varieties of peanut. This assay was sensitive enough to detect 5 pg purified cashew DNA as well as cashew DNA in a spiked chocolate cookie sample containing 0.01% (100 mg/kg) cashew.