Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

PubMed

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1991-01-01

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). Images PMID:2041773

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

PubMed Central

McGregor, W. Glenn; Wei, Dong; Maher, Veronica M.; McCormick, J. Justin

1999-01-01

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ∼37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ∼2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand. PMID:9858539

Cancer therapy and replication stress: forks on the road to perdition.

PubMed

Kotsantis, Panagiotis; Jones, Rebecca M; Higgs, Martin R; Petermann, Eva

2015-01-01

Deregulated DNA replication occurs in cancer where it contributes to genomic instability. This process is a target of cytotoxic therapies. Chemotherapies exploit high DNA replication in cancer cells by modifying the DNA template or by inhibiting vital enzymatic activities that lead to slowing or stalling replication fork progression. Stalled replication forks can be converted into toxic DNA double-strand breaks resulting in cell death, i.e., replication stress. While likely crucial for many cancer treatments, replication stress is poorly understood due to its complexity. While we still know relatively little about the role of replication stress in cancer therapy, technical advances in recent years have shed new light on the effect that cancer therapeutics have on replication forks and the molecular mechanisms that lead from obstructed fork progression to cell death. This chapter will give an overview of our current understanding of replication stress in the context of cancer therapy. © 2015 Elsevier Inc. All rights reserved.

Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

PubMed Central

Georgescu, Roxana E; Langston, Lance; Yao, Nina Y; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; Agarwal, Tani; O’Donnell, Mike E

2015-01-01

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ε to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ε rapidly replaces it. Pol δ–PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ε and stabilizing Pol ε. Comparison of Pol ε and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ε on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ε on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ε on the leading strand. PMID:24997598

Dynamics of Leading-strand Lesion Skipping by the Replisome

PubMed Central

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

PubMed Central

Kosiyatrakul, Settapong T.

2015-01-01

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis. PMID:26195664

Porcine circovirus: transcription and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

PubMed

Piette, J; Calberg-Bacq, C M; Lopez, M; van de Vorst, A

1984-04-05

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

PubMed

Kunkel, Thomas A; Burgers, Peter M J

2017-08-01

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

PubMed

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

PubMed

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units.

PubMed

Lancrey, Astrid; Safa, Layal; Chatain, Jean; Delagoutte, Emmanuelle; Riou, Jean-François; Alberti, Patrizia; Saintomé, Carole

2018-03-01

Replication protein A (RPA) is a single-stranded DNA binding protein involved in replication and in telomere maintenance. During telomere replication, G-quadruplexes (G4) can accumulate on the lagging strand template and need to be resolved. It has been shown that human RPA is able to unfold a single G4. Nevertheless, the G-strand of human telomeres is prone to fold into higher-order structures formed by contiguous G-quadruplexes. To understand how RPA deals with these structures, we studied its interaction with telomeric G-strands folding into an increasing number of contiguous G4s. The aim of this study was to determine whether the efficiency of binding/unfolding of hRPA to telomeric G-strands depends on the number of G4 units. Our data show that the number n of contiguous G4 units (n ≥ 2) does not affect the efficiency of hRPA to coat transiently exposed single-stranded telomeric G-strands. This feature may be essential in preventing instability due to G4 structures during telomere replication. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Ultrastructure of the replication sites of positive-strand RNA viruses

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

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

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

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

PubMed Central

Schauer, Grant D.; O’Donnell, Michael E.

2017-01-01

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand. PMID:28069954

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

PubMed

Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

2014-10-28

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

PubMed Central

Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

2014-01-01

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

PubMed

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

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

Kolonko, Nadine; Bannach, Oliver; Aschermann, Katja

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified bymore » affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.« less

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

Human Mitochondrial DNA Replication

PubMed Central

Holt, Ian J.; Reyes, Aurelio

2012-01-01

Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

In vitro replication of poliovirus

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

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

Structural Basis of Mitochondrial Transcription Initiation.

PubMed

Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

2017-11-16

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice.

PubMed

Hirose, Misa; Schilf, Paul; Gupta, Yask; Zarse, Kim; Künstner, Axel; Fähnrich, Anke; Busch, Hauke; Yin, Junping; Wright, Marvin N; Ziegler, Andreas; Vallier, Marie; Belheouane, Meriem; Baines, John F; Tautz, Diethard; Johann, Kornelia; Oelkrug, Rebecca; Mittag, Jens; Lehnert, Hendrik; Othman, Alaa; Jöhren, Olaf; Schwaninger, Markus; Prehn, Cornelia; Adamski, Jerzy; Shima, Kensuke; Rupp, Jan; Häsler, Robert; Fuellen, Georg; Köhling, Rüdiger; Ristow, Michael; Ibrahim, Saleh M

2018-04-12

Mutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e., the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of an AKR/J mouse strain (B6-mt AKR ) in a C57BL/6 J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to that of wild-type C57BL/6 J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair the metabolic health and lifespan in mice.

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Human CST has independent functions during telomere duplex replication and C-strand fill-in

PubMed Central

Wang, Feng; Stewart, Jason A.; Kasbek, Christopher; Zhao, Yong; Wright, Woodring E.; Price, Carolyn M.

2012-01-01

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems. PMID:23142664

Eukaryotic DNA Replication Fork.

PubMed

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

PubMed Central

Kuzminov, Andrei

1999-01-01

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

Single molecular biology: coming of age in DNA replication.

PubMed

Liu, Xiao-Jing; Lou, Hui-Qiang

2017-09-20

DNA replication is an essential process of the living organisms. To achieve precise and reliable replication, DNA polymerases play a central role in DNA synthesis. Previous investigations have shown that the average rates of DNA synthesis on the leading and lagging strands in a replisome must be similar to avoid the formation of significant gaps in the nascent strands. The underlying mechanism has been assumed to be coordination between leading- and lagging-strand polymerases. However, Kowalczykowski's lab members recently performed single molecule techniques in E. coli and showed the real-time behavior of a replisome. The leading- and lagging-strand polymerases function stochastically and independently. Furthermore, when a DNA polymerase is paused, the helicase slows down in a self-regulating fail-safe mechanism, akin to a ''dead-man's switch''. Based on the real-time single-molecular observation, the authors propose that leading- and lagging-strand polymerases synthesize DNA stochastically within a Gaussian distribution. Along with the development and application of single-molecule techniques, we will witness a new age of DNA replication and other biological researches.

Loss of NEIL3 DNA glycosylase markedly increases replication associated double strand breaks and enhances sensitivity to ATR inhibitor in glioblastoma cells

PubMed Central

Klattenhoff, Alex W.; Thakur, Megha; Chu, Christopher S.; Ray, Debolina; Habib, Samy L.; Kidane, Dawit

2017-01-01

DNA endonuclease eight-like glycosylase 3 (NEIL3) is one of the DNA glycosylases that removes oxidized DNA base lesions from single-stranded DNA (ssDNA) and non-B DNA structures. Approximately seven percent of human tumors have an altered NEIL3 gene. However, the role of NEIL3 in replication-associated repair and its impact on modulating treatment response is not known. Here, we report that NEIL3 is localized at the DNA double-strand break (DSB) sites during oxidative DNA damage and replication stress. Loss of NEIL3 significantly increased spontaneous replication-associated DSBs and recruitment of replication protein A (RPA). In contrast, we observed a marked decrease in Rad51 on nascent DNA strands at the replication fork, suggesting that HR-dependent repair is compromised in NEIL3-deficient cells. Interestingly, NEIL3-deficient cells were sensitive to ataxia–telangiectasia and Rad3 related protein (ATR) inhibitor alone or in combination with PARP1 inhibitor. This study elucidates the mechanism by which NEIL3 is critical to overcome oxidative and replication-associated genotoxic stress. Our findings may have important clinical implications to utilize ATR and PARP1 inhibitors to enhance cytotoxicity in tumors that carry altered levels of NEIL3. PMID:29348879

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

PubMed

Tone, Takahiro; Takeuchi, Ari; Makino, Osamu

2012-01-01

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

PubMed

Zakian, V A; Brewer, B J; Fangman, W L

1979-08-01

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

PubMed Central

Nichol Edamura, Kerrie; Leonard, Michelle R.; Pearson, Christopher E.

2005-01-01

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation. PMID:15625623

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

The (not so) immortal strand hypothesis.

PubMed

Tomasetti, Cristian; Bozic, Ivana

2015-03-01

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an "immortal" DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. Copyright © 2015. Published by Elsevier B.V.

The (not so) Immortal Strand Hypothesis

PubMed Central

Tomasetti, Cristian; Bozic, Ivana

2015-01-01

Background Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Principal Findings Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We to find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Significance Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide strong evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. PMID:25700960

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination.

PubMed

Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro; van Drunen, Ellen; Hickson, Ian D; Essers, Jeroen; Kanaar, Roland

2011-11-10

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.

PubMed Central

Walter, N G; Strunk, G

1994-01-01

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency. Images PMID:8058737

Replication-associated mutational asymmetry in the human genome.

PubMed

Chen, Chun-Long; Duquenne, Lauranne; Audit, Benjamin; Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Huvet, Maxime; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Arneodo, Alain; Thermes, Claude

2011-08-01

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

PubMed Central

Komoda, Keisuke; Naito, Satoshi; Ishikawa, Masayuki

2004-01-01

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses. PMID:14769932

Self-replication of chemical systems based on recognition within a double or a triple helix - A realistic hypothesis

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia

1992-01-01

A scenario is proposed for the non-enzymatic self-replication of short RNA molecules. The self-replication of an oligopyrimidine strand is considered and the process of template-directed synthesis based on recognition within a double helix is discussed. Replication mechanisms are suggested for selected oligonucleotides. The mechanisms are based on Watson-Crick base pairing between complementary nucleotides as well as Hoogsteen base pairing between a duplex and the complementary third strand. It is suggested that self-replication based on these mechanisms may be accomplished but may result in a substantial amount of misinformation transfer when mixed oligonucleotides are used.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

1995-04-11

A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

1995-01-01

Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

Use of a highly sensitive strand-specific quantitative PCR to identify abortive replication in the mouse model of respiratory syncytial virus disease

PubMed Central

2010-01-01

Background The BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics. Results In the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells. Conclusions This is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions. PMID:20860795

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

Detection of hepatitis C virus sequences in brain tissue obtained in recurrent hepatitis C after liver transplantation.

PubMed

Vargas, Hugo E; Laskus, Tomasz; Radkowski, Marek; Wilkinson, Jeff; Balan, Vijay; Douglas, David D; Harrison, M Edwyn; Mulligan, David C; Olden, Kevin; Adair, Debra; Rakela, Jorge

2002-11-01

Patients with chronic hepatitis C frequently report tiredness, easy fatigability, and depression. The aim of this study is to determine whether hepatitis C virus (HCV) replication could be found in brain tissue in patients with hepatitis C and depression. We report two patients with recurrent hepatitis C after liver transplantation who also developed severe depression. One patient died of multiorgan failure and the other, septicemia caused by Staphylococcus aureussis. Both patients had evidence of severe hepatitis C recurrence with features of cholestatic fibrosing hepatitis. We were able to study samples of their central nervous system obtained at autopsy for evidence of HCV replication. The presence of HCV RNA-negative strand, which is the viral replicative form, was determined by strand-specific Tth-based reverse-transcriptase polymerase chain reaction. Viral sequences were compared by means of single-strand conformation polymorphism and direct sequencing. HCV RNA-negative strands were found in subcortical white matter from one patient and cerebral cortex from the other patient. HCV RNA-negative strands amplified from brain tissue differed by several nucleotide substitutions from serum consensus sequences in the 5' untranslated region. These findings support the concept of HCV neuroinvasion, and we speculate that it may provide a biological substrate to neuropsychiatric disorders observed in patients with chronic hepatitis C. The exact lineage of cells permissive for HCV replication and the possible interaction between viral replication and cerebral function that may lead to depression remain to be elucidated.

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome

PubMed Central

Lewis, Jacob S.; Spenkelink, Lisanne M.; Schauer, Grant D.; Hill, Flynn R.; Georgescu, Roxanna E.; O’Donnell, Michael E.; van Oijen, Antoine M.

2017-01-01

The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1–Tof1–Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication. PMID:28923950

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

PubMed Central

Daròs, J A; Marcos, J F; Hernández, C; Flores, R

1994-01-01

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7809126

GENETICS OF HUMAN CELL LINES

PubMed Central

Djordjevic, B.; Szybalski, Waclaw

1960-01-01

The human cell line D98S can be cultivated indefinitely in the presence of up to 3 x 10–5 M 5-bromodeoxyuridine (BUDR), without loss of cell viability. During this time, BUDR is incorporated into both strands of the DNA molecules, replacing up to 45 per cent of the thymidine and thereby rendering the cells highly sensitive to UV light and to x-rays. Cells grown for a limited period of time in the presence of 5-iododeoxyuridine (IUDR) become UV-sensitized, while prolonged cultivation with IUDR results in the loss of cell viability. The properties of the BUDR label permitted the demonstration that: (a) human DNA replicates in a "semiconservative" manner; (b) the degree of radiosensitization of BUDR-treated cells depends on whether the DNA has been substituted in one strand only ("unifilarly") or in both strands ("bifilarly"); (c) functional human DNA is produced during partial inhibition of protein synthesis. The potential applicability of this new rational principle of radiosensitization to the radiotherapy of neoplastic diseases is discussed. PMID:13723177

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

The complete mitochondrial genome of the mudsnail Cipangopaludina cathayensis (Gastropoda: Viviparidae).

PubMed

Yang, Huirong; Zhang, Jia-En; Luo, Hao; Luo, Mingzhu; Guo, Jing; Deng, Zhixin; Zhao, Benliang

2016-05-01

We present the complete mitochondrial genome of Cipangopaludina cathayensis in this study. The mitochondrial genome is 17,157 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes. All of them are encoded on the heavy strand except 7 tRNA genes on the light strand. Overall nucleotide compositions of the light strand are 44.51% of A, 26.74% of T, 20.48% of C and 8.28% of G. All the protein-coding genes start with ATG initiation codon except ATP6 with ATA and ND4 with TTG, and 2 types of termination codons are TAA (ATP6, ND2, COX1, COX2, ATP8, ND1, ND6, Cytb, COX3, ND4) and TAG (ND4L, ND5, ND3). There are 29 intergenic spacers and 5 gene overlaps. The tandem repeat sequences are observed in COX2, tRNA(Asp), ATP6, tRNA(Cys), S-rRNA, ND1, Cytb, ND4 and COX3 genes. Gene arrangement and distribution are different from the typical vertebrates. The absence of D-loop is consistent with the Gastropoda, but at least one lengthy non-coding region is essential regulatory element for the initiation of transcription and replication.

Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

PubMed

Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

2013-04-01

The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

PubMed

Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

2013-06-14

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

Complex Dynamic Development of Poliovirus Membranous Replication Complexes

PubMed Central

Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

2012-01-01

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

PubMed Central

Langston, Lance; O’Donnell, Mike

2017-01-01

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand. DOI: http://dx.doi.org/10.7554/eLife.23449.001 PMID:28346143

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

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

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidencemore » that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.« less

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

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

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.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

PubMed

Audry, Julien; Maestroni, Laetitia; Delagoutte, Emmanuelle; Gauthier, Tiphaine; Nakamura, Toru M; Gachet, Yannick; Saintomé, Carole; Géli, Vincent; Coulon, Stéphane

2015-07-14

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres. © 2015 The Authors.

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

PubMed

Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El-Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

2017-07-14

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo . © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Genome instabilities arising from ribonucleotides in DNA.

PubMed

Klein, Hannah L

2017-08-01

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

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

Laughlin, C.A.; Myers, M.W.; Risin, D.L.

1979-04-15

We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious butmore » interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.« less

Absence of MutSbeta leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

PubMed Central

Slean, Meghan M.; Panigrahi, Gagan B.; Castel, Arturo López; Pearson, August B.; Tomkinson, Alan E.; Pearson, Christopher E.

2016-01-01

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats. PMID:27155933

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

PubMed

Morrow, Carl A; Nguyen, Michael O; Fower, Andrew; Wong, Io Nam; Osman, Fekret; Bryer, Claire; Whitby, Matthew C

2017-06-06

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

PubMed

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory proteins guiding distinct BER sub-pathways.

Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses.

PubMed

Ferlin, Juliette; Farhat, Rayan; Belouzard, Sandrine; Cocquerel, Laurence; Bertin, Antoine; Hober, Didier; Dubuisson, Jean; Rouillé, Yves

2018-06-20

GBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and Arf5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or Arf5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication.

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

Noise-induced bistability in the quasi-neutral coexistence of viral RNAs under different replication modes.

PubMed

Sardanyés, Josep; Arderiu, Andreu; Elena, Santiago F; Alarcón, Tomás

2018-05-01

Evolutionary and dynamical investigations into real viral populations indicate that RNA replication can range between the two extremes represented by so-called 'stamping machine replication' (SMR) and 'geometric replication' (GR). The impact of asymmetries in replication for single-stranded (+) sense RNA viruses has been mainly studied with deterministic models. However, viral replication should be better described by including stochasticity, as the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasi-neutral coexistence scenario, with a line of fixed points involving different strands' equilibrium ratios depending on the initial conditions. Recent research into the quasi-neutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alter the mean-field scenario, and one of the two species outcompetes the other. In this article, we study this phenomenon for viral RNA replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNAs, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication. © 2018 The Author(s).

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

PubMed

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. PMID:28515305

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces

PubMed Central

Romero, Héctor; Zavala, Alejandro; Musto, Héctor

2000-01-01

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C.trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted. PMID:10773076

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces.

PubMed

Romero, H; Zavala, A; Musto, H

2000-05-15

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C. trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

PubMed Central

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

PubMed Central

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

PubMed

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

CTC1-mediated C-strand fill-in is an essential step in telomere length maintenance

PubMed Central

Feng, Xuyang; Hsu, Shih-Jui; Kasbek, Christopher; Chaiken, Mary

2017-01-01

Abstract To prevent progressive telomere shortening as a result of conventional DNA replication, new telomeric DNA must be added onto the chromosome end. The de novo DNA synthesis involves elongation of the G-rich strand of the telomere by telomerase. In human cells, the CST complex (CTC1-STN1-TEN1) also functions in telomere replication. CST first aids in duplication of the telomeric dsDNA. Then after telomerase has extended the G-rich strand, CST facilitates fill-in synthesis of the complementary C-strand. Here, we analyze telomere structure after disruption of human CTC1 and demonstrate that functional CST is essential for telomere length maintenance due to its role in mediating C-strand fill-in. Removal of CTC1 results in elongation of the 3΄ overhang on the G-rich strand. This leads to accumulation of RPA and telomeric DNA damage signaling. G-overhang length increases with time after CTC1 disruption and at early times net G-strand growth is apparent, indicating telomerase-mediated G-strand extension. In contrast, C-strand length decreases continuously, indicating a deficiency in C-strand fill-in synthesis. The lack of C-strand maintenance leads to gradual shortening of the telomeric dsDNA, similar to that observed in cells lacking telomerase. Thus, telomerase-mediated G-strand extension and CST-mediated C-strand fill-in are equally important for telomere length maintenance. PMID:28334750

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

PubMed Central

Donaldson, Janet R; Courcelle, Charmain T; Courcelle, Justin

2004-01-01

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo. PMID:15126385

Stem cell identity and template DNA strand segregation.

PubMed

Tajbakhsh, Shahragim

2008-12-01

The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

PubMed

Jõers, Priit; Lewis, Samantha C; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J; Jacobs, Howard T

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

Mitochondrial Transcription Terminator Family Members mTTF and mTerf5 Have Opposing Roles in Coordination of mtDNA Synthesis

PubMed Central

Jõers, Priit; Lewis, Samantha C.; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J.; Jacobs, Howard T.

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis. PMID:24068965

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

PubMed

Awate, Sanket; Brosh, Robert M

2017-06-08

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism

PubMed Central

Awate, Sanket; Brosh, Robert M.

2017-01-01

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. PMID:28594346

Subnuclear systems for synthesis of simian virus 40 DNA in vitro.

PubMed Central

Edenberg, H J; Waqar, M A; Huberman, J A

1976-01-01

We have developed two subnuclear systems for synthesis of DNA of simian virus 40 in vitro. We prepare chromatin from infected cells by the method of Hancock [(1974) J. Mol. Biol. 86, 649-663]; these "chromatin bodies" can be disrupted and large debris can be pelleted, leaving a supernatant ("soluble system"). Both chromatin bodies and the soluble system incorporate deoxyribonucleoside triphosphates into nucleoprotein complexes that contain simian virus 40 DNA. The DNA labeled in short pulses sediments in neutral sucrose gradients slightly faster than mature simian virus 40 DNA, as expected for replicating intermediate. When rebanded in alkaline sucrose gradients, about half of the radioactivity is found in short strands (200-300 nucleotides) and half in longer strands (up to full viral size). When these systems are supplemented with a cytoplasmic preparation from HeLa cells, synthesis is stimulated about 5-fold, and the short strands are converted into strands of up to full viral length as well as into covalently closed circles. These subnuclear DNA-replicating systems should be useful for biochemical fractionation and characterization of some of the proteins required for DNA replication. PMID:188037

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed Central

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-01-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure. Images PMID:207893

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-05-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

PubMed Central

Chenon, Mélanie; Andreani, Jessica; Guerois, Raphaël; Jupin, Isabelle; Bressanelli, Stéphane

2013-01-01

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction. PMID:23966860

THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

PubMed Central

González Besteiro, Marina A.; Gottifredi, Vanesa

2014-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction*

PubMed Central

Kuong, Kawai J.; Kuzminov, Andrei

2012-01-01

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA+ cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction. PMID:22621921

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

PubMed

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen-Induced DNA-DNA Cross-Links in a Four-Stranded DNA Structure.

PubMed

Martin, Peter R; Couvé, Sophie; Zutterling, Caroline; Albelazi, Mustafa S; Groisman, Regina; Matkarimov, Bakhyt T; Parsons, Jason L; Elder, Rhoderick H; Saparbaev, Murat K

2017-12-12

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

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

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ*

PubMed Central

Xu, Yang; Wu, Xiling; Her, Chengtao

2015-01-01

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress. PMID:26055704

RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

PubMed

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

PubMed

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-04-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

A conserved MCM single-stranded DNA binding element is essential for replication initiation

PubMed Central

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-01-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

Force-induced rupture of double-stranded DNA in the absence and presence of covalently bonded anti-tumor drugs: Insights from molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Upadhyaya, Anurag; Nath, Shesh; Kumar, Sanjay

2018-06-01

DNA intra-strand cross-link (ICL) agents are widely used in the treatment of cancer. ICLs are thought to form a link between the same strand (intra-strand) or complimentary strand (inter-strand) and thereby increase the stability of DNA, which forbids the processes like replication and transcription. As a result, cell death occurs. In this work, we have studied the enhanced stability of a double stranded DNA in the presence of ICLs and compared our findings with the results obtained in the absence of these links. Using atomistic simulations with explicit solvent, a force is applied along and perpendicular to the direction of the helix and we measured the rupture force and the unzipping force of DNA-ICL complexes. Our results show that the rupture and the unzipping forces increase significantly in the presence of these links. The ICLs bind to the minor groove of DNA, which enhance the DNA stabilisation. Such information may be used to design alternative drugs that can stall replication and transcription that are critical to a growing number of anticancer drug discovery efforts.

RNA-dependent RNA polymerases from flaviviruses and Picornaviridae.

PubMed

Lescar, Julien; Canard, Bruno

2009-12-01

Flaviviruses and picornaviruses are positive-strand RNA viruses that encode the RNA-dependent RNA polymerase (RdRp) required for replicating the viral genome in infected cells. Because of their specific and essential role in the virus life cycle, RdRps are prime targets for antiviral drugs. Recent structural data have shed light on the different strategies used by RdRps from flaviviruses and Picornaviridae to initiate RNA polymerization. New details about the catalytic mechanism, the role of metal ions, how these RdRps interact with other nonstructural (NS) viral and host-cell proteins as well as with the viral RNA genome have also been published. These advances contribute to give a more complete picture of the 3D structure and mechanism of a membrane-bound viral replication complex for these two classes of medically important human pathogens.

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

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

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

PubMed Central

Shinbrot, Eve; Henninger, Erin E.; Weinhold, Nils; Covington, Kyle R.; Göksenin, A. Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M.; Gibbs, Richard A.; Sander, Chris; Pursell, Zachary F.

2014-01-01

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

PubMed

Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

2014-10-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. ©2014 American Association for Cancer Research.

BRCA2 and RAD51 promote double-strand break formation and cell death in response to Gemcitabine

PubMed Central

Jones, Rebecca M.; Kotsantis, Panagiotis; Stewart, Grant S.; Groth, Petra; Petermann, Eva

2014-01-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSBs) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitise cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here we investigate why, paradoxically, mutations in HR genes protect cells from killing by Gemcitabine. Using DNA replication and -damage assays in mammalian cells, we show that even short Gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at Gemcitabine-stalled forks are converted into DSBs and thus contribute to Gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumours. PMID:25053826

The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity*

PubMed Central

Manhart, Carol M.; McHenry, Charles S.

2013-01-01

The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5′-ends, an event that can be blocked by attaching a steric block to the 5′-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding. PMID:23264623

Characterization of the complete mitochondrial genome of the hybrid Epinephelus moara♀ × Epinephelus lanceolatus♂, and phylogenetic analysis in subfamily epinephelinae

NASA Astrophysics Data System (ADS)

Gao, Fengtao; Wei, Min; Zhu, Ying; Guo, Hua; Chen, Songlin; Yang, Guanpin

2017-06-01

This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.

Influence of oxidized purine processing on strand directionality of mismatch repair.

PubMed

Repmann, Simone; Olivera-Harris, Maite; Jiricny, Josef

2015-04-17

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

Animal Mitochondrial DNA Replication

PubMed Central

Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

2016-01-01

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

PubMed

Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

2015-07-28

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

Single-stranded DNA and RNA origami.

PubMed

Han, Dongran; Qi, Xiaodong; Myhrvold, Cameron; Wang, Bei; Dai, Mingjie; Jiang, Shuoxing; Bates, Maxwell; Liu, Yan; An, Byoungkwon; Zhang, Fei; Yan, Hao; Yin, Peng

2017-12-15

Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

PubMed

Kodaira, K I; Taketo, A

1978-06-01

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2014-01-01

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. PMID:25054883

Mapping yeast origins of replication via single-stranded DNA detection.

PubMed

Feng, Wenyi; Raghuraman, M K; Brewer, Bonita J

2007-02-01

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

The RNA synthesis machinery of negative-stranded RNA viruses

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

Ortín, Juan, E-mail: jortin@cnb.csic.es; Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure ofmore » their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.« less

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

PubMed

Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

2010-05-01

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

PubMed

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

PubMed Central

Foster, David A.; Hantzopoulos, Petros; Zubay, Geoffrey

1982-01-01

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion. Images PMID:6809958

SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

PubMed

Lam, Yung C; Akhter, Shamima; Gu, Peili; Ye, Jing; Poulet, Anaïs; Giraud-Panis, Marie-Josèphe; Bailey, Susan M; Gilson, Eric; Legerski, Randy J; Chang, Sandy

2010-07-07

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

PubMed

Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

2010-06-04

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

PubMed

González Besteiro, Marina A; Gottifredi, Vanesa

2015-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress. Copyright © 2014 Elsevier B.V. All rights reserved.

UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

PubMed Central

Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

2012-01-01

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

Evolution of the Division of Labor between Genes and Enzymes in the RNA World

PubMed Central

Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

2014-01-01

The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA. PMID:25474573

Evolution of the division of labor between genes and enzymes in the RNA world.

PubMed

Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

2014-12-01

The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA.

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

PubMed Central

Diaz, Arturo; Gallei, Andreas

2012-01-01

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell. PMID:22090102

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.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

PubMed

Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

PubMed

Ogram, Sushma A; Boone, Christopher D; McKenna, Robert; Flanegan, James B

2014-09-01

The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol). Copyright © 2014 Elsevier Inc. All rights reserved.

Experimentally Guided Models Reveal Replication Principles that Shape the Mutation Distribution of RNA Viruses

DTIC Science & Technology

2015-01-30

intracel- lular replication. Two classic replication modes have been described for single-stranded RNA viruses: the ‘stamping machine’ mode ( Stent ...Journal of Theoretical Biology 218:309–321. doi: 10.1006/jtbi.2002.3078. Stent GS. 1963. Molecular Biology of Bacterial Viruses. San Francisco, Calif: W H

Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

DTIC Science & Technology

2001-06-01

enzymatic apparatus needed to initiate DNA replication on recombination intermediates. Escherichia coli PriA protein was found to play a critical function in...the transition from recombination to DNA replication . PriA specifically binds to forked DNA structures created by recombination or replication fork

Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

PubMed Central

Prasai, Kanchanjunga; Robinson, Lucy C.; Scott, Rona S.; Tatchell, Kelly

2017-01-01

Abstract The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. PMID:28549155

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

PubMed

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks*

PubMed Central

Kim, Hyun-Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, Gurjit Singh; Reinert, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavita; Kong, Kimi; Burma, Sandeep; Lee, Suk-Hee; Hromas, Robert A.

2017-01-01

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks. PMID:28049724

Emergence of Distinct Brome Mosaic Virus Recombinants Is Determined by the Polarity of the Inoculum RNA

PubMed Central

Kwon, Sun-Jung

2012-01-01

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(−) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (−) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3′ untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3′ UTR due to end-to-end template switching by BMV replicase during (−)-strand synthesis. In contrast, when the polarity of the inoculum was (−), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms. PMID:22357282

Causes and Consequences of Replication Stress

PubMed Central

Zeman, Michelle K.; Cimprich, Karlene A.

2015-01-01

Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

Nuclear Proteins Hijacked by Mammalian Cytoplasmic Plus Strand RNA Viruses

PubMed Central

Lloyd, Richard E.

2015-01-01

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. PMID:25818028

Method and apparatus for testing a forward-moving strand

DOEpatents

Ducommun, Joel; Vulliens, Philippe

1980-01-01

In a method for testing a continuously forward-moving strand a light beam which passes along a plane that extends approximately perpendicularly to the longitudinal axis of the strand is introduced into the strand. The brightness value is measured on a place of the strand exterior which is distal from the light incidence place by means of at least one photoelectronic element disposed directly on the strand exterior and the measured result is evaluated in a gating circuit which is electrically connected to the photoelectronic element.

A viscous solvent enables information transfer from gene-length nucleic acids in a model prebiotic replication cycle

NASA Astrophysics Data System (ADS)

He, Christine; Gállego, Isaac; Laughlin, Brandon; Grover, Martha A.; Hud, Nicholas V.

2017-04-01

Many hypotheses concerning the nature of early life assume that genetic information was once transferred through the template-directed synthesis of RNA, before the emergence of coded enzymes. However, attempts to demonstrate enzyme-free, template-directed synthesis of nucleic acids have been limited by 'strand inhibition', whereby transferring information from a template strand in the presence of its complementary strand is inhibited by the stability of the template duplex. Here, we use solvent viscosity to circumvent strand inhibition, demonstrating information transfer from a gene-length template (>300 nt) within a longer (545 bp or 3 kb) duplex. These results suggest that viscous environments on the prebiotic Earth, generated periodically by water evaporation, could have facilitated nucleic acid replication—particularly of long, structured sequences such as ribozymes. Our approach works with DNA and RNA, suggesting that viscosity-mediated replication is possible for a range of genetic polymers, perhaps even for informational polymers that may have preceded RNA.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

PubMed Central

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

PubMed

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

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

Neto, J.L. Siqueira; Instituto de Biologia, UNICAMP, Campinas, SP; Lira, C.B.B.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 ismore » a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.« less

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

PubMed

Holsclaw, Julie Korda; Sekelsky, Jeff

2017-05-01

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways. Copyright © 2017 by the Genetics Society of America.

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

PubMed Central

Sasaki, Kaori; Ose, Toyoyuki; Okamoto, Naoaki; Maenaka, Katsumi; Tanaka, Taku; Masai, Hisao; Saito, Mihoko; Shirai, Tsuyoshi; Kohda, Daisuke

2007-01-01

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3′-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3′-OH is essential for the 3′-terminal recognition. In contrast, the direct interaction with 3′-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3′ end. Thus, the N-terminal domain of PriA recognizes the 3′-end base in a base-non-selective manner, in addition to the deoxyribose and 5′-side phosphodiester group, of the 3′-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA. PMID:17464287

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

NASA Astrophysics Data System (ADS)

Gaspar, Miguel; Shenk, Thomas

2006-02-01

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

PubMed

Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

2015-09-01

Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

PubMed

Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

2016-12-20

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

PubMed Central

Alfano, C; McMacken, R

1988-01-01

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands. Images PMID:2847118

Transcription coupled repair deficiency protects against human mutagenesis and carcinogenesis: Personal Reflections on the 50th anniversary of the discovery of xeroderma pigmentosum.

PubMed

Cleaver, James E

2017-10-01

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Cosegregation of chromosomes containing immortal DNA strands in cells that cycle with asymmetric stem cell kinetics.

PubMed

Merok, Joshua R; Lansita, Janice A; Tunstead, James R; Sherley, James L

2002-12-01

A long-standing intriguing hypothesis in cancer biology is that adult stem cells avoid mutations from DNA replication errors by a unique pattern of chromosome segregation. At each asymmetric cell division, adult stem cells have been postulated to selectively retain a set of chromosomes that contain old template DNA strands (i.e., "immortal DNA strands"). Using cultured cells that cycle with asymmetric cell kinetics, we confirmed both the existence of immortal DNA strands and the cosegregation of chromosomes that bear them. Our findings also lead us to propose a role for immortal DNA strands in tissue aging as well as cancer.

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae.

PubMed

Prasai, Kanchanjunga; Robinson, Lucy C; Scott, Rona S; Tatchell, Kelly; Harrison, Lynn

2017-07-27

The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

PubMed

Kogoma, T

1997-06-01

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

DNA Replication During Conjugal Transfer of R1162

DTIC Science & Technology

2002-01-01

transport out of the cell. This idea is stimulated by the similarity between the intermediates of single-stranded phage DNA replication and conjugative...the tra gene clusters ( Miele et al., 1991). The protein is not required for replication of the plasmid, and it seems to be dispensable for transfer...donor, in order to reattach to the transport mechanism prior to the start of a new round of transfer. Rolling circle replication itself might also be

Evolutionary dynamics of adult stem cells: comparison of random and immortal-strand segregation mechanisms.

PubMed

Tannenbaum, Emmanuel; Sherley, James L; Shakhnovich, Eugene I

2005-04-01

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) "immortal DNA strand" co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

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

Lloyd, Richard E., E-mail: rlloyd@bcm.edu

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizesmore » recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.« less

Enzyme-Free Replication with Two or Four Bases.

PubMed

Richert, Clemens; Hänle, Elena

2018-05-20

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies of their genes via semiconservative replication. How this process started before polymerase enzymes had been evolved is unclear. Enzyme-free copying of short stretches of DNA or RNA sequence has been demonstrated, using activated nucleotides, but not replication. We have developed a methodology for replication. It involves extension with reversible termination, enzyme-free ligation, and strand capture and allowed us to monitor nucleotide incorporation for an entire helical turn of DNA, both during a first and a second round of copying. When tracking replication mass spectrometrically, we found that with all four bases (A/C/G/T) an 'error catastrophe' occurs, with the correct sequence being 'overwhelmed' by incorrect ones. When only C and G were used, approx. half of all daughter strands had the mass of the correct sequence after 20 nonenzymatic copying steps. We conclude that enzyme-free replication is more likely to be successful with the two strongly pairing bases, rather than all four bases of the genetic alphabet. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase.

PubMed

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon; White, Charles I; Gallego, Maria E

2018-03-16

Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

PubMed Central

Charbon, Godefroid; Bjørn, Louise; Mendoza-Chamizo, Belén; Frimodt-Møller, Jakob; Løbner-Olesen, Anders

2014-01-01

In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised. PMID:25389264

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.

Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

PubMed Central

Mikkelsen, Jacob Giehm; Lund, Anders H.; Dybkær, Karen; Duch, Mogens; Pedersen, Finn Skou

1998-01-01

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed. PMID:9499117

Mechanisms generating long range correlation in nucleotide composition of the Borrelia Burgdorferi genome

NASA Astrophysics Data System (ADS)

Mackiewicz, P.; Gierlik, A.; Kowalczuk, M.; Szczepanik, D.; Dudek, M. R.; Cebrat, S.

1999-12-01

We have analysed protein coding and intergenic sequences in the Borrelia burgdorferi (the Lyme disease bacterium) genome using different kinds of DNA walks. Genes occupying the leading strand of DNA have significantly different nucleotide composition from genes occupying the lagging strand. Nucleotide compositional bias of the two DNA strands reflects the aminoacid composition of proteins. 96% of genes coding for ribosomal proteins lie on the leading DNA strand, which suggests that the positions of these as well as other genes are non-random. In the B. burgdorferi genome, the asymmetry in intergenic DNA sequences is lower than the asymmetry in the third positions in codons. All these characters of the B. burgdorferi genome suggest that both replication-associated mutational pressure and recombination mechanisms have established the specific structure of the genome and now any recombination leading to inversion of a gene in respect to the direction of replication is forbidden. This property of the genome allows us to assume that it is in a steady state, which enables us to fix some parameters for simulations of DNA evolution.

Lack of close relationship between three strains of human rhinoviruses as determined by their RNA sequences.

PubMed

Yin, F H; Lonberg-Holm, K; Chan, S P

1973-07-01

The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA.

Telomeres and replicative senescence: Is it only length that counts?

PubMed

von Zglinicki, T

2001-07-26

Telomeres are well established as a major 'replicometer', counting the population doublings in primary human cell cultures and ultimately triggering replicative senescence. However, neither is the pace of this biological clock inert, nor is there a fixed threshold telomere length acting as the universal trigger of replicative senescence. The available data suggest that opening of the telomeric loop and unscheduled exposure of the single-stranded G-rich telomeric overhang might act like a semaphore to signal senescent cell cycle arrest. Short telomere length, telomeric single-strand breaks, low levels of loop-stabilizing proteins, or other factors may trigger this opening of the loop. Thus, both telomere shortening and the ultimate signalling into senescence are able to integrate different environmental and genetic factors, especially oxidative stress-mediated damage, which might otherwise become a thread to genomic stability.

Transcription and Recombination: When RNA Meets DNA

PubMed Central

Aguilera, Andrés; Gaillard, Hélène

2014-01-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

The replisome uses mRNA as a primer after colliding with RNA polymerase.

PubMed

Pomerantz, Richard T; O'Donnell, Mike

2008-12-11

Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.

Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases

PubMed Central

Charvin, G.; Bensimon, D.; Croquette, V.

2003-01-01

Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of ≈2.9 s–1, Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of ≈2.4 s–1. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid. PMID:12902541

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2003-05-01

Alkylating minor groove DNA binder adozelesin is capable of inhibiting DNA replication in treated cells through a trans-acting mechanism. The trans... replication in vitro. Using purified proteins in DNA replication initiation assays, we found that RPA purified from cells treated with adozelesin in not...adozelesin has the same single-stranded DNA binding activity and support nucleotide excision repair as normal RPA, but is not able to support SV40 DNA

Interaction of the Tumor Suppressor p53 with Replication Protein A.

DTIC Science & Technology

1996-08-01

The DNA replication factor RPA physically associates with the tumor suppressor protein p53, an interaction that could be important for the function...binding single-stranded DNA, this mutant of RPA fails to support DNA replication . Therefore the region of RPA which interacts with p53 is essential for...of p53, p21/WAFl/CIPl, inhibits the cell-cycle by associating with cyclin-cdk kinases. It also inhibits DNA replication by interacting with a

Lipids and RNA virus replication.

PubMed

Konan, Kouacou V; Sanchez-Felipe, Lorena

2014-12-01

Most viruses rely heavily on their host machinery to successfully replicate their genome and produce new virus particles. Recently, the interaction of positive-strand RNA viruses with the lipid biosynthetic and transport machinery has been the subject of intense investigation. In this review, we will discuss the contribution of various host lipids and related proteins in RNA virus replication and maturation. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

PubMed

Krajacić, Mladen; Ivancic-Jelecki, Jelena; Forcic, Dubravko; Vrdoljak, Anto; Skorić, Dijana

2007-03-09

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

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

The immortal strand hypothesis: still non-randomly segregating opinions.

PubMed

Wakeman, Jane A; Hmadcha, Abdelkrim; Soria, Bernat; McFarlane, Ramsay J

2012-06-01

Abstract Cairns first suggested a mechanism for protecting the genomes of stem cells (SCs) from replicative errors some 40 years ago when he proposed the immortal strand hypothesis, which argued for the inheritance of a so-called immortal strand by an SC following asymmetric SC divisions. To date, the existence of immortal strands remains contentious with published evidence arguing in favour of and against the retention of an immortal strand by asymmetrically dividing SCs. The conflicting evidence is derived from a diverse array of studies on adult SC types and is predominantly based on following the fate of labelled DNA strands during asymmetric cell division events. Here, we review current data, highlighting limitations of such labelling techniques, and suggest how interpretation of such data may be improved in the future.

The origin of replicators and reproducers

PubMed Central

Szathmáry, Eörs

2006-01-01

Replicators are fundamental to the origin of life and evolvability. Their survival depends on the accuracy of replication and the efficiency of growth relative to spontaneous decay. Infrabiological systems are built of two coupled autocatalytic systems, in contrast to minimal living systems that must comprise at least a metabolic subsystem, a hereditary subsystem and a boundary, serving respective functions. Some scenarios prefer to unite all these functions into one primordial system, as illustrated in the lipid world scenario, which is considered as a didactic example in detail. Experimentally produced chemical replicators grow parabolically owing to product inhibition. A selection consequence is survival of everybody. The chromatographized replicator model predicts that such replicators spreading on surfaces can be selected for higher replication rate because double strands are washed away slower than single strands from the surface. Analysis of real ribozymes suggests that the error threshold of replication is less severe by about one order of magnitude than thought previously. Surface-bound dynamics is predicted to play a crucial role also for exponential replicators: unlinked genes belonging to the same genome do not displace each other by competition, and efficient and accurate replicases can spread. The most efficient form of such useful population structure is encapsulation by reproducing vesicles. The stochastic corrector model shows how such a bag of genes can survive, and what the role of chromosome formation and intragenic recombination could be. Prebiotic and early evolution cannot be understood without the models of dynamics. PMID:17008217

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

Evolutionary dynamics of adult stem cells: Comparison of random and immortal-strand segregation mechanisms

NASA Astrophysics Data System (ADS)

Tannenbaum, Emmanuel; Sherley, James L.; Shakhnovich, Eugene I.

2005-04-01

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) “immortal DNA strand” co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

The complete mitochondrial genome of the Antarctic springtail Cryptopygus antarcticus (Hexapoda: Collembola)

PubMed Central

Carapelli, Antonio; Comandi, Sara; Convey, Peter; Nardi, Francesco; Frati, Francesco

2008-01-01

Background Mitogenomics data, i.e. complete mitochondrial genome sequences, are popular molecular markers used for phylogenetic, phylogeographic and ecological studies in different animal lineages. Their comparative analysis has been used to shed light on the evolutionary history of given taxa and on the molecular processes that regulate the evolution of the mitochondrial genome. A considerable literature is available in the fields of invertebrate biochemical and ecophysiological adaptation to extreme environmental conditions, exemplified by those of the Antarctic. Nevertheless, limited molecular data are available from terrestrial Antarctic species, and this study represents the first attempt towards the description of a mitochondrial genome from one of the most widespread and common collembolan species of Antarctica. Results In this study we describe the mitochondrial genome of the Antarctic collembolan Cryptopygus antarcticus Willem, 1901. The genome contains the standard set of 37 genes usually present in animal mtDNAs and a large non-coding fragment putatively corresponding to the region (A+T-rich) responsible for the control of replication and transcription. All genes are arranged in the gene order typical of Pancrustacea. Three additional short non-coding regions are present at gene junctions. Two of these are located in positions of abrupt shift of the coding polarity of genes oriented on opposite strands suggesting a role in the attenuation of the polycistronic mRNA transcription(s). In addition, remnants of an additional copy of trnL(uag) are present between trnS(uga) and nad1. Nucleotide composition is biased towards a high A% and T% (A+T = 70.9%), as typically found in hexapod mtDNAs. There is also a significant strand asymmetry, with the J-strand being more abundant in A and C. Within the A+T-rich region, some short sequence fragments appear to be similar (in position and primary sequence) to those involved in the origin of the N-strand replication of the Drosophila mtDNA. Conclusion The mitochondrial genome of C. antarcticus shares several features with other pancrustacean genomes, although the presence of unusual non-coding regions is also suggestive of molecular rearrangements that probably occurred before the differentiation of major collembolan families. Closer examination of gene boundaries also confirms previous observations on the presence of unusual start and stop codons, and suggests a role for tRNA secondary structures as potential cleavage signals involved in the maturation of the primary transcript. Sequences potentially involved in the regulation of replication/transcription are present both in the A+T-rich region and in other areas of the genome. Their position is similar to that observed in a limited number of insect species, suggesting unique replication/transcription mechanisms for basal and derived hexapod lineages. This initial description and characterization of the mitochondrial genome of C. antarcticus will constitute the essential foundation prerequisite for investigations of the evolutionary history of one of the most speciose collembolan genera present in Antarctica and other localities of the Southern Hemisphere. PMID:18593463

Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application.

PubMed

Hu, Jianming; Liu, Kuancheng

2017-03-21

Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

PubMed

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

PubMed Central

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

PubMed

Neuman, Benjamin W

2016-11-01

Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains. Copyright © 2016 Elsevier B.V. All rights reserved.

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy.

PubMed

Wiegand, Thomas; Cadalbert, Riccardo; Gardiennet, Carole; Timmins, Joanna; Terradot, Laurent; Böckmann, Anja; Meier, Beat H

2016-11-02

DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

PubMed Central

Diamant, Noam; Hendel, Ayal; Vered, Ilan; Carell, Thomas; Reißner, Thomas; de Wind, Niels; Geacinov, Nicholas; Livneh, Zvi

2012-01-01

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity. PMID:21908406

Transcription and recombination: when RNA meets DNA.

PubMed

Aguilera, Andrés; Gaillard, Hélène

2014-08-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

PubMed

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-06-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

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.

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.

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase

PubMed Central

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon

2018-01-01

Abstract Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication. PMID:29346668

A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

PubMed Central

Thomas, Jacob; Lee, Catherine A.; Grossman, Alan D.

2013-01-01

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability. PMID:23326247

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein: characterization of binding specificity and DNA replication activity.

PubMed Central

Takai, T; Nishita, Y; Iguchi-Ariga, S M; Ariga, H

1994-01-01

We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication. Images PMID:7838710

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

PubMed Central

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-01-01

ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

PubMed

Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

2013-09-16

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

PubMed

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-02-16

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.

From structure to mechanism—understanding initiation of DNA replication

PubMed Central

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

PubMed Central

Mitchell, Angela M.; Hirsch, Matthew L.; Li, Chengwen

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses. PMID:24198403

Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication.

PubMed

Mitchell, Angela M; Hirsch, Matthew L; Li, Chengwen; Samulski, R Jude

2014-01-01

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses.

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.

PubMed

Makeyev, E V; Bamford, D H

2000-01-04

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses. Copyright © 2017 American Society for Microbiology.

Studies with infections fragments of phage DNA. Final report, January 1, 1970--June 30, 1976

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

Schachtele, C. F.

The minute, virulent and structurally intricate Bacillus subtilis bacteriophage phi 29 was utilized to study in vivo viral development. Purified strands of phi 29 DNA were used to analyze transcription of the viral genome. Early mRNA hybridizes to the light DNA strand which controls DNA replication and other early functions. Late mRNA hybridizes to the heavy DNA strand which codes for phage structural proteins. The temporal sequence of specific viral protein synthesis was analyzed by gel electrophoresis and was shown to directly correlate with the RNA transcription pattern. The genes carried by phi 29 have been marked with ts andmore » sus mutations and mapped by appropriate crosses yielding a linear map of 17 cistrons. Fragments of the phi 29 DNA were shown to retain their biological activity and marker rescue studies indicated that gene transfer could be performed with pieces having a molecular weight of less than 1 million daltons. Mutant infection under nonpermissive conditions and the analysis of precursor structures has allowed the formation of a tentative morphogenetic pathway leading to the formation of infectious particles. Work with phi 29 has established this virus as an advantageous model system for studying a variety of problems in molecular biology and approximately a dozen laboratories in the country and abroad are working with this phage.« less

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

PubMed

Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

2018-05-25

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Lack of a Close Relationship Between Three Strains of Human Rhinoviruses as Determined by Their RNA Sequences 1

PubMed Central

Yin, Fay H.; Lonberg-Holm, K.; Chan, S. P.

1973-01-01

The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA. PMID:4126194

The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation.

PubMed

James, A P; Kilbey, B J

1977-10-01

The mechanism of UV-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61 percent at survival levels of 90 and 77 percent, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective.

Cloning of nascent monkey DNA synthesized early in the cell cycle.

PubMed

Kaufmann, G; Zannis-Hadjopoulos, M; Martin, R G

1985-04-01

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

PubMed Central

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Insights into the Initiation of Eukaryotic DNA Replication.

PubMed

Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

2015-01-01

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication

PubMed Central

Dang, Que; Hu, Wei-Shau

2001-01-01

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer. PMID:11134294

Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

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

Kodadek, T.; Gamper, H.

The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. Theymore » demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.« less

Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

NASA Astrophysics Data System (ADS)

Abid Ali, Ferdos; Renault, Ludovic; Gannon, Julian; Gahlon, Hailey L.; Kotecha, Abhay; Zhou, Jin Chuan; Rueda, David; Costa, Alessandro

2016-02-01

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

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

Gonadal Mosaicism Induced by Chemical Treatment of Sperm in Drosophila melanogaster

PubMed Central

Lindsley, Dan L.; Hardy, Robert W.; Ripoll, Pedro; Lindsley, Dart

2016-01-01

Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding—incomplete to date—of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germline of the treated male’s offspring. We describe a model for early embryonic development and establishment of the germline of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germline. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germlines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between 0.65 and 0.80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate. PMID:26163187

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

Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

PubMed

Caldecott, K W

2001-05-01

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

PubMed Central

Long, Quanxin; Yan, Ran; Hu, Jieli; Cai, Dawei; Kim, Elena S.; Zhang, Hu; Liu, Yuanjie

2017-01-01

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell’s DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B. PMID:29287110

Gause's Principle and the Effect of Resource Partitioning on the Dynamical Coexistence of Replicating Templates

PubMed Central

Szilágyi, András; Zachar, István; Szathmáry, Eörs

2013-01-01

Models of competitive template replication, although basic for replicator dynamics and primordial evolution, have not yet taken different sequences explicitly into account, neither have they analyzed the effect of resource partitioning (feeding on different resources) on coexistence. Here we show by analytical and numerical calculations that Gause's principle of competitive exclusion holds for template replicators if resources (nucleotides) affect growth linearly and coexistence is at fixed point attractors. Cases of complementary or homologous pairing between building blocks with parallel or antiparallel strands show no deviation from the rule that the nucleotide compositions of stably coexisting species must be different and there cannot be more coexisting replicator species than nucleotide types. Besides this overlooked mechanism of template coexistence we show also that interesting sequence effects prevail as parts of sequences that are copied earlier affect coexistence more strongly due to the higher concentration of the corresponding replication intermediates. Template and copy always count as one species due their constraint of strict stoichiometric coupling. Stability of fixed-point coexistence tends to decrease with the length of sequences, although this effect is unlikely to be detrimental for sequences below 100 nucleotides. In sum, resource partitioning (niche differentiation) is the default form of competitive coexistence for replicating templates feeding on a cocktail of different nucleotides, as it may have been the case in the RNA world. Our analysis of different pairing and strand orientation schemes is relevant for artificial and potentially astrobiological genetics. PMID:23990769

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

PubMed Central

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-01-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5. PMID:23598996

From structure to mechanism-understanding initiation of DNA replication.

PubMed

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

The double-stranded, closed-circular, replicative form (RF) of M13 DNA is present in high copy numbers in infected cells, and its physical characteristics are essentially identical to those of closed-circular plasmid DNAs. Any of the methods commonly used to purify plasmid DNA can therefore be used to isolate M13 RF DNA. This protocol describes the isolation of M13 RF DNA by alkaline lysis from small volumes (1-2 mL) of infected bacterial cultures. The yield of DNA (1-4 mg, depending on the size of the M13 clone) is more than enough for most purposes in molecular cloning. However, should more DNA be needed, the procedure can easily be scaled up. © 2017 Cold Spring Harbor Laboratory Press.

Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

PubMed Central

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

PubMed

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

Localized DNA melting and structural pertubations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo.

PubMed Central

Gille, H; Messer, W

1991-01-01

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation. Images PMID:2026151

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PubMed

Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

2007-11-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

PubMed Central

Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

2007-01-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins. PMID:18081428

Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

PubMed

Kim, Ye Eun; Higgs, Paul G

2016-11-01

It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

PubMed

Berglund, Anna-Karin; Navarrete, Clara; Engqvist, Martin K M; Hoberg, Emily; Szilagyi, Zsolt; Taylor, Robert W; Gustafsson, Claes M; Falkenberg, Maria; Clausen, Anders R

2017-02-01

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

PubMed

Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

2006-08-30

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

PubMed Central

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-01-01

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends. PMID:22354040

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.

PubMed

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-04-18

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Trio of Tryptophans Aid in Camptothecin Therapy | Center for Cancer Research

Cancer.gov

On occasion, a normally desirable feature becomes not so desirable, making it necessary to find ways to work around it. The double helical nature of DNA helps it attain a compact, supercoiled state, which is essential for lengthy strands of DNA to reside in the nucleus as part of the chromosomes. During DNA replication and gene transcription, however, cellular machinery needs to access individual DNA strands; thus, the supercoiled, knotted state becomes undesirable. Just as a tangled telephone cord has to be manipulated, the DNA strands must be separated, unwound, and eventually joined together again.

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

PubMed

Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2012-01-01

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

PubMed

van der Schaar, H M; Melia, C E; van Bruggen, J A C; Strating, J R P M; van Geenen, M E D; Koster, A J; Bárcena, M; van Kuppeveld, F J M

2016-01-01

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

Understanding DNA replication by the bacteriophage T4 replisome.

PubMed

Benkovic, Stephen J; Spiering, Michelle M

2017-11-10

The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the following: its 8-protein composition, their individual and synergistic activities, and assembly in vitro and in vivo into a replisome capable of coordinated leading/lagging strand DNA synthesis. We conclude with a brief comparison with other replisomes with emphasis on how coordinated DNA replication is achieved. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

ssDNA damage dependence from singlet oxygen concentration at photodynamic interaction

NASA Astrophysics Data System (ADS)

Klimenko, V. V.; Kaydanov, N. E.; Emelyanov, A. K.; Bogdanov, A. A.

2017-11-01

Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

PubMed

Nonin-Lecomte, Sylvie; Dardel, Frédéric; Lestienne, Patrick

2005-08-01

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

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

Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

1982-12-01

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-daltonmore » DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.« less

Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

PubMed

Safa, Layal; Delagoutte, Emmanuelle; Petruseva, Irina; Alberti, Patrizia; Lavrik, Olga; Riou, Jean-François; Saintomé, Carole

2014-08-01

Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Structural Insights into the Coupling of Virion Assembly and Rotavirus Replication

PubMed Central

Trask, Shane D.; McDonald, Sarah M.; Patton, John T.

2013-01-01

Preface Viral replication is rapid and robust, but it is far from a chaotic process. Instead, successful production of infectious progeny requires that events occur in the correct place and at the correct time. Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, seems to govern its replication through ordered disassembly and assembly of a triple-layered icosahedral capsid. In recent years, high-resolution structural data have provided unprecedented insight into these events. In this Review, we explore the current understanding of rotavirus replication and how it compares to other Reoviridae family members. PMID:22266782

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

PubMed Central

Sedletska, Yuliya; Radicella, J. Pablo; Sage, Evelyne

2013-01-01

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences. PMID:23945941

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

PubMed

Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub

2017-01-01

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

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

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

PubMed

Cotmore, S F; Tattersall, P

1998-11-01

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Damage and Repair of DNA in 5-Bromodeoxyuridine-Labeled Chinese Hamster Cells Exposed to Fluorescent Light

PubMed Central

Ben-Hur, E.; Elkind, M. M.

1972-01-01

Illumination of Chinese hamster cells with fluorescent light after 5-bromodeoxyuridine incorporation leads to extensive single-strand breakage in the DNA of the exposed cells. The rate of production of single-strand breaks is dependent on the extent to which thymine is replaced by 5-bromouracil. At least some of the breaks observed with alkaline gradients are probably produced in vivo and are probably not contingent upon alkaline hydrolysis since breakage can be demonstrated with neutral gradients also. Cells are able to rejoin most of the single-strand breaks within 60 min; however, damage to the DNA-containing material (the “complex”) initially released from cells is repaired more slowly. Cysteamine protects against single-strand breakage with a dose-modifying factor of 2.8. A comparison is made between the production of single-strand breaks by fluorescent light and X-rays, and the significance of such breaks relative to cell survival is discussed. PMID:5063839

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

PubMed

Belmonte, Frances R; Martin, James L; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A; Kaufman, Brett A

2016-04-28

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

PubMed Central

Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

2016-01-01

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

Genetics Home Reference: Warsaw breakage syndrome

MedlinePlus

... helicase. Helicases are enzymes that attach (bind) to DNA and temporarily unwind the two spiral strands (double helix) of the DNA molecule. This unwinding is necessary for copying ( replicating ) ...

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

The complete mitochondrial genome sequence of Neovison vison (Carnivora: Mustelidae).

PubMed

Sun, Wei-Li; Wang, Shao-Jing; Wang, Zhuo; Liu, Han-Lu; Zhong, Wei; Yang, Ya-Han; Li, Guang-Yu

2016-05-01

The phylogenetic and taxonomic position of the American mink Neovison vison have long been unclear. In this paper, the complete mitogenome of N. vison was sequenced and characterized. The total length was 16,594 bp and typically consists of 37 genes, including 13 protein-coding genes, 2 rRNAs, 22 tRNA, a large control region (CR) and a light-strand replication origin (OL). Gene contents, locations, and arrangements were identical to those of typical vertebrate. The overall base composition is 33.6%, 25.4%, 27.8% and 13.3% for A, C, T and G, respectively, with a moderate bias on AT content (61.4%). This result is expected to provide useful molecular data and contribute to further taxonomic and phylogenetic studies of Mustelidae and Carnivora.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

PubMed Central

Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

2018-01-01

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes. PMID:25771806

Replication Stress: A Lifetime of Epigenetic Change

PubMed Central

Khurana, Simran; Oberdoerffer, Philipp

2015-01-01

DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

Picornaviral Polymerase Structure, Function, and Fidelity Modulation

PubMed Central

Peersen, Olve B.

2017-01-01

Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3Dpol. Over the past decade we have made tremendous advances in our understanding of 3Dpol structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases. PMID:28163093

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

PubMed Central

Venuti, A; Di Russo, C; del Grosso, N; Patti, A M; Ruggeri, F; De Stasio, P R; Martiniello, M G; Pagnotti, P; Degener, A M; Midulla, M

1985-01-01

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome. Images PMID:2997478

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.

PubMed

Beattie, Thomas R; Bell, Stephen D

2011-01-01

Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.

Interatomic Coulombic Decay Effects in Theoretical DNA Recombination Systems Involving Protein Interaction Sites

NASA Astrophysics Data System (ADS)

Vargas, E. L.; Rivas, D. A.; Duot, A. C.; Hovey, R. T.; Andrianarijaona, V. M.

2015-03-01

DNA replication is the basis for all biological reproduction. A strand of DNA will ``unzip'' and bind with a complimentary strand, creating two identical strands. In this study, we are considering how this process is affected by Interatomic Coulombic Decay (ICD), specifically how ICD affects the individual coding proteins' ability to hold together. ICD mainly deals with how the electron returns to its original state after excitation and how this affects its immediate atomic environment, sometimes affecting the connectivity between interaction sites on proteins involved in the DNA coding process. Biological heredity is fundamentally controlled by DNA and its replication therefore it affects every living thing. The small nature of the proteins (within the range of nanometers) makes it a good candidate for research of this scale. Understanding how ICD affects DNA molecules can give us invaluable insight into the human genetic code and the processes behind cell mutations that can lead to cancer. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae.

PubMed

Daròs, José-Antonio

2016-10-01

Eggplant latent viroid (ELVd) is the only species of the genus Elaviroid (family Avsunviroidae). All the viroids in the family Avsunviroidae contain hammerhead ribozymes in the strands of both polarities, and are considered to replicate in the chloroplasts of infected cells. This family includes two other genera: Avsunviroid and Pelamoviroid. ELVd consists of a single-stranded, circular, non-coding RNA of 332-335 nucleotides that folds in a branched quasi-rod-like minimum free-energy conformation. RNAs of complementary polarity exist in infected cells and are considered to be replication intermediates. Plus (+) polarity is assigned arbitrarily to the strand that accumulates at a higher concentration in infected tissues. HOST: To date, ELVd has only been shown to infect eggplant (Solanum melongena L.), the species in which it was discovered. A very narrow host range seems to be a common property in members of the family Avsunviroidae. ELVd infections of eggplants are apparently symptomless. ELVd is transmitted mechanically and by seed. http://subviral.med.uottawa.ca. © 2015 BSPP and John Wiley & Sons Ltd.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

PubMed Central

Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

2016-01-01

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

Probe and method for DNA detection

DOEpatents

Yeh, Hsin-Chih; Werner, James Henry; Sharma, Jaswinder Kumar; Martinez, Jennifer Suzanne

2013-07-02

A hybridization probe containing two linear strands of DNA lights up upon hybridization to a target DNA using silver nanoclusters that have been templated onto one of the DNA strands. Hybridization induces proximity between the nanoclusters on one strand and an overhang on the other strand, which results in enhanced fluorescence emission from the nanoclusters.

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

PubMed Central

Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

1992-01-01

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID:1328676

Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells

PubMed Central

Perelygina, Ludmila; Adebayo, Adebola; Metcalfe, Maureen; Icenogle, Joseph

2015-01-01

Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1–4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV. PMID:26177032

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

PubMed

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication. Copyright © 2018 American Society for Microbiology.

Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

PubMed Central

Qiu, Shihong; Ogino, Minako; Luo, Ming

2015-01-01

ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses. PMID:26512087

Isolation of Protein-Associated Circular DNA from Healthy Cattle Serum

PubMed Central

Funk, Mathis; Gunst, Karin; Lucansky, Vincent; Müller, Hermann; zur Hausen, Harald

2014-01-01

Three replication-competent single-stranded DNA molecules sharing nucleotide similarity to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 2.36 were isolated from healthy bovine serum. PMID:25169856

Crystal structure of an Okazaki fragment at 2-A resolution

NASA Technical Reports Server (NTRS)

Egli, M.; Usman, N.; Zhang, S. G.; Rich, A.

1992-01-01

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

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

Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs.

PubMed

Sanders, Ashley D; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Lansdorp, Peter M

2017-06-01

The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5'-3' orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU + strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair Defective Escherichia coli.

PubMed

Foster, Patricia L; Niccum, Brittany A; Popodi, Ellen; Townes, Jesse P; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu

2018-06-15

Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defective E. coli strains yielded ≈30,000 base-pair substitutions, revealing mutational patterns across the entire chromosome. The base-pair substitution spectrum was dominated by A:T > G:C transitions, which occurred predominantly at the center base of 5'N A C3'+5'G T N3' triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for base-pair substitutions, and the rate at which these occurred increased with run length. Comparison with ≈2000 base-pair substitutions accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an mmr ndk double mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone or less well corrected by proofreading than was leading strand synthesis. Copyright © 2018, Genetics.

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.

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events.

PubMed

Seier, Tracey; Padgett, Dana R; Zilberberg, Gal; Sutera, Vincent A; Toha, Noor; Lovett, Susan T

2011-06-01

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

PubMed Central

Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

2017-01-01

Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

"Breaking up is hard to do": the formation and resolution of sister chromatid intertwines.

PubMed

Baxter, Jonathan

2015-02-13

The absolute necessity to resolve every intertwine between the two strands of the DNA double helix provides a massive challenge to the cellular processes that duplicate and segregate chromosomes. Although the overwhelming majority of intertwines between the parental DNA strands are resolved during DNA replication, there are numerous chromosomal contexts where some intertwining is maintained into mitosis. These mitotic sister chromatid intertwines (SCIs) can be found as; short regions of unreplicated DNA, fully replicated and intertwined sister chromatids--commonly referred to as DNA catenation--and as sister chromatid linkages generated by homologous recombination-associated processes. Several overlapping mechanisms, including intra-chromosomal compaction, topoisomerase action and Holliday junction resolvases, ensure that all SCIs are removed before they can prevent normal chromosome segregation. Here, I discuss why some DNA intertwines persist into mitosis and review our current knowledge of the SCI resolution mechanisms that are employed in both prokaryotes and eukaryotes, including how deregulating SCI formation during DNA replication or disrupting the resolution processes may contribute to aneuploidy in cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

PubMed

Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E; Bogerd, Hal P; Tsai, Kevin; Heaton, Nicholas S; Cullen, Bryan R

2017-09-13

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Signal amplification of padlock probes by rolling circle replication.

PubMed Central

Banér, J; Nilsson, M; Mendel-Hartvig, M; Landegren, U

1998-01-01

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe. PMID:9801302

hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases.

PubMed

Ashton, Nicholas W; Paquet, Nicolas; Shirran, Sally L; Bolderson, Emma; Kariawasam, Ruvini; Touma, Christine; Fallahbaghery, Azadeh; Gamsjaeger, Roland; Cubeddu, Liza; Botting, Catherine; Pollock, Pamela M; O'Byrne, Kenneth J; Richard, Derek J

2017-06-01

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

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

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

PubMed Central

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

2016-01-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

The life story of hydrogen peroxide II: a periodic pH and thermochemical drive for the RNA world

PubMed Central

Ball, Rowena; Brindley, John

2015-01-01

It is now accepted that primordial non-cellular RNA communities must have been subject to a periodic drive in order to replicate and prosper. We have proposed the oxidation of thiosulfate by hydrogen peroxide as this drive. This reaction system behaves as (i) a thermochemical and (ii) a pH oscillator, and in this work, we unify (i) and (ii) for the first time. We report thermally self-consistent, dynamical simulations in which the system transitions smoothly from nearly isothermal pH to fully developed thermo-pH oscillatory regimes. We use this oscillator to drive simulated replication of a 39-bp RNA species. Production of replicated duplex under thermo-pH drive was significantly enhanced compared with that under purely thermochemical drive, effectively allowing longer strands to replicate. Longer strands are fitter, with more potential to evolve enzyme activity and resist degradation. We affirm that concern over the alleged toxicity of hydrogen peroxide to life is largely misplaced in the current context, we survey its occurrence in the solar system to motivate its inclusion as a biosignature in the search for life on other worlds and highlight that pH oscillations in a spatially extended, bounded system manifest as the fundamental driving force of life: a proton gradient. PMID:26202683

Role of Escherichia coli dnaA gene and its integrative suppression in M13 Coliphage DNA synthesis

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

Mitra, S.; Stallions, D.R.

An F/sup +/ derivative of Escherichia coli E508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508(ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also,more » but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dnaA function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dnaA function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.« less

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

PubMed

Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

2013-10-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

PubMed Central

Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine

2013-01-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses. PMID:23885072

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest.

PubMed

Kehrli, Keffy; Phelps, Michael; Lazarchuk, Pavlo; Chen, Eleanor; Monnat, Ray; Sidorova, Julia M

2016-11-18

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1-3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

PubMed Central

Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

2017-01-01

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis

PubMed Central

Zhang, Zhenlu; He, Guijuan; Catanzaro, Nicholas; Wu, Zujian; Xie, Lianhui

2018-01-01

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection. PMID:29649282

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

PubMed

Huh, Yang Hoon; Cohen, Justin; Sherley, James L

2013-10-15

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

PubMed Central

Possoz, Christophe; Durand, Adeline; Desfontaines, Jean-Michel; Barre, François-Xavier; Leach, David R. F.

2018-01-01

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations. PMID:29522563

Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands

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

Bates, R.C.; Snyder, C.E.; Banerjee, P.T.

1984-02-01

Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. The uniqueness of minus strand encapsidation is reexamined for the autonomous parvoviruses. Although it was found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNAmore » when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.« less

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque.

PubMed

Majerowicz, Selma; Grief, Christopher; Ferguson, Debora; Airano, Renata C; Baptista, Marcia L; Pinto, Marcelo A; Barth, Ortrud Monika

2004-10-01

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Getting it done at the ends: Pif1 family DNA helicases and telomeres.

PubMed

Geronimo, Carly L; Zakian, Virginia A

2016-08-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5'-3' DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. Copyright © 2016. Published by Elsevier B.V.

Getting it done at the ends: Pif1 family DNA helicases and telomeres

PubMed Central

Geronimo, Carly L.; Zakian, Virginia A.

2017-01-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. PMID:27233114

Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA

PubMed Central

Hoberg, Emily; Szilagyi, Zsolt; Taylor, Robert W.; Gustafsson, Claes M.; Falkenberg, Maria

2017-01-01

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication. PMID:28207748

Three stages in the evolution of the genetic code

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1993-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity those amino acids emerging later in a translation process are derived. Codon number and chemical complexity indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage 1 use purine-rich codons, while all the amino acids introduced in the second stage, in contrast, use pyrimidines in the third position of their codons. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non-enzymatic replication and interactions of hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids, which gradually decreased during their evolution. Amino acids independently available from prebiotic synthesis were thus correlated to purine-rich codons. Implications on the prebiotic replication are discussed also in the light of recent codon usage data.

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.

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

PubMed Central

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.

2016-01-01

ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. PMID:27390781

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism

PubMed Central

Gubaev, Airat; Weidlich, Daniela; Klostermeier, Dagmar

2016-01-01

The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors. PMID:27557712

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease ‘driver’ mutations

PubMed Central

Suárez, Martha Y.; Villagrán; Miller, John H.

2015-01-01

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease ‘driver’ mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands. PMID:26310834

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease 'driver' mutations.

PubMed

Villagrán, Martha Y Suárez; Miller, John H

2015-08-27

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease 'driver' mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands.

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

PubMed

Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

PubMed

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer.

PubMed

Izhar, Lior; Goldsmith, Moshe; Dahan, Ronny; Geacintov, Nicholas; Lloyd, Robert G; Livneh, Zvi

2008-09-12

Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.

A distinct first replication cycle of DNA introduced in mammalian cells

PubMed Central

Chandok, Gurangad S.; Kapoor, Kalvin K.; Brick, Rachel M.; Sidorova, Julia M.; Krasilnikova, Maria M.

2011-01-01

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells. PMID:21062817

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

PubMed

Reid, David W; Campos, Rafael K; Child, Jessica R; Zheng, Tianli; Chan, Kitti Wing Ki; Bradrick, Shelton S; Vasudevan, Subhash G; Garcia-Blanco, Mariano A; Nicchitta, Christopher V

2018-04-01

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways. IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention. Copyright © 2018 American Society for Microbiology.

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

PubMed

Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

2017-04-17

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

Bypass of a Nick by the Replisome of Bacteriophage T7*

PubMed Central

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.

2011-01-01

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase·polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick. PMID:21701044

Bypass of a nick by the replisome of bacteriophage T7.

PubMed

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C

2011-08-12

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

PubMed

Zgaga, Z

1991-08-01

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen

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

Piette, J.; Decuyper-Debergh, D.; Gamper, H.

Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively. The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli. The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA. A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts. Numerous mutant clones partially or totallymore » deficient in beta-galactosidase were plaque-purified and amplified. The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure. All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations. The predominant mutation was a T.A----G.C transversion.« less

Signal replication in a DNA nanostructure

NASA Astrophysics Data System (ADS)

Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

2017-01-01

Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

DNA replication depends on photosynthetic electron transport in cyanobacteria.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Kanesaki, Yu; Narikawa, Rei; Chibazakura, Taku; Ikeuchi, Masahiko; Yoshikawa, Hirofumi

2013-07-01

The freshwater cyanobacterium Synechococcus elongatus PCC 7942 exhibits light-dependent growth. Although it has been reported that DNA replication also depends on light irradiation in S. elongatus 7942, the involvement of the light in the regulation of DNA replication remains unclear. To elucidate the regulatory pathway of DNA replication by light, we studied the effect of several inhibitors, including two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on DNA replication in S. elongatus 7942. DCMU inhibited only DNA replication initiation, whereas DBMIB blocked both the initiation and progression of DNA replication. These results suggest that DNA replication depends on the photosynthetic electron transport activity and initiation and progression of DNA replication are regulated in different ways. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

PubMed Central

Gibb, Bryan; Ye, Ling F.; Gergoudis, Stephanie C.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

2014-01-01

Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance [1], [2], [3]. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA. PMID:24498402

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

PubMed

Reyes-Ruiz, José M; Osuna-Ramos, Juan F; Cervantes-Salazar, Margot; Lagunes Guillen, Anel E; Chávez-Munguía, Bibiana; Salas-Benito, Juan S; Del Ángel, Rosa M

2018-02-01

Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells. Copyright © 2017 Elsevier Inc. All rights reserved.

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

PubMed Central

Janska, Agnieszka; Goswami, Panchali; Renault, Ludovic; Abid Ali, Ferdos; Kotecha, Abhay; Costa, Alessandro

2017-01-01

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG–Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression. PMID:28373564

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1992-01-01

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression. Images PMID:1475192

Base-unpaired regions in supercoiled replicative form DNA of coliphage M13

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

Dasgupta, S.; Allison, D.P.; Snyder, C.E.

Superhelical covalently closed circular replicative form DNA (RF I) of coliphage M13 appears as a relaxed molecule that has a base-unpaired region in the form of a bubble (100 to 200 base pairs long) seen in electron micrographs when spread in the presence of formaldehyde and formamide or after pretreatment with glyoxal. S1 endonuclease, specific for single-stranded DNA, converts superhelical M13 RF I DNA, but not nonsuperhelical M13 RF I to a significant extent, into unit-length linear molecules by sequential nicking of two strands. The locations of S1 nuclease-susceptible sites and glyoxal-fixed base-unpaired regions were both related to the fivemore » A-T-rich regions in M13 RF DNA. While S1 nuclease does not show preference for any of these sites, glyoxal-fixed bubbles occur predominantly at the major A-T-rich region in M13 RF DNA.« less

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

DNA Repair at Telomeres: Keeping the Ends Intact

PubMed Central

Webb, Christopher J.; Wu, Yun; Zakian, Virginia A.

2013-01-01

The molecular era of telomere biology began with the discovery that telomeres usually consist of G-rich simple repeats and end with 3′ single-stranded tails. Enormous progress has been made in identifying the mechanisms that maintain and replenish telomeric DNA and the proteins that protect them from degradation, fusions, and checkpoint activation. Although telomeres in different organisms (or even in the same organism under different conditions) are maintained by different mechanisms, the disparate processes have the common goals of repairing defects caused by semiconservative replication through G-rich DNA, countering the shortening caused by incomplete replication, and postreplication regeneration of G tails. In addition, standard DNA repair mechanisms must be suppressed or modified at telomeres to prevent their being recognized and processed as DNA double-strand breaks. Here, we discuss the players and processes that maintain and regenerate telomere structure. PMID:23732473

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

PubMed Central

Huh, Yang Hoon; Cohen, Justin; Sherley, James L.

2013-01-01

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs “know” the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency. PMID:24082118

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

PubMed

Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

2017-06-20

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

PubMed Central

Soustelle, Christine; Vedel, Michèle; Kolodner, Richard; Nicolas, Alain

2002-01-01

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis. PMID:12072452

Creating complex molecular topologies by configuring DNA four-way junctions

NASA Astrophysics Data System (ADS)

Liu, Di; Chen, Gang; Akhter, Usman; Cronin, Timothy M.; Weizmann, Yossi

2016-10-01

The realization of complex topologies at the molecular level represents a grand challenge in chemistry. This necessitates the manipulation of molecular interactions with high precision. Here we show that single-stranded DNA (ssDNA) knots and links can be created by utilizing the inherent topological properties that pertain to the DNA four-way junction, at which the two helical strands form a node and can be configured conveniently and connected for complex topological construction. Using this strategy, we produced series of ssDNA topoisomers with the same sequences. By finely designing the curvature and torsion, double-stranded DNA knots were accessed by hybridizing and ligating the complementary strands with the knotted ssDNA templates. Furthermore, we demonstrate the use of a constructed ssDNA knot both to probe the topological conversion catalysed by DNA topoisomerase and to study the DNA replication under topological constraint.

Reliable method for generating double-stranded DNA vectors containing site-specific base modifications.

PubMed

Brégeon, Damien; Doetsch, Paul W

2004-11-01

Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.

Structure of L-A Virus: A Specialized Compartment for the Transcription and Replication of Double-stranded RNA

PubMed Central

Castón, José R.; Trus, Benes L.; Booy, Frank P.; Wickner, Reed B.; Wall, Joseph S.; Steven, Alasdair C.

1997-01-01

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment—the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10–15 Å across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40–45 Å, compared with 25–30 Å in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall. PMID:9281577

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

PubMed

Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

2015-05-22

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes. Copyright © 2018 American Society for Microbiology.

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization

PubMed Central

Limoli, Charles L.; Giedzinski, Erich; Bonner, William M.; Cleaver, James E.

2002-01-01

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (γ-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of γ-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10–15%) up to fluences of 10 J⋅m−2 before saturating at higher fluences. Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of γ-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of γ-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and γ-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation. PMID:11756691

Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1.

PubMed

Oakley, Greg G; Tillison, Kristin; Opiyo, Stephen A; Glanzer, Jason G; Horn, Jeffrey M; Patrick, Steve M

2009-08-11

Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

INITIATION AND REGULATION OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION

PubMed Central

Noton, Sarah L.; Fearns, Rachel

2015-01-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. PMID:25683441

Initiation and regulation of paramyxovirus transcription and replication.

PubMed

Noton, Sarah L; Fearns, Rachel

2015-05-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

PubMed

Toledo, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

2013-11-21

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

Engineering resistance against viroid

USDA-ARS?s Scientific Manuscript database

Viroids, the smallest infectious agents endowed with autonomous replication, are tiny single-stranded circular RNAs (~250-400 nt) without protein-coding ability that, despite their simplicity, infect and often cause disease in herbaceous and woody plants of economic relevance. To mitigate the result...

HSP70 induction during baculovirus infection

USDA-ARS?s Scientific Manuscript database

Baculoviruses are arthropod-specific double-stranded DNA viruses that have been employed as bio-insecticides against crop pests and to produce heterologous proteins in baculovirus expression systems. Although a consensus has emerged on the dominant molecular events driving baculovirus replication i...

Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro.

PubMed

Previsani, N; Fontana, S; Hirt, B; Beard, P

1997-10-01

Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of double-stranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32P-labeled single-stranded MVM DNA to 32P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.

Erratum: Correction of “Pridgeon, J. W., Zhao, L., Becnel, J. J., Strickman, D. A., Clark, G. G., and Linthicum, K. J. 2008. Topically applied AaeIAP1 double-stranded RNA kills female adults of Aedes aegypti."

USDA-ARS?s Scientific Manuscript database

The coauthors of previously published work correct details from a 2008 publication. Specifically, it was incorrectly indicated in the methods section for data presented in Tables 2 and 3 that this experiment was the result of three replicates. These data were not the result of three replicate experi...

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

PubMed

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

2016-04-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. © 2016 American Society of Plant Biologists. All rights reserved.

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

PubMed

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

PubMed

Bharti, Sanjay Kumar; Sommers, Joshua A; Awate, Sanket; Bellani, Marina A; Khan, Irfan; Bradley, Lynda; King, Graeme A; Seol, Yeonee; Vidhyasagar, Venkatasubramanian; Wu, Yuliang; Abe, Takuye; Kobayashi, Koji; Shin-Ya, Kazuo; Kitao, Hiroyuki; Wold, Marc S; Branzei, Dana; Neuman, Keir C; Brosh, Robert M

2018-05-21

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N/sup 6/-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon

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

Basu, A.K.; Niedernhofer, L.J.; Essigmann, J.M.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N/sup 6/-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d(GCT(epsilon A)GC) was chemically synthesized by the phosphotriester method. Physical studies involving fluorescence, circular dichroism , and /sup 1/H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed inmore » the genome of an M13mp19 insertion mutant. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer (5'-/sup 32/P)d-(GCT(epsilon A)GC), after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.« less

DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

NASA Technical Reports Server (NTRS)

Carra, Claudio; Cucinotta, Francis A.

2007-01-01

Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus

PubMed Central

Neamah, Maan M.; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F.

2017-01-01

Abstract DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. PMID:28475766

Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

PubMed

Hizume, Kohji; Kominami, Hiroaki; Kobayashi, Kei; Yamada, Hirofumi; Araki, Hiroyuki

2017-05-16

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

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.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2016-06-07

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2016-01-01

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

Transposition-mediated DNA re-replication in maize

PubMed Central

Zhang, Jianbo; Zuo, Tao; Wang, Dafang; Peterson, Thomas

2014-01-01

Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability. We show here that this restriction can be bypassed through alternative transposition, a transposition reaction that utilizes the termini of two separate, nearby transposable elements (TEs). Our results suggest that alternative transposition during S phase can induce re-replication of the TEs and their flanking sequences. The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths. These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.03724.001 PMID:25406063

SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair.

PubMed

Cabello-Lobato, Maria Jose; Wang, Siyue; Schmidt, Christine Katrin

2017-12-01

SAMHD1 (sterile α motif and histidine (H) aspartate (D) domain-containing protein 1) is known for its antiviral activity of hydrolysing deoxynucleotides required for virus replication. Daddacha et al. identify a hydrolase-independent, moonlighting function of SAMHD1 that facilitates homologous recombination of DNA double-strand breaks (DSBs) by promoting recruitment of C-terminal binding protein interacting protein (CTIP), a DNA-end resection factor, to damaged DNA. These findings could benefit anticancer treatment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Computational biology approach to uncover hepatitis C virus helicase operation.

PubMed

Flechsig, Holger

2014-04-07

Hepatitis C virus (HCV) helicase is a molecular motor that splits nucleic acid duplex structures during viral replication, therefore representing a promising target for antiviral treatment. Hence, a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity. Despite extensive investigations performed in the past, a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved. Here we review investigations that have been previously performed by us for HCV helicase. Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation. We also discuss observations from that study in the light of recent experimental studies that confirm our findings.

APOBEC3B edits HBV DNA and inhibits HBV replication during reverse transcription.

PubMed

Chen, Yanmeng; Hu, Jie; Cai, Xuefei; Huang, Yao; Zhou, Xing; Tu, Zeng; Hu, Jieli; Tavis, John E; Tang, Ni; Huang, Ailong; Hu, Yuan

2018-01-01

Hepatitis B virus is a partially double-stranded DNA virus that replicates by reverse transcription, which occurs within viral core particles in the cytoplasm. The cytidine deaminase APOBEC3B is a cellular restriction factor for HBV. Recently, it was reported that APOBEC3B can edit HBV cccDNA in the nucleus, causing its degradation. However, whether and how it can edit HBV core-associated DNAs during reverse transcription is unclear. Our studies to address this question revealed the following: First, silencing endogenous APOBEC3B in an HBV infection system lead to upregulation of HBV replication. Second, APOBEC3B can inhibit replication of HBV isolates from genotypes (gt) A, B, C, and D as determined by employing transfection of plasmids expressing isolates from four different HBV genotypes. For HBV inhibition, APOBEC3B-mediated inhibition of replication primarily depends on the C-terminal active site of APOBEC3B. In addition, employing the HBV RNaseH-deficient D702A mutant and a polymerase-deficient YMHA mutant, we demonstrated that APOBEC3B can edit both the HBV minus- and plus-strand DNAs, but not the pregenomic RNA in core particles. Furthermore, we found by co-immunoprecipitation assays that APOBEC3B can interact with HBV core protein in an RNA-dependent manner. Our results provide evidence that APOBEC3B can interact with HBV core protein and edit HBV DNAs during reverse transcription. These data suggest that APOBEC3B exerts multifaceted antiviral effects against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

ICTV Virus Taxonomy Profile: Virgaviridae

USDA-ARS?s Scientific Manuscript database

The family Virgaviridae is comprised of plant-infecting viruses with rod-shaped particles, single stranded RNA genomes with 3' terminal tRNA-like structures, and replication proteins typical of alphalike viruses. Differences in the number of genome components, genome organization and transmission m...

Mutagenic cost of ribonucleotides in bacterial DNA

PubMed Central

Schroeder, Jeremy W.; Randall, Justin R.; Hirst, William G.; O’Donnell, Michael E.; Simmons, Lyle A.

2017-01-01

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genome-wide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand- and sequence-context–dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE. PMID:29078353

Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon

PubMed Central

Martin, Sandra L.; Bushman, Frederic D.

2001-01-01

Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a “nucleic acid chaperone,” promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription. PMID:11134335

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

PubMed Central

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

PubMed

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

PubMed Central

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

PubMed

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

An oligonucleotide microarray to characterize multidrug resistant plasmids

USDA-ARS?s Scientific Manuscript database

Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the host organism like antibiotic drug resistance. Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replic...

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

PubMed Central

Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

2015-01-01

ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. PMID:26699641

Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins.

PubMed

Foulk, Michael S; Urban, John M; Casella, Cinzia; Gerbi, Susan A

2015-05-01

Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand-independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na(+) instead of K(+) in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq. © 2015 Foulk et al.; Published by Cold Spring Harbor Laboratory Press.

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

PubMed

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

RNA secondary structures of the bacteriophage phi6 packaging regions.

PubMed

Pirttimaa, M J; Bamford, D H

2000-06-01

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Virus Assembly and Maturation

NASA Astrophysics Data System (ADS)

Johnson, John E.

2004-03-01

We use two techniques to look at three-dimensional virus structure: electron cryomicroscopy (cryoEM) and X-ray crystallography. Figure 1 is a gallery of virus particles whose structures Timothy Baker, one of my former colleagues at Purdue University, used cryoEM to determine. It illustrates the variety of sizes of icosahedral virus particles. The largest virus particle on this slide is the Herpes simplex virus, around 1200Å in diameter; the smallest we examined was around 250Å in diameter. Viruses bear their genomic information either as positive-sense DNA and RNA, double-strand DNA, double-strand RNA, or negative-strand RNA. Viruses utilize the various structure and function "tactics" seen throughout cell biology to replicate at high levels. Many of the biological principles that we consider general were in fact discovered in the context of viruses ...

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-01-01

Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

Three stages during the evolution of the genetic code. [Abstract only

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1994-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity and a small codon number those amino acids emerging later in a translation process are derived. Both criteria indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage one use purines rich codons, thus purines have been retained in their third codon position. All the amino acids introduced in the second stage, in contrast, use pyrimidines in this codon position. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non enzymatic replication and interactions of DNA hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids which gradually decreased during their evolution. Amino acids independently available form prebiotic synthesis were thus correlated to purine rich codons. Conclusions on prebiotic replication are discussed also in the light of recent codon usage data.

Correlated Template-Switching Events during Minus-Strand DNA Synthesis: a Mechanism for High Negative Interference during Retroviral Recombination

PubMed Central

Anderson, Jeffrey A.; Teufel, Ronald J.; Yin, Philip D.; Hu, Wei-Shau

1998-01-01

Two models for the mechanism of retroviral recombination have been proposed: forced copy choice (minus-strand recombination) and strand displacement-assimilation (plus-strand recombination). Each minus-strand recombination event results in one template switch, whereas each plus-strand recombination event results in two template switches. Recombinant proviruses with one and more than one template switches were previously observed. Recombinants with one template switch were generated by minus-strand recombination, while recombinants containing more than one template switch may have been generated by plus-strand recombination or by correlated minus-strand recombination. We recently observed that retroviral recombination exhibits high negative interference whereby the frequency of recombinants containing multiple template-switching events is higher than expected. To delineate the mechanism that generates recombinants with more than one template switch, we devised a system that permits only minus-strand recombination. Two highly homologous vectors, WH204 and WH221, containing eight different restriction site markers were used. The primer binding site (PBS) of WH221 was deleted; although reverse transcription cannot initiate from WH221 RNA, it can serve as a template for DNA synthesis in heterozygotic virions. After one round of retroviral replication, the structures of the recombinant proviruses were examined. Recombinants containing two, three, four, and five template switches were observed at 1.4-, 10-, 65-, and 50-fold-higher frequencies, respectively, than expected. This indicates that minus-strand recombination events are correlated and can generate proviruses with multiple template switches efficiently. The frequencies of recombinants containing multiple template switches were similar to those observed in the previous system, which allowed both minus- and plus-strand recombination. Thus, the previously reported high negative interference during retroviral recombination can be caused by correlated template switches during minus-strand DNA synthesis. In addition, all examined recombinants contained an intact PBS, indicating that most of the plus-strand DNA transfer occurs after completion of the strong-stop DNA. PMID:9445017

Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

PubMed

Nagy, Peter D; Pogany, Judit; Xu, Kai

2016-03-03

Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

Ribozyme-catalysed RNA synthesis using triplet building blocks.

PubMed

Attwater, James; Raguram, Aditya; Morgunov, Alexey S; Gianni, Edoardo; Holliger, Philipp

2018-05-15

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme. © 2018, Attwater et al.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

PubMed

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Management of fluorescent lamps in controlled environment chambers

NASA Technical Reports Server (NTRS)

Romer, Mark

1994-01-01

Management of fluorescent lights is recommended to (1) maintain uniformity of light intensity over time and (2) permit reproducibility of lighting conditions during experimental replications. At the McGill Phytotron, the lighting intensity can be controlled to desired level because any individual pair of the 40 lamps in each chamber can be set to be 'on' at any particular time. A lamp canopy service history is maintained for each experiment permitting accurate replication of lighting conditions for subsequent replicate trials.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

PubMed Central

Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

2014-01-01

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

Saliva of TBP digests double stranded RNAs

USDA-ARS?s Scientific Manuscript database

The tarnished plant bug (TPB), Lygus lineolaris, is a generalist pest of many crops including cotton, strawberries, and alfalfa. While studying gene expression and gene knockdown through RNA interference, my lab was frustrated by an observation. We were unable to replicate knock down effects observe...

Human RECQL5: guarding the crossroads of DNA replication and transcription and providing backup capability.

PubMed

Popuri, Venkateswarlu; Tadokoro, Takashi; Croteau, Deborah L; Bohr, Vilhelm A

2013-01-01

DNA helicases are ubiquitous enzymes that catalyze unwinding of duplex DNA and function in all metabolic processes in which access to single-stranded DNA is required, including DNA replication, repair, recombination and RNA transcription. RecQ helicases are a conserved family of DNA helicases that display highly specialized and vital roles in the maintenance of genome stability. Mutations in three of the five human RecQ helicases, BLM, WRN and RECQL4 are associated with the genetic disorders Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome that are characterized by chromosomal instability, premature aging and predisposition to cancer. The biological role of human RECQL5 is only partially understood and RECQL5 has not yet been associated with any human disease. Illegitimate recombination and replication stress are hallmarks of human cancers and common instigators for genomic instability and cell death. Recql5 knockout mice are cancer prone and show increased chromosomal instability. Recql5-deficient mouse embryonic fibroblasts are sensitive to camptothecin and display elevated levels of sister chromatid exchanges. Unlike other human RecQ helicases, RECQL5 is recruited to single-stranded DNA breaks and is also proposed to play an essential role in RNA transcription. Here, we review the established roles of RECQL5 at the cross roads of DNA replication, recombination and transcription, and propose that human RECQL5 provides important backup functions in the absence of other DNA helicases.

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

PubMed Central

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

Drosophila mitochondrial DNA: a novel gene order.

PubMed Central

Clary, D O; Goddard, J M; Martin, S C; Fauron, C M; Wolstenholme, D R

1982-01-01

Part of the replication origin-containing A+T-rich region of the Drosophila yakuba mtDNA molecule and segments on either side of this region have been sequenced, and the genes within them identified. The data confirm that the small and large rRNA genes lie in tandem adjacent to that side of the A+T-rich region which is replicated first, and establish that a tRNAval gene lies between the two rRNA genes and that URF1 follows the large rRNA gene. The data further establish that the genes for tRNAile, tRNAgln, tRNAf-met and URF2 lie in the order given, on the opposite side of the A+T-rich region to the rRNA genes and, except for tRNAgln, are contained in the opposite strand to the rRNA, tRNAval and URF1 genes. This is in contrast to mammalian mtDNAs where all of these genes are located on the side of the replication origin which is replicated last, within the order tRNAphe, small (12S) rRNA, tRNAval, large (16S) rRNA, tRNAleu, URF1, tRNAile, tRNAgln, tRNAf-met and URF2, and, except tRNAgln, are all contained in the same (H) strand. In D. yakuba URF1 and URF2, the triplet AGA appears to specify an amino acid, which is again different from the situation found in mammalian mtDNAs, where AGA is used only as a rare termination codon. PMID:6294611

Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

PubMed

Shiraki, K

2017-11-01

Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy. Copyright 2017 Clarivate Analytics.

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

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

PubMed Central

Liu, Y H; Symons, R H

1998-01-01

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids. PMID:9630248

Cdc45-induced loading of human RPA onto single-stranded DNA

PubMed Central

Tessmer, Ingrid; Prus, Piotr; Schlott, Bernhard; Pospiech, Helmut

2017-01-01

Abstract Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8–10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork. PMID:28100698

Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

PubMed

Park, Miey; Kim, Min-Soo; Lee, Kyung-Min; Hwang, Sue-Yun; Ahn, Tae In

2009-01-01

A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

Yeast exonuclease 5 is essential for mitochondrial genome maintenance.

PubMed

Burgers, Peter M; Stith, Carrie M; Yoder, Bonita L; Sparks, Justin L

2010-03-01

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

PubMed

Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

2017-03-01

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera).

PubMed

Park, Bokri; Kim, Yonggyun

2011-06-01

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses

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

Chauhan, Vinita S.; Furr, Samantha R.; Sterka, David G.

2010-05-10

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we havemore » confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.« less

Epigenetic Instability due to Defective Replication of Structured DNA

PubMed Central

Sarkies, Peter; Reams, Charlie; Simpson, Laura J.; Sale, Julian E.

2010-01-01

Summary The accurate propagation of histone marks during chromosomal replication is proposed to rely on the tight coupling of replication with the recycling of parental histones to the daughter strands. Here, we show in the avian cell line DT40 that REV1, a key regulator of DNA translesion synthesis at the replication fork, is required for the maintenance of repressive chromatin marks and gene silencing in the vicinity of DNA capable of forming G-quadruplex (G4) structures. We demonstrate a previously unappreciated requirement for REV1 in replication of G4 forming sequences and show that transplanting a G4 forming sequence into a silent locus leads to its derepression in REV1-deficient cells. Together, our observations support a model in which failure to maintain processive DNA replication at G4 DNA in REV1-deficient cells leads to uncoupling of DNA synthesis from histone recycling, resulting in localized loss of repressive chromatin through biased incorporation of newly synthesized histones. PMID:21145480

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

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

Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov; Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu; Krupovic, Mart, E-mail: krupovic@pasteur.fr

2015-05-15

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangiblemore » clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes. - Highlights: • Eukaryotic virome dramatically differs from the viromes of bacteria and archaea. • Eukaryotic virome is dominated by RNA viruses and retroelements. • All classes of eukaryotic viruses evolved by gene module exchange. • Prokaryotic ancestry is traceable for core gene modules of most eukaryotic viruses. • Evolutionary histories of viruses and transposable elements are tightly linked.« less

Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

PubMed

Tijssen, Peter; Pénzes, Judit J; Yu, Qian; Pham, Hanh T; Bergoin, Max

2016-10-01

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy. Copyright © 2016. Published by Elsevier Inc.

ICTV Virus Taxonomy Profile: Avsunviroidae.

PubMed

Di Serio, Francesco; Li, Shi-Fang; Matoušek, Jaroslav; Owens, Robert A; Pallás, Vicente; Randles, John W; Sano, Teruo; Verhoeven, Jacobus Th J; Vidalakis, Georgios; Flores, Ricardo; Ictv Report Consortium

2018-05-01

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

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

C Harrison; G Meinke; H Kwun

2011-12-31

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 {angstrom} crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistentmore » with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be {approx} 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.« less

Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe

PubMed Central

Lenglez, Sandrine; Hermand, Damien; Decottignies, Anabelle

2010-01-01

Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites. PMID:20688779

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

PubMed Central

Nishikiori, Masaki; Dohi, Koji; Mori, Masashi; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

2006-01-01

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity. PMID:16912296

Modulation of in vitro transformation and the early and late modes of DNA replication of uv-irradiation Syrian hamster cells by caffeine

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

Doniger, J.; DiPaolo, J.A.

1981-09-01

The effect of caffeine on post-uv DNA replication was studied to determine its relevance to carcinogenesis. The level of uv-induced transformed colonies of Syrian hamster embryo cells (HEC) was increased up to fivefold when caffeine was added to cells between 0 and 6 h post-uv. The greatest increase was observed when the interval between uv irradiation and caffeine addition was 4 h. Two modes of DNA replication occurred after uv irradiation. During the early mode (0 to 3 h post-uv) the size of nascent strands, as measured by alkaline sucrose sedimentation, was smaller than those in nonirradiated cells, whereas duringmore » the late mode they recovered to normal size. Caffeine inhibited the rate of elongation of nascent strands during the early mode. When caffeine was added immediately after uv irradiation, the conversion of the early mode to the late mode was inhibited. Studies on the effects of caffeine have now been extended to the late mode. While caffeine has little effect with the fd elements beginning from the 10th day after irradiation is connected with their proliferation but not with the migration out from lymphoid organs.« less

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus.

PubMed

Neamah, Maan M; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F; Marina, Alberto; Ayora, Silvia; Penadés, José R

2017-06-20

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

PubMed

Ribeck, Noah; Saleh, Omar A

2013-01-01

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

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

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

PubMed Central

Mahy, B. W. J.; Bromley, P. A.

1970-01-01

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus. PMID:5480408

Salivary gland hypertrophy viruses (SGHVs): a novel group of insect pathogenic viruses

USDA-ARS?s Scientific Manuscript database

Salivary gland hypertrophy viruses (SGHVs) are a unique, unclassified group of entomopathogenic, double-stranded DNA viruses that have been reported from three genera of Diptera. These viruses replicate in nuclei of salivary gland cells in adult flies, inducing gland enlargement with little obvious ...

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

PubMed

Yang, Liping; Wang, Rong; Yang, Shixing; Ma, Zexu; Lin, Shaoli; Nan, Yuchen; Li, Qisheng; Tang, Qiyi; Zhang, Yan-Jin

2018-05-01

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV. IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics. Copyright © 2018 American Society for Microbiology.

Software Development to Assist in the Processing and Analysis of Data Obtained Using Fiber Bragg Grating Interrogation Systems

NASA Technical Reports Server (NTRS)

Hicks, Rebecca

2009-01-01

A fiber Bragg grating is a portion of a core of a fiber optic strand that has been treated to affect the way light travels through the strand. Light within a certain narrow range of wavelengths will be reflected along the fiber by the grating, while light outside that range will pass through the grating mostly undisturbed. Since the range of wavelengths that can penetrate the grating depends on the grating itself as well as temperature and mechanical strain, fiber Bragg gratings can be used as temperature and strain sensors. This capability, along with the light-weight nature of the fiber optic strands in which the gratings reside, make fiber optic sensors an ideal candidate for flight testing and monitoring in which temperature and wing strain are factors. The purpose of this project is to research the availability of software capable of processing massive amounts of data in both real-time and post-flight settings, and to produce software segments that can be integrated to assist in the task as well.

Optically Controlled Signal Amplification for DNA Computation.

PubMed

Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

2015-10-16

The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF → ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF → ON or ON → OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

PubMed Central

Recolin, Bénédicte; Van Der Laan, Siem; Maiorano, Domenico

2012-01-01

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation. PMID:22187152

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

PubMed

Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

2015-05-08

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Rolling-circle amplification under topological constraints

PubMed Central

Kuhn, Heiko; Demidov, Vadim V.; Frank-Kamenetskii, Maxim D.

2002-01-01

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics. PMID:11788721

The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination†

PubMed Central

Besmer, Eva; Market, Eleonora; Papavasiliou, F. Nina

2006-01-01

Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. PMID:16705187

Regulation of Replication Fork Advance and Stability by Nucleosome Assembly

PubMed Central

Prado, Felix; Maya, Douglas

2017-01-01

The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging. PMID:28125036

Synchronization of DNA array replication kinetics

NASA Astrophysics Data System (ADS)

Manturov, Alexey O.; Grigoryev, Anton V.

2016-04-01

In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

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

Takeda, N.; Kuhn, R.J.; Yang, C.F.

1986-10-01

An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T/sub 1/-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized /sup 32/P-RNA. Incubation ofmore » preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.« less

Molecular mechanism of DNA association with single-stranded DNA binding protein

PubMed Central

Maffeo, Christopher

2017-01-01

Abstract During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA–SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB–ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion. PMID:29059392

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

PubMed

Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

PubMed Central

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.

PubMed

Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P

2017-02-01

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

PubMed

Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

2011-01-02

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

PubMed

Rybak, Paulina; Hoang, Agnieszka; Bujnowicz, Lukasz; Bernas, Tytus; Berniak, Krzysztof; Zarębski, Mirosław; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy

2016-08-02

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair.

PubMed

Kumar, Charanya; Williams, Gregory M; Havens, Brett; Dinicola, Michelle K; Surtees, Jennifer A

2013-06-12

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3' non-homologous tail removal (3' NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR. Copyright © 2013 Elsevier Ltd. All rights reserved.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair

PubMed Central

Kumar, Charanya; Williams, Gregory M.; Havens, Brett; Dinicola, Michelle; Surtees, Jennifer A.

2013-01-01

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3’ non-homologous tail removal (3’NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3’ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3’NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3’NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3’ NHTR. PMID:23458407

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

PubMed

Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

2014-10-01

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

PubMed Central

Georgescu, Roxana; Yuan, Zuanning; Bai, Lin; de Luna Almeida Santos, Ruda; Sun, Jingchuan; Zhang, Dan; Yurieva, Olga; Li, Huilin; O’Donnell, Michael E.

2017-01-01

The eukaryotic CMG (Cdc45, Mcm2–7, GINS) helicase consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin. PMID:28096349

Survey of diagnostic tools for detection of viroids and impacts of test results on the seed industry

USDA-ARS?s Scientific Manuscript database

Viroids are unencapsidated, single-stranded, covalently closed circular, highly structured noncoding RNAs of 239 – 401 nucleotides that are replicated by host enzymes and cause disease in several economically important crop plants. Although viroids are primarily and easily transmitted mechanically t...

HIGH-TEMPERATURE INDUCIBLE CELL-FREE TRANSCRIPTION AND REPLICATION OF DOUBLE-STRANDED RNAS WITHIN THE PARASITIC PROTOZOAN CRYPTOSPORIDIUM PARVUM. (R825148)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Integrating PCR Theory and Bioinformatics into a Research-oriented Primer Design Exercise

ERIC Educational Resources Information Center

Robertson, Amber L.; Phillips, Allison R.

2008-01-01

Polymerase chain reaction (PCR) is a conceptually difficult technique that embodies many fundamental biological processes. Traditionally, students have struggled to analyze PCR results due to an incomplete understanding of the biological concepts (theory) of DNA replication and strand complementarity. Here we describe the design of a novel…

Potato spindle tuber viroid detection in phloem exudates and guttation fluid of tomato plants (Solanum lycopersicum)

USDA-ARS?s Scientific Manuscript database

Potato spindle tuber viroid (PSTVd) is a single-stranded, non protein-encoding, covalently-closed circular RNA molecule (359nt) that infects many horticultural and agricultural crops. PSTVd is mechanically transmitted, replicates in the nucleus, and moves cell-to-cell through plasmodesmata. Though i...

Single slit interference made easy with a strand of hair and a laser

NASA Astrophysics Data System (ADS)

Messer, Rebecca

2018-01-01

Students can easily measure the width of a strand of their own hair with a monochromatic light source such as a laser. This inexpensive activity engages students in an application of single slit diffraction using Babinet's principle.

Performance of prestressed girders cast with LWSCC.

DOT National Transportation Integrated Search

2012-08-01

Bond of prestressing steel has been a much debated topic since the 1950s. Limited data are available on the transfer and development length of strands cast in self : consolidating concrete (SCC) and even less for strands cast in light weight, self-co...

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

PubMed

Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

2015-12-23

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Acute inactivation of the replicative helicase in human cells triggers MCM8–9-dependent DNA synthesis

PubMed Central

Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy; Bhowmick, Rahul; Hickson, Ian D.; Kanemaki, Masato T.

2017-01-01

DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8–9 complex, a paralog of the MCM2–7 replicative helicase. We show that MCM8–9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8–9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8–9 as an alternative replicative helicase. PMID:28487407

Identification of proteins that may directly interact with human RPA.

PubMed

Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

2010-11-01

RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

DNA replication after mutagenic treatment in Hordeum vulgare.

PubMed

Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

2016-12-01

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

PubMed

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

The Fanconi Anemia Pathway in Replication Stress and DNA Crosslink Repair

PubMed Central

Jones, Mathew JK.; Huang, Tony T.

2013-01-01

Interstand crosslinks (ICLs) are DNA lesions where the bases of opposing DNA strands are covalently linked, inhibiting critical cellular processes such as transcription and replication. Chemical agents that generate ICLs cause chromosomal abnormalities including breaks, deletions and rearrangements, making them highly genotoxic compounds. This toxicity has proven useful for chemotherapeutic treatment against a wide variety of cancer types. The majority of our understanding of ICL repair in humans has been uncovered thorough analysis of the rare genetic disorder Fanconi anemia, in which patients are extremely sensitive to crosslinking agents. Here, we discuss recent insights into ICL repair gained through new ICL repair assays and highlight the role of the Fanconi Anemia repair pathway during replication stress. PMID:22744751

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

PubMed

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.

PubMed

Hutinet, Geoffrey; Besle, Arthur; Son, Olivier; McGovern, Stephen; Guerois, Raphaël; Petit, Marie-Agnès; Ochsenbein, Françoise; Lecointe, François

2018-01-01

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo , but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro , Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo , though Sak4 and its SSB are unable to promote strand exchange in vitro . Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.

Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein

PubMed Central

Hutinet, Geoffrey; Besle, Arthur; Son, Olivier; McGovern, Stephen; Guerois, Raphaël; Petit, Marie-Agnès; Ochsenbein, Françoise; Lecointe, François

2018-01-01

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves. PMID:29740405

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

PubMed

Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

2017-02-01

The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

Role of Escherichia coli dnaG function in coliphage M13 DNA synthesis

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

Dasgupta, S.; Mitra, S.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

PubMed Central

Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

2016-01-01

ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. PMID:26739056

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

PubMed

Advani, S J; Weichselbaum, R R; Whitley, R J; Roizman, B

2002-09-01

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Defects in Mitochondrial DNA Replication and Human Disease

PubMed Central

Copeland, William C.

2011-01-01

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease. PMID:22176657

Structural basis for DNA binding by replication initiator Mcm10

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

Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin

2009-06-30

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae resultmore » in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.« less

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

PubMed Central

Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

2016-01-01

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp.

PubMed

Ongvarrasopone, Chalermporn; Chomchay, Ekapol; Panyim, Sakol

2010-10-01

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp. Copyright © 2010 Elsevier B.V. All rights reserved.