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Sample records for complex dna repair

  1. Complexities of the DNA Base Excision Repair Pathway for Repair of Oxidative DNA Damage

    PubMed Central

    Mitra, Sankar; Boldogh, Istvan; Izumi, Tadahide; Hazra, Tapas K.

    2016-01-01

    Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase β or replicative polymerases δ and ε. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation. PMID:11746753

  2. DNA repair

    SciTech Connect

    Friedberg, E.C.; Hanawalt, P.C. )

    1988-01-01

    Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.

  3. Stalled transcription complexes promote DNA repair at a distance

    PubMed Central

    Haines, Nia M.; Kim, Young-In T.; Smith, Abigail J.; Savery, Nigel J.

    2014-01-01

    Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected. PMID:24554077

  4. Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end.

    PubMed

    Yoo, S; Kimzey, A; Dynan, W S

    1999-07-01

    Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively. PMID:10391954

  5. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

    SciTech Connect

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-09-07

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

  6. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    SciTech Connect

    Jones, IM

    2002-01-09

    of this pilot project was to obtain preliminary data on genetic variation in DNA repair function in human cells that might encourage our efforts to establish a research program to relate DNA repair function to complex DNA repair genotype and ultimately to cancer risk of radiation exposure.

  7. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    SciTech Connect

    Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P.

    2011-08-29

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  8. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    SciTech Connect

    W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

    2011-12-31

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  9. Structural biology of DNA repair: spatial organisation of the multicomponent complexes of nonhomologous end joining.

    PubMed

    Ochi, Takashi; Sibanda, Bancinyane Lynn; Wu, Qian; Chirgadze, Dimitri Y; Bolanos-Garcia, Victor M; Blundell, Tom L

    2010-01-01

    Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods-X-ray crystallography, electron microscopy and small angle X-ray scattering-can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere. PMID:20862368

  10. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  11. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  12. Architecture of the human XPC DNA repair and stem cell coactivator complex

    PubMed Central

    He, Yuan; Grob, Patricia; Fong, Yick W.; Nogales, Eva; Tjian, Robert

    2015-01-01

    The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair. PMID:26627236

  13. Architecture of the human XPC DNA repair and stem cell coactivator complex.

    PubMed

    Zhang, Elisa T; He, Yuan; Grob, Patricia; Fong, Yick W; Nogales, Eva; Tjian, Robert

    2015-12-01

    The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair. PMID:26627236

  14. Structure of UvrA nucleotide excision repair protein in complex with modified DNA

    PubMed Central

    Jaciuk, Marcin; Nowak, Elżbieta; Skowronek, Krzysztof; Tańska, Anna; Nowotny, Marcin

    2012-01-01

    One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion. PMID:21240268

  15. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  16. DNA Mismatch Repair

    PubMed Central

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  17. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair[C][W

    PubMed Central

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-01-01

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway. PMID:20551348

  18. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  19. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages. PMID:17141556

  20. Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin

    PubMed Central

    Ziani, Salim; Nagy, Zita; Alekseev, Sergey; Soutoglou, Evi; Egly, Jean-Marc

    2014-01-01

    In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment. PMID:25154395

  1. Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin.

    PubMed

    Ziani, Salim; Nagy, Zita; Alekseev, Sergey; Soutoglou, Evi; Egly, Jean-Marc; Coin, Frédéric

    2014-09-01

    In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment. PMID:25154395

  2. The PBAF chromatin remodeling complex represses transcription and promotes rapid repair at DNA double-strand breaks

    PubMed Central

    Kakarougkas, Andreas; Downs, Jessica A; Jeggo, Penny A

    2015-01-01

    Transcription in the vicinity of DNA double-strand breaks (DSBs) is suppressed via a process involving ataxia telangiectasia mutated protein (ATM) and H2AK119 ubiquitylation.1 We discuss recent findings that components of the Polybromo and Brahma-related gene 1 (BRG1)-associated factor (PBAF) remodeling complex and the polycomb repressive complex (PRC1/2) are also required.2 Failure to activate transcriptional suppression impedes a rapid DSB repair process. PMID:27308404

  3. Cdk1-dependent regulation of the Mre11 complex couples DNA repair pathways to cell cycle progression

    PubMed Central

    Simoneau, Antoine; Robellet, Xavier; Ladouceur, Anne-Marie; D’Amours, Damien

    2014-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways ensuring the repair of DNA double-stranded breaks (DSBs) in eukaryotes. It has long been known that cell cycle stage is a major determinant of the type of pathway used to repair DSBs in vivo. However, the mechanistic basis for the cell cycle regulation of the DNA damage response is still unclear. Here we show that a major DSB sensor, the Mre11–Rad50–Xrs2 (MRX) complex, is regulated by cell cycle-dependent phosphorylation specifically in mitosis. This modification depends on the cyclin-dependent kinase Cdc28/Cdk1, and abrogation of Xrs2 and Mre11 phosphorylation results in a marked preference for DSB repair through NHEJ. Importantly, we show that phosphorylation of the MRX complex after DNA damage and during mitosis are regulated independently of each other by Tel1/ATM and Cdc28/Cdk1 kinases. Collectively, our results unravel an intricate network of phosphoregulatory mechanisms that act through the MRX complex to modulate DSB repair efficiency during mitosis. PMID:24553123

  4. Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein.

    PubMed

    Yoo, S; Dynan, W S

    1999-12-15

    Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested. PMID:10572166

  5. ATM protein is indispensable to repair complex-type DNA double strand breaks induced by high LET heavy ion irradiation.

    NASA Astrophysics Data System (ADS)

    Sekine, Emiko; Yu, Dong; Fujimori, Akira; Anzai, Kazunori; Okayasu, Ryuichi

    ATM (ataxia telangiectasia-mutated) protein responsible for a rare genetic disease with hyperradiosensitivity, is the one of the earliest repair proteins sensing DNA double-strand breaks (DSB). ATM is known to phosphorylate DNA repair proteins such as MRN complex (Mre11, Rad50 and NBS1), 53BP1, Artemis, Brca1, gamma-H2AX, and MDC. We studied the interactions between ATM and DNA-PKcs, a crucial NHEJ repair protein, after cells exposure to high and low LET irradiation. Normal human (HFL III, MRC5VA) and AT homozygote (AT2KY, AT5BIVA, AT3BIVA) cells were irradiated with X-rays and high LET radiation (carbon ions: 290MeV/n initial energy at 70 keV/um, and iron ions: 500MeV/n initial energy at 200KeV/um), and several critical end points were examined. AT cells with high LET irradiation showed a significantly higher radiosensitivity when compared with normal cells. The behavior of DNA DSB repair was monitored by immuno-fluorescence techniques using DNA-PKcs (pThr2609, pSer2056) and ATM (pSer1981) antibodies. In normal cells, the phosphorylation of DNA-PKcs was clearly detected after high LET irradiation, though the peak of phosphorylation was delayed when compared to X-irradiation. In contrast, almost no DNA-PKcs phosphorylation foci were detected in AT cells irradiated with high LET radiation. A similar result was also observed in normal cells treated with 10 uM ATM kinase specific inhibitor (KU55933) one hour before irradiation. These data suggest that the phosphorylation of DNA-PKcs with low LET X-rays is mostly ATM-independent, and the phosphorylation of DNA-PKcs with high LET radiation seems to require ATM probably due to its complex nature of DSB induced. Our study indicates that high LET heavy ion irradiation which we can observe in the space environment would provide a useful tool to study the fundamental mechanism associated with DNA DSB repair.

  6. Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

    PubMed

    Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum

    2016-04-19

    DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942

  7. Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

    PubMed Central

    Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum

    2016-01-01

    DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 “opens” up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion “opening” is slow (˜5–10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/“twisting”) of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise “twist-open” mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942

  8. [A Nobel Prize for DNA repair].

    PubMed

    Jordan, Bertrand

    2016-01-01

    This year's Nobel Prize for chemistry recognizes the seminal contributions of three researchers who discovered the existence and the basic mechanisms of DNA repair: base excision repair, mismatch repair, and nucleotide excision repair. They have since been joined by many scientists elucidating diverse aspects of these complex mechanisms that now constitute a thriving research field with many applications, notably for understanding oncogenesis and devising more effective therapies. PMID:26850617

  9. RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

    PubMed

    Lescale, Chloé; Abramowski, Vincent; Bedora-Faure, Marie; Murigneux, Valentine; Vera, Gabriella; Roth, David B; Revy, Patrick; de Villartay, Jean-Pierre; Deriano, Ludovic

    2016-01-01

    XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. PMID:26833222

  10. RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair

    PubMed Central

    Lescale, Chloé; Abramowski, Vincent; Bedora-Faure, Marie; Murigneux, Valentine; Vera, Gabriella; Roth, David B.; Revy, Patrick; de Villartay, Jean-Pierre; Deriano, Ludovic

    2016-01-01

    XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2c/c mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2c/c XLF−/− p53−/− mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. PMID:26833222

  11. Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

    PubMed

    Gicquel, Etienne; Souchard, Jean-Pierre; Magnusson, Fay; Chemaly, Jad; Calsou, Patrick; Vicendo, Patricia

    2013-08-01

    Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids. PMID:23835850

  12. Electron crystal structure of the transcription factor and DNA repair complex, core TFIIH.

    PubMed

    Chang, W H; Kornberg, R D

    2000-09-01

    Core TFIIH from yeast, made up of five subunits required both for RNA polymerase II transcription and nucleotide excision DNA repair, formed 2D crystals on charged lipid layers. Diffraction from electron micrographs of the crystals in negative stain extended to about 13 angstrom resolution, and 3D reconstruction revealed several discrete densities whose volumes corresponded well with those of individual TFIIH subunits. The structure is based on a ring of three subunits, Tfb1, Tfb2, and Tfb3, to which are appended several functional moieties: Rad3, bridged to Tfb1 by SsI1; SsI2, known to interact with Tfb2; and Kin28, known to interact with Tfb3. PMID:11007479

  13. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

    PubMed

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R; Baranello, Laura; Levens, David; Kraemer, Kenneth H; Stefanini, Miria

    2016-04-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

  14. Preferential DNA repair in expressed genes.

    PubMed Central

    Hanawalt, P C

    1987-01-01

    Potentially deleterious alterations to DNA occur nonrandomly within the mammalian genome. These alterations include the adducts produced by many chemical carcinogens, but not the UV-induced cyclobutane pyrimidine dimer, which may be an exception. Recent studies in our laboratory have shown that the excision repair of pyrimidine dimers and certain other lesions is nonrandom in the mammalian genome, exhibiting a distinct preference for actively transcribed DNA sequences. An important consequence of this fact is that mutagenesis and carcinogenesis may be determined in part by the activities of the relevant genes. Repair may also be processive, and a model is proposed in which excision repair is coupled to transcription at the nuclear matrix. Similar but freely diffusing repair complexes may account for the lower overall repair efficiencies in the silent domains of the genome. Risk assessment in relation to chemical carcinogenesis requires assays that determine effective levels of DNA damage for producing malignancy. The existence of nonrandom repair in the genome casts into doubt the reliability of overall indicators of DNA binding and lesion repair for such determinations. Furthermore, some apparent differences between the intragenomic repair heterogeneity in rodent cells and that in human cells mandate a reevaluation of rodent test systems for human risk assessment. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response. Images FIGURE 1. FIGURE 1. PMID:3447906

  15. Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

    SciTech Connect

    Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K.; Bradbury, E.M.; Garcia, A.E.

    1997-07-01

    This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.

  16. Mutagenic DNA repair in enterobacteria

    SciTech Connect

    Sedgwick, S.G. ); Chao Ho; Woodgate, R. )

    1991-09-01

    Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD{prime} fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD{prime} antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium u mu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umuDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention.

  17. Structure of p15PAF-PCNA complex and implications for clamp sliding during DNA replication and repair

    NASA Astrophysics Data System (ADS)

    de Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B.; Molina, Rafael; Cordeiro, Tiago N.; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J.

    2015-03-01

    The intrinsically disordered protein p15PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15PAF-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  18. DNA repair in cultured keratinocytes

    SciTech Connect

    Liu, S.C.; Parsons, S.; Hanawalt, P.C.

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  19. HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

    PubMed Central

    Fang, Qingming; Inanc, Burcu; Schamus, Sandy; Wang, Xiao-hong; Wei, Leizhen; Brown, Ashley R.; Svilar, David; Sugrue, Kelsey F.; Goellner, Eva M.; Zeng, Xuemei; Yates, Nathan A.; Lan, Li; Vens, Conchita; Sobol, Robert W.

    2014-01-01

    Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance. PMID:25423885

  20. Rethinking transcription coupled DNA repair.

    PubMed

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity. PMID:25596348

  1. Emerging roles of DNA-PK besides DNA repair.

    PubMed

    Kong, Xianming; Shen, Ying; Jiang, Na; Fei, Xin; Mi, Jun

    2011-08-01

    The DNA-dependent protein kinase (DNA-PK) is a DNA-activated serine/threonine protein kinase, and abundantly expressed in almost all mammalian cells. The roles of DNA-PK in DNA-damage repair pathways, including non-homologous end-joining (NHEJ) repair and homologous recombinant (HR) repair, have been studied intensively. However, the high levels of DNA-PK in human cells are somewhat paradoxical in that it does not impart any increased ability to repair DNA damage. If DNA-PK essentially exceeds the demand for DNA damage repair, why do human cells universally express such high levels of this huge complex? DNA-PK has been recently reported to be involved in metabolic gene regulation in response to feeding/insulin stimulation; our studies have also suggested a role of DNA-PK in the regulation of the homeostasis of cell proliferation. These novel findings expand our horizons about the importance of DNA-PK. PMID:21514376

  2. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  3. DNA repair in Mycoplasma gallisepticum

    PubMed Central

    2013-01-01

    Background DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a “minimal cell” concept. Results In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. Conclusions Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase. PMID:24148612

  4. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  5. Energy and Technology Review: Unlocking the mysteries of DNA repair

    SciTech Connect

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  6. Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

    SciTech Connect

    Noda, Taichi; Takahashi, Akihisa; Kondo, Natsuko; Mori, Eiichiro; Okamoto, Noritomo; Nakagawa, Yosuke; Ohnishi, Ken; Zdzienicka, Malgorzata Z.; Thompson, Larry H.; Helleday, Thomas; Asada, Hideo; and others

    2011-01-07

    The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

  7. Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells

    PubMed Central

    Cattoglio, Claudia; Zhang, Elisa T.; Grubisic, Ivan; Chiba, Kunitoshi; Fong, Yick W.; Tjian, Robert

    2015-01-01

    The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex. PMID:25901318

  8. Repair of DNA-containing pyrimidine dimers

    SciTech Connect

    Grossman, L.; Caron, P.R.; Mazur, S.J.; Oh, E.Y.

    1988-08-01

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references.

  9. Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes.

    PubMed

    Mohrenweiser, Harvey W; Wilson, David M; Jones, Irene M

    2003-05-15

    Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care. PMID:12714187

  10. Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

    PubMed

    Chapman, Timothy M; Gillen, Kevin J; Wallace, Claire; Lee, Maximillian T; Bakrania, Preeti; Khurana, Puneet; Coombs, Peter J; Stennett, Laura; Fox, Simon; Bureau, Emilie A; Brownlees, Janet; Melton, David W; Saxty, Barbara

    2015-10-01

    Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 μM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 μM. PMID:26318993

  11. Rad54B targeting to DNA double-strand break repair sites requires complex formation with S100A11.

    PubMed

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand; Melle, Christian

    2008-07-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21(WAF1/CIP1) was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  12. Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

    PubMed Central

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand

    2008-01-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  13. The growing complexity of HIF-1α's role in tumorigenesis: DNA repair and beyond.

    PubMed

    Rohwer, N; Zasada, C; Kempa, S; Cramer, T

    2013-08-01

    Lack of oxygen (hypoxia) is a central hallmark of cancer and a pivotal driving force of malignant progression. Transcriptional activators of the hypoxia-inducible factor α (HIFα) family represent the principal molecular mediators of hypoxia under both physiological and pathophysiological conditions. While HIF-2α is expressed in a tissue- and cell-type-restricted manner, stabilization of HIF-1α was reported in tumours of widely different origin, and functional analyses led to the perception of HIF-1α as an oncoprotein. In this review, we aim to acknowledge HIFα's growing complexity by outlining its functional relevance for genomic integrity and tumour heterogeneity, two features of paramount importance for basic and clinical oncology. Pharmaceutical companies around the globe are ambitiously hunting for HIF-1α-inhibiting compounds, some of which are currently being evaluated in phase 1 trials. To avoid the rather disappointing clinical efficacy emblematic of most targeted therapeutics, potential resistance mechanisms of, as well as potential combination partners for, HIF-1α-inhibiting drugs should be evaluated. In this regard, the interrelation of HIF-1α with genomic integrity and tumour heterogeneity offers ample possibilities, potentially resulting in more efficient clinical translation of HIF-1α's pathobiology. PMID:23160373

  14. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  15. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

    NASA Technical Reports Server (NTRS)

    Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

    2003-01-01

    The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

  16. Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

    PubMed

    Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa; Akopiants, Konstantin; Zhou, Tong; Yannone, Steven M; Ramsden, Dale A; Hartman, Matthew C T; Povirk, Lawrence F

    2016-05-01

    DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. PMID:27049455

  17. Mutant huntingtin impairs Ku70-mediated DNA repair

    PubMed Central

    Enokido, Yasushi; Tamura, Takuya; Ito, Hikaru; Arumughan, Anup; Komuro, Akihiko; Shiwaku, Hiroki; Sone, Masaki; Foulle, Raphaele; Sawada, Hirohide; Ishiguro, Hiroshi; Ono, Tetsuya; Murata, Miho; Kanazawa, Ichiro; Tomilin, Nikolai; Tagawa, Kazuhiko; Wanker, Erich E.

    2010-01-01

    DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt–expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington’s disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology. PMID:20439996

  18. Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

    PubMed

    Liu, Zhihui; Lam, Norris; Thiele, Carol J

    2015-09-29

    The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs. PMID:26296975

  19. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  20. Functional characterization of dna repair proteins

    SciTech Connect

    Wilson, D M III

    2000-02-16

    Genetic material (DNA) is susceptible to spontaneous decomposition, to attack by reactive chemicals produced naturally in cells, and to assault by environmental and food mutagens. DNA modifications can lead to permanent genetic changes that promote human disease. To combat the deleterious effects of DNA damage, organisms are equipped with DNA repair systems. The focus of our investigations has been to elucidate the details of mammalian DNA repair. These studies have provided important insights into the relationship of DNA repair to human disease and the genetic factors that contribute to individual susceptibility to the harmful effects of environmental mutagens (e.g. ionizing radiation), and have established a framework for designing more effective anti-cancer treatment schemes. To investigate questions related to DNA repair, its mechanism(s) and linkage to disease development, the authors are employing an array of molecular, cellular, biochemical and structural approaches. These complementary experimental techniques have permitted a detailed analysis into various processes of human DNA repair.

  1. Protein oxidation, UVA and human DNA repair.

    PubMed

    Karran, Peter; Brem, Reto

    2016-08-01

    Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency - with particular reference to NER and skin cancer risk. PMID:27324272

  2. Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

    PubMed Central

    Gadaleta, Mariana C.; Iwasaki, Osamu; Noguchi, Chiaki; Noma, Ken-Ichi; Noguchi, Eishi

    2015-01-01

    DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors. PMID:25916713

  3. DNA Damage and Repair in Vascular Disease.

    PubMed

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease. PMID:26442438

  4. Role of Deubiquitinating Enzymes in DNA Repair

    PubMed Central

    2015-01-01

    Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling. PMID:26644404

  5. The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex*

    PubMed Central

    Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E.; Jaspers, Nicolaas G. J.; Kaptein, Robert; Hoeijmakers, Jan H. J.; Boelens, Rolf

    2015-01-01

    The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. PMID:26085086

  6. Recognition and repair of chemically heterogeneous structures at DNA ends.

    PubMed

    Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D; Tumbale, Percy; Williams, R Scott

    2015-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  7. Recognition and repair of chemically heterogeneous structures at DNA ends

    PubMed Central

    Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.; Tumbale, Percy; Williams, R. Scott

    2014-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  8. Robustness of DNA repair through collective rate control.

    PubMed

    Verbruggen, Paul; Heinemann, Tim; Manders, Erik; von Bornstaedt, Gesa; van Driel, Roel; Höfer, Thomas

    2014-01-01

    DNA repair and other chromatin-associated processes are carried out by enzymatic macromolecular complexes that assemble at specific sites on the chromatin fiber. How the rate of these molecular machineries is regulated by their constituent parts is poorly understood. Here we quantify nucleotide-excision DNA repair in mammalian cells and find that, despite the pathways' molecular complexity, repair effectively obeys slow first-order kinetics. Theoretical analysis and data-based modeling indicate that these kinetics are not due to a singular rate-limiting step. Rather, first-order kinetics emerge from the interplay of rapidly and reversibly assembling repair proteins, stochastically distributing DNA lesion repair over a broad time period. Based on this mechanism, the model predicts that the repair proteins collectively control the repair rate. Exploiting natural cell-to-cell variability, we corroborate this prediction for the lesion-recognition factor XPC and the downstream factor XPA. Our findings provide a rationale for the emergence of slow time scales in chromatin-associated processes from fast molecular steps and suggest that collective rate control might be a widespread mode of robust regulation in DNA repair and transcription. PMID:24499930

  9. Mechanism of DNA loading by the DNA repair helicase XPD.

    PubMed

    Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Penedo, J Carlos; White, Malcolm F; Naismith, James H

    2016-04-01

    The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase fromThermoplasma acidophilumhas been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD fromSulfolobus acidocaldiariusthat lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD. PMID:26896802

  10. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  11. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. PMID:27247237

  12. DNA Triplet Repeat Expansion and Mismatch Repair

    PubMed Central

    Iyer, Ravi R.; Pluciennik, Anna; Napierala, Marek; Wells, Robert D.

    2016-01-01

    DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSβ and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway. PMID:25580529

  13. Molecular mechanisms of DNA repair inhibition by caffeine

    SciTech Connect

    Selby, C.P.; Sancar, A. )

    1990-05-01

    Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

  14. DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

    PubMed Central

    Genois, Marie-Michelle; Paquet, Eric R.; Laffitte, Marie-Claude N.; Maity, Ranjan; Rodrigue, Amélie

    2014-01-01

    SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance. PMID:24600040

  15. Antibody specific for a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-07-11

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  16. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

    Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312

  17. Mitochondrial DNA repair: a novel therapeutic target for heart failure.

    PubMed

    Marín-García, José

    2016-09-01

    Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS) and Ca(2+) handling to stress responses, cell survival and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular, cancer, premature aging and cardiovascular diseases (CVD), including myocardial ischemia, cardiomyopathy and heart failure (HF). Mitochondria contain their own genome organized into DNA-protein complexes, called "mitochondrial nucleoids," along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription and repair. Although the mammalian organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to various types of mtDNA damage. These include accumulation of mtDNA point mutations and/or deletions and decreased mtDNA copy number, which will impair mitochondrial function and finally, may lead to CVD including HF. PMID:26940911

  18. Nuclear position dictates DNA repair pathway choice

    PubMed Central

    Lemaître, Charlène; Grabarz, Anastazja; Tsouroula, Katerina; Andronov, Leonid; Furst, Audrey; Pankotai, Tibor; Heyer, Vincent; Rogier, Mélanie; Attwood, Kathleen M.; Kessler, Pascal; Dellaire, Graham; Klaholz, Bruno; Reina-San-Martin, Bernardo; Soutoglou, Evi

    2014-01-01

    Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus. PMID:25366693

  19. DNA damage checkpoint, damage repair, and genome stability.

    PubMed

    Liu, Wei-Feng; Yu, Shan-Shan; Chen, Guan-Jun; Li, Yue-Zhong

    2006-05-01

    Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed herein are the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity. PMID:16722332

  20. International congress on DNA damage and repair: Book of abstracts

    SciTech Connect

    Not Available

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

  1. DNA repair variants and breast cancer risk.

    PubMed

    Grundy, Anne; Richardson, Harriet; Schuetz, Johanna M; Burstyn, Igor; Spinelli, John J; Brooks-Wilson, Angela; Aronson, Kristan J

    2016-05-01

    A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P < 0.05), although no associations were observed for other DNA repair SNPs. Interactions of six SNPs in multiple DNA repair pathways with physical activity were evident prior to correction for FDR, following which there was support for only one of the interaction terms (P < 0.05). No consistent associations between variants in DNA repair genes and breast cancer risk or their modification by breast cancer risk factors were observed. Environ. Mol. Mutagen. 57:269-281, 2016. © 2016 Wiley Periodicals, Inc. PMID:27060854

  2. Repair of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    The genetic information of cells continuously undergoes damage induced by intracellular processes including energy metabolism, DNA replication and transcription, and by environmental factors such as mutagenic chemicals and UV and ionizing radiation. This causes numerous DNA lesions, including double strand breaks (DSBs). Since cells cannot escape this damage or normally function with a damaged genome, several DNA repair mechanisms have evolved. Although most "single-stranded" DNA lesions are rapidly removed from DNA without permanent damage, DSBs completely break the DNA molecule, presenting a real challenge for repair mechanisms, with the highest risk among DNA lesions of incorrect repair. Hence, DSBs can have serious consequences for human health. Therefore, in this chapter, we will refer only to this type of DNA damage. In addition to the biochemical aspects of DSB repair, which have been extensively studied over a long period of time, the spatio-temporal organization of DSB induction and repair, the importance of which was recognized only recently, will be considered in terms of current knowledge and remaining questions.

  3. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  4. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  5. Hsp90: A New Player in DNA Repair?

    PubMed

    Pennisi, Rosa; Ascenzi, Paolo; di Masi, Alessandra

    2015-01-01

    Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis), transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR) pathways that include: (i) cell cycle arrest; (ii) transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii) triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted. PMID:26501335

  6. Targeting backup DNA repair in cancer.

    PubMed

    2015-06-01

    New research shows that DNA polymerase θ is a key player in PARP-mediated DNA damage repair and essential for the survival of cancer cells where homologous recombination is compromised. Polθ could be a biomarker for PARP-inhibitor response, and is a potential therapeutic target for overcoming resistance to these drugs. PMID:25851856

  7. Surviving the sun: Repair and bypass of DNA UV lesions

    PubMed Central

    Yang, Wei

    2011-01-01

    Structural studies of UV-induced lesions and their complexes with repair proteins reveal an intrinsic flexibility of DNA at lesion sites. Reduced DNA rigidity stems primarily from the loss of base stacking, which may manifest as bending, unwinding, base unstacking, or flipping out. The intrinsic flexibility at UV lesions allows efficient initial lesion recognition within a pool of millions to billions of normal DNA base pairs. To bypass the damaged site by translesion synthesis, the specialized DNA polymerase η acts like a molecular “splint” and reinforces B-form DNA by numerous protein–phosphate interactions. Photolyases and glycosylases that specifically repair UV lesions interact directly with UV lesions in bent DNA via surface complementation. UvrA and UvrB, which recognize a variety of lesions in the bacterial nucleotide excision repair pathway, appear to exploit hysteresis exhibited by DNA lesions and conduct an ATP-dependent stress test to distort and separate DNA strands. Similar stress tests are likely conducted in eukaryotic nucleotide excision repair. PMID:21898645

  8. DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP

    PubMed Central

    Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

    2009-01-01

    Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

  9. The Awakening of DNA Repair at Yale

    PubMed Central

    Hanawalt, Philip C.

    2013-01-01

    As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease. PMID:24348216

  10. Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks.

    PubMed

    Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D

    2016-05-01

    The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template. PMID:26625977

  11. DNA repair responses in human skin cells

    SciTech Connect

    Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

    1981-07-01

    Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

  12. DNA Repair and Personalized Breast Cancer Therapy

    PubMed Central

    Li, Shu-Xia; Sjolund, Ashley; Harris, Lyndsay; Sweasy, Joann B.

    2010-01-01

    Personalized cancer therapy is likely to be one of the next big advances in our search for a cure for cancer. To be able to treat people in an individualized manner, researchers need to know a great deal about their genetic constitution and the DNA repair status of their tumors. Specific knowledge is required regarding the polymorphisms individuals carry and how these polymorphisms influence responses to therapy. Researchers are actively engaged in biomarker discovery and validation for this purpose. In addition, the design of clinical trials must be reassessed to include new information on biomarkers and drug responses. In this review, we focus on personalized breast cancer therapy. The hypothesis we focus upon in this review is that there is connection between the DNA repair profile of individuals, their breast tumor subtypes, and their responses to cancer therapy. We first briefly review cellular DNA repair pathways that are likely to be impacted by breast cancer therapies. Next, we review the phenotypes of breast tumor subtypes with an emphasis on how a DNA repair deficiency might result in tumorigenesis itself and lead to the chemotherapeutic responses that are observed. Specific examples of breast tumor subtypes and their responses to cancer therapy are given, and we discuss possible DNA repair mechanisms that underlie the responses of tumors to various chemotherapeutic agents. Much is known about breast cancer subtypes and the way each of these subtypes responds to chemotherapy. In addition, we discuss novel design of clinical trials that incorporates rapidly emerging information on biomarkers. PMID:20872853

  13. Recombination and DNA Repair in Helicobacter pylori

    PubMed Central

    Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.

    2013-01-01

    All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection. PMID:21682641

  14. Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

    PubMed Central

    Bernstein, Carol; Bernstein, Harris

    2015-01-01

    Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy. PMID:25987950

  15. Protein-protein interactions in DNA mismatch repair.

    PubMed

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  16. Regulation of DNA repair by parkin

    SciTech Connect

    Kao, Shyan-Yuan

    2009-05-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  17. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  18. DNA damage and repair after high LET radiation

    NASA Astrophysics Data System (ADS)

    O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

    Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

  19. The RecQ DNA helicases in DNA Repair

    PubMed Central

    Bernstein, Kara A.; Gangloff, Serge; Rothstein, Rodney

    2014-01-01

    The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent data have shown that the RecQ helicases function during two distinct steps during DNA repair; DNA end resection and resolution of double Holliday junctions (dHJs). RecQ functions in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication, meiosis and at specific genomic loci such as telomeres. PMID:21047263

  20. DNA repair in hyperthermophilic and hyperradioresistant microorganisms.

    PubMed

    Ishino, Yoshizumi; Narumi, Issay

    2015-06-01

    The genome of a living cell is continuously under attack by exogenous and endogenous genotoxins. Especially, life at high temperature inflicts additional stress on genomic DNA, and very high rates of potentially mutagenic DNA lesions, including deamination, depurination, and oxidation, are expected. However, the spontaneous mutation rates in hyperthermophiles are similar to that in Escherichia coli, and it is interesting to determine how the hyperthermophiles preserve their genomes under such grueling environmental conditions. In addition, organisms with extremely radioresistant phenotypes are targets for investigating special DNA repair mechanisms in extreme environments. Multiple DNA repair mechanisms have evolved in all organisms to ensure genomic stability, by preventing impediments that result in genome destabilizing lesions. PMID:26056771

  1. Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks.

    PubMed

    Zhao, Junhua; Jain, Aklank; Iyer, Ravi R; Modrich, Paul L; Vasquez, Karen M

    2009-07-01

    DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA-RPA and XPC-RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta interacted with XPA-RPA or XPC-RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair. PMID:19468048

  2. Electrically monitoring DNA repair by photolyase

    NASA Astrophysics Data System (ADS)

    DeRosa, Maria C.; Sancar, Aziz; Barton, Jacqueline K.

    2005-08-01

    Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the π-stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV/blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA. Author contributions: M.C.D. and J.K.B. designed research; M.C.D. performed research; A.S. contributed new reagents/analytic tools; M.C.D. analyzed data; and M.C.D. and J.K.B. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Abbreviations: T<>T, thymine dimer; CT, charge transport.

  3. Monoclonal antibody to single-stranded DNA: a potential tool for DNA repair studies.

    PubMed

    Cooke, M S; Patel, K; Ahmad, J; Holloway, K; Evans, M D; Lunec, J

    2001-06-01

    Growing evidence suggests that DNA repair capacity is an important factor in cancer risk and is therefore essential to assess. Immunochemical assays are amenable to the detection of repair products in complex matrices, such as urine, facilitating noninvasive measurements, although diet and extra-DNA sources of lesion can confound interpretation. The production of single-stranded, lesion-containing DNA oligomers characterises nucleotide excision repair (NER) and hence defines the repair pathway from which a lesion may be derived. Herein we describe the characterisation of a monoclonal antibody which recognises guanine moieties in single-stranded DNA. Application of this antibody in ELISA, demonstrated such oligomers in supernatants from repair-proficient cells post-insult. Testing of urine samples from volunteers demonstrated a relationship between oligomer levels and two urinary DNA damage products, thymine dimers and 8-oxo-2'-deoxyguanosine, supporting our hypothesis that NER gives rise to lesion-containing oligomers which are specific targets for the investigation of DNA repair. PMID:11374895

  4. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells. PMID:26581212

  5. DNA double strand break repair, aging and the chromatin connection.

    PubMed

    Gorbunova, Vera; Seluanov, Andrei

    2016-06-01

    Are DNA damage and mutations possible causes or consequences of aging? This question has been hotly debated by biogerontologists for decades. The importance of DNA damage as a possible driver of the aging process went from being widely recognized to then forgotten, and is now slowly making a comeback. DNA double strand breaks (DSBs) are particularly relevant to aging because of their toxicity, increased frequency with age and the association of defects in their repair with premature aging. Recent studies expand the potential impact of DNA damage and mutations on aging by linking DNA DSB repair and age-related chromatin changes. There is overwhelming evidence that increased DNA damage and mutations accelerate aging. However, an ultimate proof of causality would be to show that enhanced genome and epigenome stability delays aging. This is not an easy task, as improving such complex biological processes is infinitely more difficult than disabling it. We will discuss the possibility that animal models with enhanced DNA repair and epigenome maintenance will be generated in the near future. PMID:26923716

  6. Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

    PubMed

    Çağlayan, Melike; Wilson, Samuel H

    2015-12-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  7. Posttranscriptional regulation of the RAD5 DNA repair gene by the Dun1 kinase and the Pan2-Pan3 poly(A)-nuclease complex contributes to survival of replication blocks.

    PubMed

    Hammet, Andrew; Pike, Brietta L; Heierhorst, Jörg

    2002-06-21

    The yeast Dun1 kinase has complex checkpoint functions including DNA damage-dependent cell cycle arrest in G(2)/M, transcriptional induction of repair genes, and regulation of postreplicative DNA repair pathways. Here we report that the Dun1 forkhead-associated domain interacts with the Pan3 subunit of the poly(A)-nuclease complex and that dun1pan2 and dun1pan3 double mutants are dramatically hypersensitive to replicational stress. This phenotype was independent of the function of Dun1 in regulating deoxyribonucleotide levels as it was also observed in strains lacking the ribonucleotide reductase inhibitor Sml1. dun1pan2 mutants initially arrested normally in response to replication blocks but died in the presence of persistent replication blocks with considerably delayed kinetics compared with mutants lacking the Rad53 kinase, indicating that the double mutation does not compromise the intra-S phase checkpoint. Interestingly, the RAD5 gene involved in error-free postreplication repair pathways was specifically up-regulated in dun1pan2 double mutants. Moreover, inducible overexpression of RAD5 mimicked the double mutant phenotype by hypersensitizing dun1 mutants to replication blocks. The data indicate that Dun1 and Pan2-Pan3 cooperate to regulate the stoichiometry and thereby the activity of postreplication repair complexes, suggesting that posttranscriptional mechanisms complement the transcriptional response in the regulation of gene expression by checkpoint signaling pathways in Saccharomyces cerevisiae. PMID:11953437

  8. The complex choreography of transcription-coupled repair.

    PubMed

    Spivak, Graciela; Ganesan, Ann K

    2014-07-01

    A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations. PMID:24751236

  9. Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2-Cdk7 complex.

    PubMed

    Paul, Somnath; Banerjee, Nilanjana; Chatterjee, Aditi; Sau, Tanmoy J; Das, Jayanta K; Mishra, Prafulla K; Chakrabarti, Partha; Bandyopadhyay, Arun; Giri, Ashok K

    2014-04-01

    Arsenic in drinking water is of critical concern in West Bengal, India, as it results in several physiological symptoms including dermatological lesions and cancers. Impairment of the DNA repair mechanism has been associated with arsenic-induced genetic damage as well as with several cancers. ERCC2 (Excision Repair Cross-Complementing rodent repair, complementation group 2), mediates DNA-repair by interacting with Cdk-activating kinase (CAK) complex, which helps in DNA proof-reading during transcription. Arsenic metabolism alters epigenetic regulation; we tried to elucidate the regulation of ERCC2 in arsenic-exposed humans. Water, urine, nails, hair and blood samples from one hundred and fifty seven exposed and eighty eight unexposed individuals were collected. Dose dependent validation was done in vitro using HepG2 and HEK-293. Arsenic content in the biological samples was higher in the exposed individuals compared with the content in unexposed individuals (p < 0.001). Bisulfite-modified methylation specific PCR showed a significant (p < 0.0001) hypomethylation of the ERCC2 promoter in the arsenic-exposed individuals. Densitometric analysis of immunoblots showed a nearly two-fold increase in expression of ERCC2 in exposed individuals, but there was an enhanced genotoxic insult as measured by micronuclei frequency. Immuno-precipitation and western blotting revealed an increased (p < 0.001) association of Cdk7 with ERCC2 in highly arsenic exposed individuals. The decrease in CAK activity was determined by observing the intensity of Ser(392) phosphorylation in p53, in vitro, which decreased with an increase in arsenic dose. Thus we infer that arsenic biotransformation leads to promoter hypomethylation of ERCC2, which in turn inhibits the normal functioning of the CAK-complex, thus affecting DNA-repair; this effect was highest among the arsenic exposed individuals with dermatological lesions. PMID:24473091

  10. Xeroderma pigmentosum and the role of DNA repair in oncogenesis.

    PubMed

    Giannelli, F

    1978-01-01

    Biochemical and genetic information on xeroderma pigmentosum (XP) has been briefly reviewed. This indicates that 80 to 90 percent of all XP patients are defective in the excision repair of pyrimidine dimers and are unable to perform the first step of this process as shown, for example, by their inability to undergo the DNA superhelical changes which accompany the initiation of excision repair in normal cells. However, in spite of its apparent biochemical homogeneity, XP is genetically heterogeneous and many genes appear to be responsible for the function of the factor defective in XP. Ten to 20 percent of all XP patients (called XP-variants) are capable of "dimer excision repair" but have difficulties in replicating UV-damaged DNA. The defects of XP and XP-variant affect also the repair of DNA damage caused by a number of chemical mutagens and carcinogens. This has important theoretical and practical implications since it indicates, for example, that the repair systems defective in XP must have broad specificity and that even XP cells not exposed to the harmful effect of light may suffer from poor repair of DNA damage. With regard to cancer, two questions have been considered. Namely, does XP provide a valid general model for UV-carcinogenesis in man and does it show how DNA damage leads to malignant transformation? The first question was answered in the affirmative in view of some clinical but, mainly, of cell biological data indicating that normal and excision defective XP cells differ, more quantitatively than qualitatively, in their response to UV-light. With regard to the second question XP seems to provide some support for various theories on carcinogenesis and, DNA repair defects may favour actinic carcinogenesis in a complex, non-univocous manner. Possibly the most important lesson imparted by XP is that, in man, the stability of the genetic material is dependent on the function of repair systems whose failure may predispose to cancer. In addition, the

  11. [Progress of enzyme in mitochondrial DNA repair system].

    PubMed

    Zhu, Ke-Jun; Wang, Zhen-Cheng; Wang, Xue-Min

    2004-03-01

    Mitochondrial DNA (mtDNA) encodes subunits of the mitochondrial electron transport system and the rRNAs and tRNAs required for constructing the mitochondrial translational machinery. Each subunit encoded by mtDNA is essential for normal oxidative phosphorylation. Thus, integrity of the mtDNA is crucial for the survival of organisms. It has long been held that there is no DNA repair in mitochondria. But in recent years,a number of repair factors have been found in mitochondrial extracts, suggesting the presence of DNA repair in mitochondria. This review summarized recent progress of enzyme in mitochondrial DNA repair processes. PMID:15640002

  12. DNA Repair-Protein Relocalization After Heavy Ion Exposure

    NASA Technical Reports Server (NTRS)

    Metting, N. F.

    1999-01-01

    Ionizing radiation is good at making DNA double strand breaks, and high linear energy transfer (LET) radiations such as heavy ion particles are particularly efficient. For this reason, the proteins belonging to repair systems that deal with double strand breaks are of particular interest. One such protein is Ku, a component in the non-homologous recombination repair system. The Ku protein is an abundant, heterodimeric DNA end-binding complex, composed of one 70 and one 86 kDa subunit. Ku protein binds to DNA ends, nicks, gaps, and regions of transition between single and double-stranded structure. These binding properties suggest an important role in DNA repair. The Ku antigen is important in this study because it is present in relatively large copy numbers and it is part of a double-strand-break repair system. More importantly, we consistently measure an apparent upregulation in situ that is not verified by whole-cell-lysate immunoblot measurements. This apparent upregulation is triggered by very low doses of radiation, thus showing a potentially useful high sensitivity. However, elucidation of the mechanism underlying this phenomenon is still to be done.

  13. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  14. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  15. The interplay between DNA repair and autophagy in cancer therapy.

    PubMed

    Zhang, Dan; Tang, Bo; Xie, Xia; Xiao, Yu-Feng; Yang, Shi-Ming; Zhang, Jian-Wei

    2015-01-01

    DNA is the prime target of anticancer treatments. DNA damage triggers a series of signaling cascades promoting cellular survival, including DNA repair, cell cycle arrest, and autophagy. The elevated basal and/or stressful levels of both DNA repair and autophagy observed in tumor cells, in contrast to normal cells, have been identified as the most important drug-responsive programs that impact the outcome of anticancer therapy. The exact relationship between DNA repair and autophagy in cancer cells remains unclear. On one hand, autophagy has been shown to regulate some of the DNA repair proteins after DNA damage by maintaining the balance between their synthesis, stabilization, and degradation. One the other hand, some evidence has demonstrated that some DNA repair molecular have a crucial role in the initiation of autophagy. In this review, we mainly discuss the interplay between DNA repair and autophagy in anticancer therapy and expect to enlighten some effective strategies for cancer treatment. PMID:25985143

  16. Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond

    PubMed Central

    Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

    2012-01-01

    Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug

  17. Deficient DNA repair capacity, a predisposing factor in breast cancer.

    PubMed Central

    Parshad, R.; Price, F. M.; Bohr, V. A.; Cowans, K. H.; Zujewski, J. A.; Sanford, K. K.

    1996-01-01

    Women with breast cancer and a family history of breast cancer and some with sporadic breast cancer are deficient in the repair of radiation-induced DNA damage compared with normal donors with no family history of breast cancer. DNA repair was measured indirectly by quantifying chromatid breaks in phytohaemagglutinin (PHA)-stimulated blood lymphocytes after either X-irradiation or UV-C exposure, with or without post treatment with the DNA repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C). We have correlated chromatid breaks with unrepaired DNA strand breaks using responses to X-irradiation of cells from xeroderma pigmentosum patients with well-characterised DNA repair defects or responses of repair-deficient mutant Chinese hamster ovary (CHO) cells with or without transfected human DNA repair genes. Deficient DNA repair appears to be a predisposing factor in familial breast cancer and in some sporadic breast cancers. PMID:8679441

  18. Cloning a Eukaryotic DNA Glycosylase Repair Gene by the Suppression of a DNA Repair Defect in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chen, Jin; Derfler, Bruce; Maskati, Azmat; Samson, Leona

    1989-10-01

    If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S. cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation. This approach may allow the isolation of a large number of eukaryotic DNA repair genes.

  19. Targeting DNA repair pathways for cancer treatment: what's new?

    PubMed Central

    Kelley, Mark R; Logsdon, Derek; Fishel, Melissa L

    2014-01-01

    Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses. PMID:24947262

  20. DNA repair in species with extreme lifespan differences

    PubMed Central

    MacRae, Sheila L.; Croken, Matthew McKnight; Calder, R.B.; Aliper, Alexander; Milholland, Brandon; White, Ryan R.; Zhavoronkov, Alexander; Gladyshev, Vadim N.; Seluanov, Andrei; Gorbunova, Vera; Zhang, Zhengdong D.; Vijg, Jan

    2015-01-01

    Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals—depending on habitats, anatomical characteristics, and life styles—varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ∼120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system. PMID:26729707

  1. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  2. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  3. Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

    PubMed Central

    Brown, Maxwell W.; Kim, Yoori; Williams, Gregory M.; Huck, John D.; Surtees, Jennifer A.; Finkelstein, Ilya J.

    2016-01-01

    DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin. PMID:26837705

  4. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  5. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  6. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  7. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  8. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  9. DNA repair of a single UV photoproduct in a designed nucleosome

    SciTech Connect

    Kosmoskil, Joseph V.; Ackerman, Eric J. ); Smerdon, Michael J.

    2001-08-28

    Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is > 50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA ( 165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

  10. TOPBP1 takes RADical command in recombinational DNA repair.

    PubMed

    Liu, Yi; Smolka, Marcus B

    2016-02-01

    TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination. PMID:26811424

  11. Pathophysiology of Bronchoconstriction: Role of Oxidatively Damaged DNA Repair

    PubMed Central

    Bacsi, Attila; Pan, Lang; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Purpose of review To provide an overview on the present understanding of roles of oxidative DNA damage repair in cell signaling underlying bronchoconstriction common to, but not restricted to various forms of asthma and chronic obstructive pulmonary disease Recent findings Bronchoconstriction is a tightening of smooth muscle surrounding the bronchi and bronchioles with consequent wheezing and shortness of breath. Key stimuli include air pollutants, viral infections, allergens, thermal and osmotic changes, and shear stress of mucosal epithelium, triggering a wide range of cellular, vascular and neural events. Although activation of nerve fibers, the role of G-proteins, protein kinases and Ca++, and molecular interaction within contracting filaments of muscle are well defined, the overarching mechanisms by which a wide range of stimuli initiate these events are not fully understood. Many, if not all, stimuli increase levels of reactive oxygen species (ROS), which are signaling and oxidatively modifying macromolecules, including DNA. The primary ROS target in DNA is guanine, and 8-oxoguanine is one of the most abundant base lesions. It is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair processes. The product, free 8-oxoG base, is bound by OGG1 with high affinity, and the complex then functions as an activator of small GTPases, triggering pathways for inducing gene expression and contraction of intracellular filaments in mast and smooth muscle cells. Summary Oxidative DNA damage repair-mediated cell activation signaling result in gene expression that “primes” the mucosal epithelium and submucosal tissues to generate mediators of airway smooth muscle contractions. PMID:26694039

  12. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    SciTech Connect

    Smith, P.J.

    1986-03-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

  13. Comet assay to measure DNA repair: approach and applications

    PubMed Central

    Azqueta, Amaya; Slyskova, Jana; Langie, Sabine A. S.; O’Neill Gaivão, Isabel; Collins, Andrew

    2014-01-01

    Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations. PMID:25202323

  14. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    PubMed

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. PMID:26861186

  15. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  16. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  17. Chromatin Structure Following UV-Induced DNA Damage—Repair or Death?

    PubMed Central

    Farrell, Andrew W.; Halliday, Gary M.; Lyons, James Guy

    2011-01-01

    In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury. PMID:22174650

  18. Participation of DNA repair in the response to 5-fluorouracil

    PubMed Central

    Wyatt, Michael D.; Wilson, David M.

    2008-01-01

    The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anti-cancer treatments. PMID:18979208

  19. A central role of TRAX in the ATM-mediated DNA repair.

    PubMed

    Wang, J-Y; Chen, S-Y; Sun, C-N; Chien, T; Chern, Y

    2016-03-31

    DNA repair is critical for the maintenance of genome stability. Upon genotoxic stress, dysregulated DNA repair may induce apoptosis. Translin-associated factor X (TRAX), which was initially identified as a binding partner of Translin, has been implicated in genome stability. However, the exact role of TRAX in DNA repair remains largely unknown. Here, we showed that TRAX participates in the ATM/H2AX-mediated DNA repair machinery by interacting with ATM and stabilizing the MRN complex at double-strand breaks. The exogenous expression of wild-type (WT) TRAX, but not a TRAX variant lacking the nuclear localization signal (NLS), rescued the vulnerability of TRAX-null mouse embryo fibroblasts (MEFs). This finding confirms the importance of the nuclear localization of TRAX in the repair of DNA damage. Compared with WT MEFs, TRAX-null MEFs exhibited impaired DNA repair (for example, reduced phosphorylation of ATM and H2AX) after treatment with ultra violet-C or γ-ray irradiation and a higher incidence of p53-mediated apoptosis. Our findings demonstrate that TRAX is required for MRN complex-ATM-H2AX signaling, which optimizes DNA repair by interacting with the activated ATM and protects cells from genotoxic stress-induced apoptosis. PMID:26096928

  20. DNA repair: From genome maintenance to biomarker and therapeutic target

    PubMed Central

    Jalal, Shadia; Earley, Jennifer N.; Turchi, John J.

    2011-01-01

    A critical link exists between an individual's ability to repair cellular DNA damage and cancer development, progression and response to therapy. Knowledge gained regarding the proteins involved and types of damage repaired by the individual DNA repair pathways has led to the development of a variety of assays aimed at determining an individual's DNA repair capacity. These assays and their use in the analysis of clinical samples has yielded useful though somewhat conflicting data. In this review article, we discuss the major DNA repair pathways, the proteins and genes required for each, assays used to assess activity and the relevant clinical studies to date. With the recent results from clinical trials targeting specific DNA repair proteins for the treatment of cancer, accurate, reproducible and relevant analysis of DNA repair takes on an even greater significance. We highlight the strengths and limitations of these DNA repair studies and assays with respect to the clinical assessment of DNA repair capacity to determine cancer development and response to therapy. PMID:21908578

  1. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair.

    PubMed

    Brocas, Clémentine; Charbonnier, Jean-Baptiste; Dhérin, Claudine; Gangloff, Serge; Maloisel, Laurent

    2010-10-01

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair. PMID:20813592

  2. Assessment of the role of DNA repair in damaged forensic samples.

    PubMed

    Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

    2014-11-01

    Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results. PMID:24792635

  3. The contribution of CMP kinase to the efficiency of DNA repair

    PubMed Central

    Tsao, Ning; Lee, Ming-Hsiang; Zhang, Wei; Cheng, Yung-Chi; Chang, Zee-Fen

    2015-01-01

    Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells. PMID:25659034

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

    PubMed

    Zahn, Karl E; Averill, April M; Aller, Pierre; Wood, Richard D; Doublié, Sylvie

    2015-04-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

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

    PubMed Central

    Zahn, Karl E.; Averill, April M.; Aller, Pierre; Wood, Richard D.; Doublié, Sylvie

    2015-01-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase θ, and reduction of its activity in mammalian cells increases sensitivity to double-strand break inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ employs a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction to the 3’-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

  6. DNA Repair and Genome Maintenance in Bacillus subtilis

    PubMed Central

    Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

  7. Repair of DNA lesions associated with triplex-forming oligonucleotides.

    PubMed

    Chin, Joanna Y; Glazer, Peter M

    2009-04-01

    Triplex-forming oligonucleotides (TFOs) are gene targeting tools that can bind in the major groove of duplex DNA in a sequence-specific manner. When bound to DNA, TFOs can inhibit gene expression, can position DNA-reactive agents to specific locations in the genome, or can induce targeted mutagenesis and recombination. There is evidence that third strand binding, alone or with an associated cross-link, is recognized and metabolized by DNA repair factors, particularly the nucleotide excision repair pathway. This review examines the evidence for DNA repair of triplex-associated lesions. PMID:19072762

  8. Specificity of damage recognition and catalysis of DNA repair.

    PubMed

    Osman, R; Fuxreiter, M; Luo, N

    2000-05-01

    A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton

  9. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

    PubMed Central

    Aparicio, Tomas; Baer, Richard; Gottesman, Max

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein–DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)–DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2–DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication. PMID:26880199

  10. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization

    SciTech Connect

    Gruskin, E.A.; Lloyd, R.S.

    1988-09-05

    In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

  11. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    PubMed Central

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

    2014-01-01

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting. PMID:25099886

  12. DNA-PK: a dynamic enzyme in a versatile DSB repair pathway

    PubMed Central

    Davis, Anthony J.; Chen, Benjamin P.C.; Chen, David J.

    2014-01-01

    DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex. PMID:24680878

  13. DNA repair systems as targets of cadmium toxicity

    SciTech Connect

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios . E-mail: theocharis@ath.forthnet.gr

    2006-06-15

    Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity.

  14. NRAGE is involved in homologous recombination repair to resist the DNA-damaging chemotherapy and composes a ternary complex with RNF8-BARD1 to promote cell survival in squamous esophageal tumorigenesis.

    PubMed

    Yang, Q; Pan, Q; Li, C; Xu, Y; Wen, C; Sun, F

    2016-08-01

    NRAGE, a neurotrophin receptor-interacting melanoma antigen-encoding gene homolog, is significantly increased in the nucleus of radioresistant esophageal tumor cell lines and is highly upregulated to promote cell proliferation in esophageal carcinomas (ECs). However, whether the overexpressed NRAGE promotes cell growth by participating in DNA-damage response (DDR) is still unclear. Here we show that NRAGE is required for efficient double-strand breaks (DSBs) repair via homologous recombination repair (HRR) and downregulation of NRAGE greatly sensitizes EC cells to DNA-damaging agents both in vitro and in vivo. Moreover, NRAGE not only regulates the stability of DDR factors, RNF8 and BARD1, in a ubiquitin-proteolytic pathway, but also chaperons the interaction between BARD1 and RNF8 via their RING domains to form a novel ternary complex. Additionally, the expression of NRAGE is closely correlated with RNF8 and BARD1 in esophageal tumor tissues. In summary, our findings reveal a novel function of NRAGE that will help to guide personalized esophageal cancer treatments by targeting NRAGE to increase cell sensitivity to DNA-damaging therapeutics in the long run. PMID:27035619

  15. Destabilizing DNA during Rejoining Enhances Fidelity of Repair.

    PubMed

    Robinson, Richard

    2015-08-01

    A new study shows that during repair of DNA, the effect of a single-strand annealing protein is to destabilize DNA duplex formation so that annealing only occurs between perfectly matched strands; the protein then clamps the strands together for repair. Read the Research Article. PMID:26271078

  16. Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination

    PubMed Central

    López-Torrejón, Gema; Martínez-Jiménez, María I.; Ayora, Silvia

    2006-01-01

    Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein–DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination. PMID:16407330

  17. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

    PubMed Central

    Milne, G T; Jin, S; Shannon, K B; Weaver, D T

    1996-01-01

    DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells. PMID:8754818

  18. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  19. DNA Damage Repair in the Context of Plant Chromatin.

    PubMed

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-08-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  20. Role of DNA repair in host immune response and inflammation.

    PubMed

    Fontes, Fabrícia Lima; Pinheiro, Daniele Maria Lopes; Oliveira, Ana Helena Sales de; Oliveira, Rayssa Karla de Medeiros; Lajus, Tirzah Braz Petta; Agnez-Lima, Lucymara Fassarella

    2015-01-01

    In recent years, the understanding of how DNA repair contributes to the development of innate and acquired immunity has emerged. The DNA damage incurred during the inflammatory response triggers the activation of DNA repair pathways, which are required for host-cell survival. Here, we reviewed current understanding of the mechanism by which DNA repair contributes to protection against the oxidized DNA damage generated during infectious and inflammatory diseases and its involvement in innate and adaptive immunity. We discussed the functional role of DNA repair enzymes in the immune activation and the relevance of these processes to: transcriptional regulation of cytokines and other genes involved in the inflammatory response; V(D)J recombination; class-switch recombination (CSR); and somatic hypermutation (SHM). These three last processes of DNA damage repair are required for effective humoral adaptive immunity, creating genetic diversity in developing T and B cells. Furthermore, viral replication is also dependent on host DNA repair mechanisms. Therefore, the elucidation of the pathways of DNA damage and its repair that activate innate and adaptive immunity will be important for a better understanding of the immune and inflammatory disorders and developing new therapeutic interventions for treatment of these diseases and for improving their outcome. PMID:25795123

  1. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    DOE PAGESBeta

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N -nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O 6more » -alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects.« less

  2. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    PubMed Central

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N-nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O6-alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects. PMID:21234336

  3. UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

    PubMed Central

    Muller, C; Calsou, P; Frit, P; Cayrol, C; Carter, T; Salles, B

    1998-01-01

    DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion. PMID:9490781

  4. DNA repair and the evolution of transformation in Bacillus subtilis. II. Role of inducible repair

    SciTech Connect

    Wojciechowski, M.F.; Hoelzer, M.A.; Michod, R.E.

    1989-03-01

    In Bacillus subtilis, DNA repair and recombination are intimately associated with competence, the physiological state in which the bacterium can bind, take up and recombine exogenous DNA. Previously, we have shown that the homologous DNA transformation rate (ratio of transformants to total cells) increases with increasing UV dosage if cells are transformed after exposure to UV radiation (UV-DNA), whereas the transformation rate decreases if cells are transformed before exposure to UV (DNA-UV). In this report, by using different DNA repair-deficient mutants, we show that the greater increase in transformation rate in UV-DNA experiments than in DNA-UV experiments does not depend upon excision repair or inducible SOS-like repair, although certain quantitative aspects of the response do depend upon these repair systems. We also show that there is no increase in the transformation rate in a UV-DNA experiment when repair and recombination proficient cells are transformed with nonhomologous plasmid DNA, although the results in a DNA-UV experiment are essentially unchanged by using plasmid DNA. We have used din operon fusions as a sensitive means of assaying for the expression of genes under the control of the SOS-like regulon in both competent and noncompetent cell subpopulations as a consequence of competence development and our subsequent experimental treatments. Results indicate that the SOS-like system is induced in both competent and noncompetent subpopulations in our treatments and so should not be a major factor in the differential response in transformation rate observed in UV-DNA and DNA-UV treatments. These results provide further support to the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in the repair of DNA damage.

  5. DNA repair in cancer: emerging targets for personalized therapy

    PubMed Central

    Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

    2014-01-01

    Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer. PMID:24600246

  6. SMC complexes: from DNA to chromosomes.

    PubMed

    Uhlmann, Frank

    2016-07-01

    SMC (structural maintenance of chromosomes) complexes - which include condensin, cohesin and the SMC5-SMC6 complex - are major components of chromosomes in all living organisms, from bacteria to humans. These ring-shaped protein machines, which are powered by ATP hydrolysis, topologically encircle DNA. With their ability to hold more than one strand of DNA together, SMC complexes control a plethora of chromosomal activities. Notable among these are chromosome condensation and sister chromatid cohesion. Moreover, SMC complexes have an important role in DNA repair. Recent mechanistic insight into the function and regulation of these universal chromosomal machines enables us to propose molecular models of chromosome structure, dynamics and function, illuminating one of the fundamental entities in biology. PMID:27075410

  7. Hereditary cancer syndromes: utilizing DNA repair deficiency as therapeutic target.

    PubMed

    Goyal, Gaurav; Fan, Tiffany; Silberstein, Peter Todd

    2016-07-01

    Human cells have numerous repair mechanisms to counteract various insults incurred on the DNA. Any mutation in these repair mechanisms can lead to accumulation of DNA errors and carcinogenesis. This review aims to discuss the therapeutic options in the two most common DNA repair deficient cancer syndromes, namely Lynch syndrome (hereditary non-polyposis colorectal cancer) and breast cancer susceptibility gene (BRCA) associated ovarian and breast cancer. Deficiency in DNA repair mechanisms renders these tumors with increased sensitivity to platinum agents. There has been increasing amount of information on the utility of the defects in DNA repair as targets for cancer therapy in these syndromes. Novel therapies like poly (ADP-ribose) polymerase (PARP) inhibitors are one of such example where the induction of double stranded breaks in DNA leads to tumoricidal effect in patients with homologous DNA repair deficiency. Interestingly, patients with DNA repair deficiencies tend to have a more favorable prognosis than sporadic malignancies. In microsatellite high colorectal cancer patients, this has been attributed to increased recruitment of CD8+ T lymphocytes in tumor microenvironment. However, these tumors are able to limit the host immune response by activation of immune checkpoints that seem like attractive targets of therapy in the future. PMID:26873719

  8. DNA repair: Dynamic defenders against cancer and aging

    SciTech Connect

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet (UV

  9. Mechanisms of interstrand DNA crosslink repair and human disorders.

    PubMed

    Hashimoto, Satoru; Anai, Hirofumi; Hanada, Katsuhiro

    2016-01-01

    Interstrand DNA crosslinks (ICLs) are the link between Watson-Crick strands of DNAs with the covalent bond and prevent separation of DNA strands. Since the ICL lesion affects both strands of the DNA, the ICL repair is not simple. So far, nucleotide excision repair (NER), structure-specific endonucleases, translesion DNA synthesis (TLS), homologous recombination (HR), and factors responsible for Fanconi anemia (FA) are identified to be involved in ICL repair. Since the presence of ICL lesions causes severe defects in transcription and DNA replication, mutations in these DNA repair pathways give rise to a various hereditary disorders. NER plays an important role for the ICL recognition and removal in quiescent cells, and defects of NER causes congential progeria syndrome, such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. On the other hand, the ICL repair in S phase requires more complicated orchestration of multiple factors, including structure-specific endonucleases, and TLS, and HR. Disturbed this ICL repair orchestration in S phase causes genome instability resulting a cancer prone disease, Fanconi anemia. So far more than 30 factors in ICL repair have already identified. Recently, a new factor, UHRF1, was discovered as a sensor of ICLs. In addition to this, numbers of nucleases that are involved in the first incision, also called unhooking, of ICL lesions have also been identified. Here we summarize the recent studies of ICL associated disorders and repair mechanism, with emphasis in the first incision of ICLs. PMID:27350828

  10. 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

  11. Dynamic binding of replication protein a is required for DNA repair.

    PubMed

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

    2016-07-01

    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

  12. The Role of the COP9 Signalosome and Neddylation in DNA Damage Signaling and Repair

    PubMed Central

    Chung, Dudley; Dellaire, Graham

    2015-01-01

    The maintenance of genomic integrity is an important process in organisms as failure to sense and repair damaged DNA can result in a variety of diseases. Eukaryotic cells have developed complex DNA repair response (DDR) mechanisms to accurately sense and repair damaged DNA. Post-translational modifications by ubiquitin and ubiquitin-like proteins, such as SUMO and NEDD8, have roles in coordinating the progression of DDR. Proteins in the neddylation pathway have also been linked to regulating DDR. Of interest is the COP9 signalosome (CSN), a multi-subunit metalloprotease present in eukaryotes that removes NEDD8 from cullins and regulates the activity of cullin-RING ubiquitin ligases (CRLs). This in turn regulates the stability and turnover of a host of CRL-targeted proteins, some of which have established roles in DDR. This review will summarize the current knowledge on the role of the CSN and neddylation in DNA repair. PMID:26437438

  13. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  14. DNA double-strand break repair inhibitors as cancer therapeutics.

    PubMed

    Srivastava, Mrinal; Raghavan, Sathees C

    2015-01-22

    Among DNA damages, double-strand breaks (DSBs) are one of the most harmful lesions to a cell. Failure in DSB repair could lead to genomic instability and cancer. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are major DSB repair pathways in higher eukaryotes. It is known that expression of DSB repair genes is altered in various cancers. Activation of DSB repair genes is one of the reasons for chemo- and radioresistance. Therefore, targeting DSB repair is an attractive strategy to eliminate cancer. Besides, therapeutic agents introduce breaks in the genome as an intermediate. Therefore, blocking the residual repair using inhibitors can potentiate the efficacy of cancer treatment. In this review, we discuss the importance of targeting DSB repair pathways for the treatment of cancer. Recent advances in the development of DSB repair inhibitors and their clinical relevance are also addressed. PMID:25579208

  15. Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair

    PubMed Central

    Li, Wentao; Selvam, Kathiresan; Ko, Tengyu; Li, Shisheng

    2014-01-01

    Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. PMID:25389266

  16. Connecting the Dots: From DNA Damage and Repair to Aging.

    PubMed

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new "dots" are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  17. Connecting the Dots: From DNA Damage and Repair to Aging

    PubMed Central

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new “dots” are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  18. Acetylation regulates DNA repair mechanisms in human cells.

    PubMed

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  19. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  20. Effect of aging and dietary restriction on DNA repair

    SciTech Connect

    Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

    1989-03-01

    DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

  1. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    SciTech Connect

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  2. Fanconi Anemia: A Signal Transduction and DNA Repair Pathway

    PubMed Central

    Kupfer, Gary M.

    2013-01-01

    Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response. PMID:24348213

  3. Specific association between the human DNA repair proteins XPA and ERCC1.

    PubMed Central

    Li, L; Elledge, S J; Peterson, C A; Bales, E S; Legerski, R J

    1994-01-01

    Processing of DNA damage by the nucleotide-excision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specifically interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that mediate this interaction. These results suggest that XPA may play a role in the localization or loading of an incision complex, composed of ERCC1 and possibly other repair factors, onto a damaged site. Images PMID:8197174

  4. A review of DNA repair and possible DNA-repair adjuvants and selected natural anti-oxidants.

    PubMed

    Emanuel, Patrick; Scheinfeld, Noah

    2007-01-01

    Few other organs have the environmental exposure-neoplasia relationship that has been observed between epithelial cutaneous malignancy and UVB exposure. A significant DNA type of defective linking of DNA nucleotides involves pyrimidine dimers. Important insight into the molecular processes that affect the response of cells to UVB have been provided by the study of rare inherited diseases characterized by DNA repair defects. Nucleotide excision repair is the best characterized of these and its importance is illustrated by the disease, xeroderma pigmentosum. This heterogenous disorder clinically characterized by malignant tumor development and molecularly by distinct alterations in the nucleotide excision repair apparatus. More recently, other DNA mechanisms have been shown to have some role in skin cancer, such as DNA-mismatch repair and double-stranded DNA breaks. Herein, we discuss the DNA-repair adjuvants a aqueous extract of Urcaria tomentosa (AC-11, Optigenex, Inc.), and T4 endonuclease V that is prepared in a liposome lotion (Dimericine, Applied Genetics Inc. Dermatics). The positive effects on the integrity DNA of other substances (from nature, heat shock proteins and cytokines) including IL-12, Polypodium leucotomos, and ubiquitin are also reviewed. Understanding DNA repair mechanisms is far from complete; further understanding will provide insight into the pathogenesis of cancer and pave the way for efficacious therapeutic agents. PMID:18328204

  5. DNA Repair Dysfunction and Neurodegeneration: Lessons From Rare Pediatric Disorders.

    PubMed

    Shabbir, Syed H

    2016-03-01

    Nucleotide excision repair disorders display a wide range of clinical syndromes and presentations, all associated at the molecular level by dysfunction of genes participating in the nucleotide excision repair pathway. Genotype-phenotype relationships are remarkably complex and not well understood. This article outlines neurodegenerative symptoms seen in nucleotide excision repair disorders and explores the role that nucleotide excision repair dysfunction can play in the pathogenesis of chronic neurodegenerative diseases. PMID:26116382

  6. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  7. The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation

    SciTech Connect

    Brandt, Patrick D.; Helt, Christopher E.; Keng, Peter C.; Bambara, Robert A. . E-mail: robert_bambara@urmc.rochester.edu

    2006-08-18

    Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.

  8. Induced DNA repair pathway in mammalian cells

    SciTech Connect

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-..beta..-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells.

  9. The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo.

    PubMed

    Malivert, Laurent; Callebaut, Isabelle; Rivera-Munoz, Paola; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

    2009-03-01

    The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair. PMID:19103754

  10. Imaging of DNA and Protein–DNA Complexes with Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein–DNA complexes, including recent advances in the visualization of protein–DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein–DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein–DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described. PMID:27278886

  11. Radiation-Induced Survivin Nuclear Accumulation is Linked to DNA Damage Repair

    SciTech Connect

    Capalbo, Gianni; Weiss, Christian; Reichert, Sebastian; Roedel, Claus

    2010-05-01

    Purpose: Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivin's cellular localization and DNA double-strand break repair. Methods and Materials: Survivin's cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA. Results: Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity. Conclusion: These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.

  12. The molecular origin of high DNA-repair efficiency by photolyase.

    PubMed

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Sancar, Aziz; Zhong, Dongping

    2015-01-01

    The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. Here, we analyse the primary reaction steps of repair of ultraviolet-damaged DNA by photolyase using femtosecond spectroscopy. With systematic mutations of the amino acids involved in binding of the flavin cofactor and the cyclobutane pyrimidine dimer substrate, we report our direct deconvolution of the catalytic dynamics with three electron-transfer and two bond-breaking elementary steps and thus the fine tuning of the biological repair function for optimal efficiency. We found that the maximum repair efficiency is not enhanced by the ultrafast photoinduced process but achieved by the synergistic optimization of all steps in the complex repair reaction. PMID:26065359

  13. The molecular origin of high DNA-repair efficiency by photolyase

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Sancar, Aziz; Zhong, Dongping

    2015-06-01

    The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. Here, we analyse the primary reaction steps of repair of ultraviolet-damaged DNA by photolyase using femtosecond spectroscopy. With systematic mutations of the amino acids involved in binding of the flavin cofactor and the cyclobutane pyrimidine dimer substrate, we report our direct deconvolution of the catalytic dynamics with three electron-transfer and two bond-breaking elementary steps and thus the fine tuning of the biological repair function for optimal efficiency. We found that the maximum repair efficiency is not enhanced by the ultrafast photoinduced process but achieved by the synergistic optimization of all steps in the complex repair reaction.

  14. Impact of Alternative DNA Structures on DNA Damage, DNA Repair, and Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2014-01-01

    Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability. PMID:24767258

  15. Differential modulation of base excision repair activities during brain ontogeny: implications for repair of transcribed DNA.

    PubMed

    Englander, Ella W; Ma, Huaxian

    2006-01-01

    DNA repair sustains fidelity of genomic replication in proliferating cells and integrity of transcribed sequences in postmitotic tissues. The repair process is critical in the brain, because high oxygen consumption exacerbates the risk for accumulation of oxidative DNA lesions in postmitotic neurons. Most oxidative DNA damage is repaired by the base excision repair (BER) pathway, which is initiated by specialized DNA glycosylases. Because the newly discovered Nei-like mammalian DNA glycosylases (NEIL1/2) proficiently excise oxidized bases from bubble structured DNA, it was suggested that NEILs favor repair of transcribed or replicated DNA. In addition, since NEILs generate 3'-phosphate termini, which are poor targets for AP endonuclease (APE1), it was proposed that APE1-dependent and independent BER sub-pathways exist in mammalian cells. We measured expression and activities of BER enzymes during brain ontogeny, i.e., during a physiologic transition from proliferative to postmitotic differentiated state. While a subset of BER enzymes, exhibited declining expression and excision activities, expression of NEIL1 and NEIL2 glycosylases increased during brain development. Furthermore, the capacity for excision of 5-hydroxyuracil from bubble structured DNA was retained in the mature rat brain suggesting a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in postmitotic brain. PMID:16257035

  16. Molecular regulation of UV-induced DNA repair.

    PubMed

    Shah, Palak; He, Yu-Ying

    2015-01-01

    Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors. PMID:25534312

  17. Molecular Regulation of UV-Induced DNA Repair

    PubMed Central

    Shah, Palak; He, Yu-Ying

    2014-01-01

    Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the U.S., as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors. PMID:25534312

  18. p53 in the DNA-Damage-Repair Process.

    PubMed

    Williams, Ashley B; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability. PMID:27048304

  19. Modeling Damage Complexity-Dependent Non-Homologous End-Joining Repair Pathway

    PubMed Central

    Li, Yongfeng; Reynolds, Pamela; O'Neill, Peter; Cucinotta, Francis A.

    2014-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF) model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET) radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed. PMID:24520318

  20. Modeling damage complexity-dependent non-homologous end-joining repair pathway.

    PubMed

    Li, Yongfeng; Reynolds, Pamela; O'Neill, Peter; Cucinotta, Francis A

    2014-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF) model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET) radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed. PMID:24520318

  1. 8-oxoguanine DNA glycosylase-1 driven DNA base excision repair: role in asthma pathogenesis

    PubMed Central

    Ba, Xueqing; Aguilera Aguirre, Leopoldo; Sur, Sanjiv; Boldogh, Istvan

    2015-01-01

    Purpose of review To provide both an overview and evidence of the potential etiology of oxidative DNA base damage and repair-signaling in chronic inflammation and histological changes associated with asthma. Recent findings Asthma is initiated/maintained by immunological, genetic/epigenetic and environmental factors. It is a world-wide health problem, as current therapies suppress symptoms rather than prevent/reverse the disease, largely due to gaps in understanding its molecular mechanisms. Inflammation, oxidative stress and DNA damage are inseparable phenomena, but their molecular roles in asthma pathogenesis are unclear. It was found that among oxidatively modified DNA bases, 8-oxoguanine (8-oxoG) is one of the most abundant, and its levels in DNA and body fluids are considered a biomarker of ongoing asthmatic processes. Free 8-oxoG forms a complex with 8-oxoguanine DNA glycosylase-1 (OGG1) and activates RAS-family GTPases that induce gene expression to mobilize innate and adaptive immune systems, along with genes regulating airway hyperplasia, hyper-responsiveness and lung remodeling in atopic and non-atopic asthma. Summary DNA’s integrity must be maintained to prevent mutation, so its continuous repair and downstream signaling “fuels” chronic inflammatory processes in asthma, and forms the basic mechanism whose elucidation will allow the development of new drug targets for the prevention/reversal of lung diseases. PMID:25486379

  2. MCM9 Is Required for Mammalian DNA Mismatch Repair.

    PubMed

    Traver, Sabine; Coulombe, Philippe; Peiffer, Isabelle; Hutchins, James R A; Kitzmann, Magali; Latreille, Daniel; Méchali, Marcel

    2015-09-01

    DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9-/- cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9-/- cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR. PMID:26300262

  3. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

    PubMed Central

    Morales, Maria E.; Derbes, Rebecca S.; Ade, Catherine M.; Ortego, Jonathan C.; Stark, Jeremy; Deininger, Prescott L.; Roy-Engel, Astrid M.

    2016-01-01

    Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair. PMID:26966913

  4. Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair.

    PubMed Central

    Dolganov, G M; Maser, R S; Novikov, A; Tosto, L; Chong, S; Bressan, D A; Petrini, J H

    1996-01-01

    In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells. PMID:8756642

  5. Purification of mammalian DNA repair protein XRCC1

    SciTech Connect

    Chen, I.

    1995-11-01

    Malfunctioning DNA repair systems lead to cancer mutations, and cell death. XRCC1 (X-ray Repair Cross Complementing) is a human DNA repair gene that has been found to fully correct the x-ray repair defect in Chinese hamster ovary (CHO) cell mutant EM9. The corresponding protein (XRCC1) encoded by this gene has been linked to a DNA repair pathway known as base excision repair, and affects the activity of DNA ligase III. Previously, an XRCC1 cDNA minigene (consisting of the uninterrupted coding sequence for XRCC1 protein followed by a decahistidine tag) was constructed and cloned into vector pET-16b for the purpose of: (1) overproduction of XRCC1 in both prokaryotic and eukaryotic cells; and (2) to facilitate rapid purification of XRCC1 from these systems. A vector is basically a DNA carrier that allows recombinant protein to be cloned and overexpressed in host cells. In this study, XRCC1 protein was overexpressed in E. coli and purified by immobilized metal affinity chromatography. Currently, the XRCC1 minigene is being inserted into a new vector [pET-26b(+)] in hopes to increase overexpression and improve purification. Once purified XRCC1 can be crystallized for structural studies, or studied in vitro for its biological function.

  6. DNA repair proficiency: A potential susceptibility factor for breast cancer

    SciTech Connect

    Helzlsouer, K.J.; Perry, H.; Harris, E.L. |

    1994-09-01

    A family study and a case-control study were conducted to examine the association between sub-optimal repair of ionizing radiation induced DNA damage and the development of breast cancer. A familial cluster of breast cancer was investigated in which breast cancer occurred in 4 of 6 sisters, some of whom were exposed to ionizing radiation from repeated chest fluoroscopic examinations during adolescence and early adulthood. DNA repair proficiency was measured among available family members and correlated with their history of radiation exposure. DNA repair proficiency was also measured among 16 breast cancer cases, 5 women with a family history of breast cancer and 12 controls. The results of the family study suggest an association between poor DNA repair proficiency and increased sensitivity to the carcinogenic effects of early radiation exposure on breast tissue. The case-control study showed that a significantly higher percentage of women with breast cancer (63%) and women with a family history of breast cancer (80%) had poor repair of ionizing radiation induced DNA damage than control women (17%) (P-value=0.02). Sub-optimal repair of DNA damage may be a host susceptibility factor predisposing individuals to breast cancer through increased sensitivity to carcinogenic damage from environmental exposures such as ionizing radiation.

  7. New approaches to biochemical radioprotection: antioxidants and DNA repair enhancement

    NASA Astrophysics Data System (ADS)

    Riklis, E.; Emerit, I.; Setlow, R. B.

    Chemical repair may be provided by radioprotective compounds present during exposure to ionizing radiation. Considering DNA as the most sensitive target it is feasible to biochemically improve protection by enhancing DNA repair mechanisms. Protection of DNA by reducing the amount of damage (by radical scavenging and chemical repair) followed by enhanced repair of DNA will provide much improved protection and recovery. Furthermore, in cases of prolonged exposure, such as is possible in prolonged space missions, or of unexpected variations in the intensity of radiation, as is possible when encountering solar flares, it is important to provide long-acting protection, and this may be provided by antioxidants and well functioning DNA repair systems. It has also become important to provide protection from the potentially damaging action of long-lived clastogenic factors which have been found in plasma of exposed persons from Hiroshima & Nagasaki, radiation accidents, radiotherapy patients and recently in ``liquidators'' - persons involved in salvage operations at the Chernobyl reactor. The clastogenic factor, which causes chromatid breaks in non-exposed plasma, might account for late effects and is posing a potential carcinogenic hazard /1/. The enzyme superoxide dismutase (SOD) has been shown to eliminate the breakage factor from cultured plasma of exposed persons /2/. Several compounds have been shown to enhance DNA repair: WR-2721 /3/, nicotinamide /4/, glutathione monoester (Riklis et al., unpublished) and others. The right combination of such compounds may prove effective in providing protection from a wide range of radiation exposures over a long period of time.

  8. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

    PubMed

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-03-18

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  9. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  10. Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

    PubMed

    Croset, Amelie; Cordelières, Fabrice P; Berthault, Nathalie; Buhler, Cyril; Sun, Jian-Sheng; Quanz, Maria; Dutreix, Marie

    2013-08-01

    One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. PMID:23761435

  11. MicroRNAs in the DNA Damage/Repair Network and Cancer

    PubMed Central

    Tessitore, Alessandra; Cicciarelli, Germana; Del Vecchio, Filippo; Gaggiano, Agata; Verzella, Daniela; Fischietti, Mariafausta; Vecchiotti, Davide; Capece, Daria; Zazzeroni, Francesca; Alesse, Edoardo

    2014-01-01

    Cancer is a multistep process characterized by various and different genetic lesions which cause the transformation of normal cells into tumor cells. To preserve the genomic integrity, eukaryotic cells need a complex DNA damage/repair response network of signaling pathways, involving many proteins, able to induce cell cycle arrest, apoptosis, or DNA repair. Chemotherapy and/or radiation therapy are the most commonly used therapeutic approaches to manage cancer and act mainly through the induction of DNA damage. Impairment in the DNA repair proteins, which physiologically protect cells from persistent DNA injury, can affect the efficacy of cancer therapies. Recently, increasing evidence has suggested that microRNAs take actively part in the regulation of the DNA damage/repair network. MicroRNAs are endogenous short noncoding molecules able to regulate gene expression at the post-transcriptional level. Due to their activity, microRNAs play a role in many fundamental physiological and pathological processes. In this review we report and discuss the role of microRNAs in the DNA damage/repair and cancer. PMID:24616890

  12. DNA repair mechanisms in dividing and non-dividing cells

    PubMed Central

    Iyama, Teruaki; Wilson, David M.

    2013-01-01

    DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye towards how these pathways may regulate the development of neurological disease. PMID:23684800

  13. DNA damage and repair in human skin in situ

    SciTech Connect

    Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

    1987-01-01

    Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

  14. An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

    PubMed

    Kowalczykowski, Stephen C

    2015-11-01

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

  15. Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis.

    PubMed

    Corcoran, Niall M; Clarkson, Michael J; Stuchbery, Ryan; Hovens, Christopher M

    2016-07-01

    The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage repair (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate, however, that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family. Targeting of these pathways, especially through PARP inhibition, is now being exploited therapeutically to effect significant clinical responses in subsets of individuals, particularly in patients with ovarian cancer or prostate cancer, including cancers with a marked metastatic burden. Targeting DNA repair-deficient tumors with drugs that take advantage of the fundamental differences between normal repair-proficient cells and repair-deficient tumors offers new avenues for treating advanced disease in the future. Clin Cancer Res; 22(13); 3132-7. ©2016 AACR. PMID:27169997

  16. DNA repair: a changing geography? (1964-2008).

    PubMed

    Maisonobe, Marion; Giglia-Mari, Giuseppina; Eckert, Denis

    2013-07-01

    This article aims to explain the current state of DNA Repair studies' global geography by focusing on the genesis of the community. Bibliometric data is used to localize scientific activities related to DNA Repair at the city level. The keyword "DNA Repair" was introduced first by American scientists. It started to spread after 1964 that is to say, after P. Howard-Flanders (Yale University), P. Hanawalt (Stanford University) and R. Setlow (Oak Ridge Laboratories) found evidence for Excision Repair mechanisms. It was the first stage in the emergence of an autonomous scientific community. In this article, we will try to assess to what extent the geo-history of this scientific field is determinant in understanding its current geography. In order to do so, we will localize the places where the first "DNA Repair" publications were signed fifty years ago and the following spatial diffusion process, which led to the current geography of the field. Then, we will focus on the evolution of the research activity of "early entrants" in relation to the activity of "latecomers". This article is an opportunity to share with DNA Repair scientists some research results of a dynamic field in Science studies: spatial scientometrics. PMID:23669398

  17. Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

    PubMed Central

    Mehta, Anuja; Haber, James E.

    2014-01-01

    DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination. PMID:25104768

  18. Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

    PubMed

    Zhang, Yaru; O'Brien, Patrick J

    2015-11-20

    Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein. PMID:26317160

  19. Exploiting DNA repair defects for novel cancer therapies.

    PubMed

    van Gent, Dik C; Kanaar, Roland

    2016-07-15

    Most human tumors accumulate a multitude of genetic changes due to defects in the DNA damage response. Recently, small-molecule inhibitors have been developed that target cells with specific DNA repair defects, providing hope for precision treatment of such tumors. Here we discuss the rationale behind these therapies and how an important bottleneck-patient selection-can be approached. PMID:27418635

  20. Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair

    PubMed Central

    Tahbaz, Nasser; Subedi, Sudip; Weinfeld, Michael

    2012-01-01

    Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H2O2-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria. PMID:22210862

  1. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period.

  2. ABH2 couples regulation of ribosomal DNA transcription with DNA alkylation repair.

    PubMed

    Li, Pishun; Gao, Shuman; Wang, Lina; Yu, Fang; Li, Jialun; Wang, Chuangui; Li, Jiwen; Wong, Jiemin

    2013-08-29

    Transcription has been linked to DNA damage. How the most highly transcribed mammalian ribosomal (rDNA) genes maintain genome integrity in the absence of transcription-coupled DNA damage repair is poorly understood. Here, we report that ABH2/ALKBH2, a DNA alkylation repair enzyme, is highly enriched in the nucleolus. ABH2 interacts with DNA repair proteins Ku70 and Ku80 as well as nucleolar proteins nucleolin, nucleophosmin 1, and upstream binding factor (UBF). ABH2 associates with and promotes rDNA transcription through its DNA repair activity. ABH2 knockdown impairs rDNA transcription and leads to increased single-stranded and double-stranded DNA breaks that are more pronounced in the rDNA genes, whereas ABH2 overexpression protects cells from methyl-methanesulfonate-induced DNA damage and inhibition of rDNA transcription. In response to massive alkylation damage, ABH2 rapidly redistributes from the nucleolus to nucleoplasm. Our study thus reveals a critical role of ABH2 in maintaining rDNA gene integrity and transcription and provides insight into the ABH2 DNA repair function. PMID:23972994

  3. Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

    PubMed Central

    Blasiak, Janusz; Synowiec, Ewelina; Salminen, Antero; Kaarniranta, Kai

    2012-01-01

    The pathogenesis of age-related macular degeneration (AMD) is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133) in the hOGG1 gene and the c.972G>C polymorphism (rs3219489) in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395) and c.–32A>G (rs3087404) polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2), XRCC1 and ERCC6 (CSB) have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis. PMID:23202958

  4. RNF4 is required for DNA double-strand break repair in vivo.

    PubMed

    Vyas, R; Kumar, R; Clermont, F; Helfricht, A; Kalev, P; Sotiropoulou, P; Hendriks, I A; Radaelli, E; Hochepied, T; Blanpain, C; Sablina, A; van Attikum, H; Olsen, J V; Jochemsen, A G; Vertegaal, A C O; Marine, J-C

    2013-03-01

    Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events. PMID:23197296

  5. Interplay of DNA repair with transcription: from structures to mechanisms

    PubMed Central

    Deaconescu, Alexandra M.; Artsimovitch, Irina; Grigorieff, Nikolaus

    2013-01-01

    Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions. PMID:23084398

  6. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen. PMID:26527082

  7. The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

    PubMed

    Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

    2011-03-01

    The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the

  8. Molecular basis for DNA strand displacement by NHEJ repair polymerases

    PubMed Central

    Bartlett, Edward J.; Brissett, Nigel C.; Plocinski, Przemyslaw; Carlberg, Tom; Doherty, Aidan J.

    2016-01-01

    The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair. PMID:26405198

  9. Molecular basis for DNA strand displacement by NHEJ repair polymerases.

    PubMed

    Bartlett, Edward J; Brissett, Nigel C; Plocinski, Przemyslaw; Carlberg, Tom; Doherty, Aidan J

    2016-03-18

    The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair. PMID:26405198

  10. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  11. Dynamics and mechanisms of DNA repair by photolyase

    PubMed Central

    Liu, Zheyun; Wang, Lijuan; Zhong, Dongping

    2015-01-01

    Photolyase, a class of flavoproteins, uses blue light to repair two types of ultraviolet-induced DNA damage, cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6–4) photoproduct (6–4PP). In this perspective, we review the recent progress on the repair dynamics and mechanisms of both types of DNA restoration by photolyases. We first report the spectroscopic characterization of flavin in various redox states and the active-site solvation dynamics in photolyases. We then systematically summarize the detailed repair dynamics of damaged DNA by photolyases and a biomimetic system through resolving all elementary steps on the ultrafast timescales, including multiple intermolecular electron- and proton-transfer reactions and bond-breaking and -making processes. We determined the unique electron tunneling pathways, identified the key functional residues and revealed the molecular origin of high repair efficiency, and thus elucidate the molecular mechanisms and repair photocycles at the most fundamental level. We finally conclude that the active sites of photolyases, unlike aqueous solution for the biomimetic system, provide a unique electrostatic environment and local flexibility and thus a dedicated synergy for all elementary dynamics to maximize the repair efficiency. This repair photomachine is the first enzyme that the entire functional evolution is completely mapped out in real time. PMID:25870862

  12. Electron Transfer Mechanisms of DNA Repair by Photolyase

    NASA Astrophysics Data System (ADS)

    Zhong, Dongping

    2015-04-01

    Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

  13. Effect of Carcinogenic Acrolein on DNA Repair and Mutagenic Susceptibility*

    PubMed Central

    Wang, Hsiang-Tsui; Hu, Yu; Tong, Dan; Huang, Jian; Gu, Liya; Wu, Xue-Ru; Chung, Fung-Lung; Li, Guo-Min; Tang, Moon-shong

    2012-01-01

    Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N2-cyclic propano-2′-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity. PMID:22275365

  14. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    PubMed Central

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness. PMID:27446204

  15. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

    PubMed

    Svendsen, Jennifer M; Smogorzewska, Agata; Sowa, Mathew E; O'Connell, Brenda C; Gygi, Steven P; Elledge, Stephen J; Harper, J Wade

    2009-07-10

    Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3' flap, 5' flap, and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular toolkit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure. PMID:19596235

  16. A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair.

    PubMed

    Fujii, Naoaki; Evison, Benjamin J; Actis, Marcelo L; Inoue, Akira

    2015-11-01

    Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. PMID:26462050

  17. The current state of eukaryotic DNA base damage and repair.

    PubMed

    Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2015-12-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

  18. The current state of eukaryotic DNA base damage and repair

    PubMed Central

    Bauer, Nicholas C.; Corbett, Anita H.; Doetsch, Paul W.

    2015-01-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

  19. Repair of mismatched basepairs in mammalian DNA

    SciTech Connect

    Taylor, J.H.; Hare, J.T.

    1991-08-01

    We have concentrated on three specific areas of our research plan. Our greatest emphasis is on the role of single strand nicks in influencing template strand selection in mismatch repair. We have found, that the ability of a nick in one strand to influence which strand is repaired is not a simple function of distance from the mismatched site but rather that an hot spot where a nick is more likely to have an influence can exist. The second line was production of single-genotype heteroduplexes in order to examine independently the repair of T/G and A/C mispairs within the same sequence context as in our mixed mispair preparations. We have shown preparations of supercoiled heteroduplex can be prepared that were exclusively T/G or exclusively A/C at the mispair site. The third effort has been to understand the difference in repair bias of different cell lines or different transfection conditions as it may relate to different repair systems in the cell. We have identified some of the sources of variation, including cell cycle position. We hope to continue this work to more precisely identify the phase of the cell cycle.

  20. Biomarkers of oxidative damage to DNA and repair.

    PubMed

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone; Risom, Lotte; Forchhammer, Lykke; Møller, Peter

    2008-10-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease. PMID:18793191

  1. Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair.

    PubMed

    Goldstein, Michael; Derheimer, Frederick A; Tait-Mulder, Jacqueline; Kastan, Michael B

    2013-10-15

    Recruitment of DNA repair factors and modulation of chromatin structure at sites of DNA double-strand breaks (DSBs) is a complex and highly orchestrated process. We developed a system that can induce DSBs rapidly at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Distinct advantages of this system over focus formation/disappearance assays for assessing DSB repair are demonstrated. Using ChIP, we found that nucleosomes are partially disassembled around DSBs during nonhomologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its arginine-glycine rich domain and is recruited to DSBs rapidly in an MRE11-NBS1-RAD50 complex-dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and the repair of the DSB, demonstrating the functional importance of nucleosome disruption in DSB repair and identifying a chromatin-remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for recruitment of replication protein A to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair. PMID:24082117

  2. Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2014-01-01

    Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

  3. The ancient and evolving roles of cohesin in DNA repair and gene expression

    PubMed Central

    Dorsett, Dale; Ström, Lena

    2012-01-01

    The cohesin complex, named for its key role in sister chromatid cohesion, also plays critical roles in DNA repair and gene regulation. It performs all three functions in single cell eukaryotes such as yeasts, and in higher organisms such as man. Minor disruption of cohesin function has significant consequences for human development, even in the absence of measurable effects on chromatid cohesion or chromosome segregation. Here we survey the roles of cohesin in DNA repair and gene regulation, and how these functions vary from yeast to man. PMID:22497943

  4. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.

    PubMed

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-05-23

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability. PMID:27211488

  5. Heterochromatin formation via recruitment of DNA repair proteins

    PubMed Central

    Kirkland, Jacob G.; Peterson, Misty R.; Still, Christopher D.; Brueggeman, Leo; Dhillon, Namrita; Kamakaka, Rohinton T.

    2015-01-01

    Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein–mediated silencing—via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity. PMID:25631822

  6. UvrD facilitates DNA repair by pulling RNA polymerase backwards.

    PubMed

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-16

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes. PMID:24402227

  7. DNA Double-Strand Break Repair at −15°C

    PubMed Central

    Dieser, Markus; Battista, John R.

    2013-01-01

    The survival of microorganisms in ancient glacial ice and permafrost has been ascribed to their ability to persist in a dormant, metabolically inert state. An alternative possibility, supported by experimental data, is that microorganisms in frozen matrices are able to sustain a level of metabolic function that is sufficient for cellular repair and maintenance. To examine this experimentally, frozen populations of Psychrobacter arcticus 273-4 were exposed to ionizing radiation (IR) to simulate the damage incurred from natural background IR sources in the permafrost environment from over ∼225 kiloyears (ky). High-molecular-weight DNA was fragmented by exposure to 450 Gy of IR, which introduced an average of 16 double-strand breaks (DSBs) per chromosome. During incubation at −15°C for 505 days, P. arcticus repaired DNA DSBs in the absence of net growth. Based on the time frame for the assembly of genomic fragments by P. arcticus, the rate of DNA DSB repair was estimated at 7 to 10 DSBs year−1 under the conditions tested. Our results provide direct evidence for the repair of DNA lesions, extending the range of complex biochemical reactions known to occur in bacteria at frozen temperatures. Provided that sufficient energy and nutrient sources are available, a functional DNA repair mechanism would allow cells to maintain genome integrity and augment microbial survival in icy terrestrial or extraterrestrial environments. PMID:24077718

  8. Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

    PubMed Central

    Tagua, Victor G.; Pausch, Marcell; Eckel, Maike; Gutiérrez, Gabriel; Miralles-Durán, Alejandro; Sanz, Catalina; Eslava, Arturo P.; Pokorny, Richard; Corrochano, Luis M.; Batschauer, Alfred

    2015-01-01

    DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B–induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi. PMID:26578805

  9. Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution.

    PubMed

    Tagua, Victor G; Pausch, Marcell; Eckel, Maike; Gutiérrez, Gabriel; Miralles-Durán, Alejandro; Sanz, Catalina; Eslava, Arturo P; Pokorny, Richard; Corrochano, Luis M; Batschauer, Alfred

    2015-12-01

    DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi. PMID:26578805

  10. DNA repair mechanisms and their clinical impact in glioblastoma.

    PubMed

    Erasimus, Hélène; Gobin, Matthieu; Niclou, Simone; Van Dyck, Eric

    2016-01-01

    Despite surgical resection and genotoxic treatment with ionizing radiation and the DNA alkylating agent temozolomide, glioblastoma remains one of the most lethal cancers, due in great part to the action of DNA repair mechanisms that drive resistance and tumor relapse. Understanding the molecular details of these mechanisms and identifying potential pharmacological targets have emerged as vital tasks to improve treatment. In this review, we introduce the various cellular systems and animal models that are used in studies of DNA repair in glioblastoma. We summarize recent progress in our knowledge of the pathways and factors involved in the removal of DNA lesions induced by ionizing radiation and temozolomide. We introduce the therapeutic strategies relying on DNA repair inhibitors that are currently being tested in vitro or in clinical trials, and present the challenges raised by drug delivery across the blood brain barrier as well as new opportunities in this field. Finally, we review the genetic and epigenetic alterations that help shape the DNA repair makeup of glioblastoma cells, and discuss their potential therapeutic impact and implications for personalized therapy. PMID:27543314

  11. Assembling an orchestra: Fanconi anemia pathway of DNA repair.

    PubMed

    Yuan, Fenghua; Song, Limin; Qian, Liangyue; Hu, Jennifer J; Zhang, Yanbin

    2010-01-01

    Fanconi anemia (FA) is a recessive genetic disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. The complete set of FA genes has only been identified recently and seems to be uniquely conserved among vertebrates. Fanconi anemia proteins have been implicated in the repair of interstrand DNA crosslinks that block DNA replication and transcription. Although all thirteen FA complementation groups show similar clinical and cellular phenotypes, approximately 85% of patients presented defective FANCA, FANCC, or FANCG. The established DNA interacting components (FANCM, FANCI, FANCD2, and FANCJ) account only for approximately 5% of all FA patients, an observation that raises doubt concerning the roles of FA proteins in DNA repair. In recent years, rapid progress in the area of FA research has provided great insights into the critical roles of FA proteins in DNA repair. However, many FA proteins do not have identifiable domains to indicate how they contribute to biological processes, particularly DNA repair. Therefore, future biochemical studies are warranted to understand the biological functions of FA proteins and their implications in human diseases. PMID:20515746

  12. MicroRNA expression and its association with DNA repair in preimplantation embryos

    PubMed Central

    TULAY, Pinar; SENGUPTA, Sioban B.

    2016-01-01

    Active DNA repair pathways are crucial for preserving genomic integrity and are likely among the complex mechanisms involved in the normal development of preimplantation embryos. MicroRNAs (miRNA), short non-coding RNAs, are key regulators of gene expression through the post-transcriptional and post-translational modification of mRNA. The association of miRNA expression with infertility or polycystic ovarian syndrome has been widely investigated; however, there are limited data regarding the importance of miRNA regulation in DNA repair during preimplantation embryo development. In this article, we review normal miRNA biogenesis and consequences of aberrant miRNA expression in the regulation of DNA repair in gametes and preimplantation embryos. PMID:26853522

  13. DNA/chitosan electrostatic complex.

    PubMed

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80). PMID:27050113

  14. Exercise as Gene Therapy: BDNF and DNA Damage Repair.

    PubMed

    Schmidt, Robin H; Nickerson, John M; Boatright, Jeffrey H

    2016-01-01

    DNA damage is a common feature of neurodegenerative illnesses, and the ability to repair DNA strand breaks and lesions is crucial for neuronal survival, reported by Jeppesen et al (Prog Neurobiol. 2011;94:166-200) and Shiwaku et al (Curr Mol Med. 2015;15:119-128). Interventions aimed at repairing these lesions, therefore, could be useful for preventing or delaying the progression of disease. One potential strategy for promoting DNA damage repair (DDR) is exercise. Although the role of exercise in DDR is not understood, there is increasing evidence that simple physical activity may impact clinical outcomes for neurodegeneration. Here, we discuss what is currently known about the molecular mechanisms of brain-derived neurotrophic factor and how these mechanisms might influence the DDR process. PMID:27488073

  15. Curcumin Triggers DNA Damage and Inhibits Expression of DNA Repair Proteins in Human Lung Cancer Cells.

    PubMed

    Ting, Chien-Yi; Wang, Hsin-Ell; Yu, Chien-Chih; Liu, Hsin-Chung; Liu, Yu-Chang; Chiang, I-Tsang

    2015-07-01

    The study goal was to evaluate the effects of curcumin on DNA damage and expression of DNA-repair proteins in human lung cancer. Thus, NCI-H460 cells were used to study the effects of curcumin on DNA damage and repair in vitro. We investigated curcumin induces DNA damage by comet the assay and 4',6-diamidino-2-phenylindole (DAPI) staining. The DNA damage/repair-related protein levels were examined and monitored by western blotting and confocal microscopy. Curcumin significantly increased the length of comet tails and DNA condensation in NCI-H460 cells. Curcumin reduced expression of DNA-repair proteins such as 14-3-3 protein sigma (14-3-3σ), O6-methylguanine-DNA methyltransferase (MGMT), breast cancer susceptibility gene 1 (BRCA1), and mediator of DNA damage checkpoint 1 (MDC1). Curcumin also increased phosphorylation of p53 and Histone H2A.X (S140) in the nuclei of NCI-H460 cells. Taken together, our findings indicated that curcumin triggered DNA damage and inhibited expression of DNA-repair-associated proteins in NCI-H460 cells. PMID:26124332

  16. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.

    PubMed

    Grin, Inga; Ishchenko, Alexander A

    2016-05-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  17. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  18. Polymorphisms in DNA repair genes, smoking, and pancreatic adenocarcinoma risk.

    PubMed

    McWilliams, Robert R; Bamlet, William R; Cunningham, Julie M; Goode, Ellen L; de Andrade, Mariza; Boardman, Lisa A; Petersen, Gloria M

    2008-06-15

    Base excision repair and nucleotide excision repair are vital responses to multiple types of DNA damage, including damage from tobacco exposure. Single-nucleotide polymorphisms (SNP) in these pathways may affect DNA repair capacity and therefore influence risk for cancer development. We performed a clinic-based, case-control study comprising 481 consecutive patients with confirmed pancreatic adenocarcinoma and 625 healthy controls. Allele and genotype frequencies for 16 SNPs in DNA repair genes ERCC1, XPD/ERCC2, XPC, XPF/ERCC4, OGG1, and XRCC1 were compared after adjusting for age, sex, and smoking history. Subgroup analysis by sex and smoking history was performed. Carriers of one or two XPF/ERCC4 minor alleles at R415Q had decreased risk of pancreatic adenocarcinoma compared with those who had two major alleles [odds ratio (OR), 0.59; 95% confidence interval (95% CI), 0.40-0.85]. Heavy smokers (>40 pack-years) had increased risk for cancer if they were carriers of at least one minor allele for XPD/ERCC2 at D312N (OR, 2.78; 95% CI, 1.28-6.04) or D711D (OR, 2.19; 95% CI, 1.01-4.73). No other significant differences in risk were identified. Minor alleles in DNA repair genes XPF/ERCC4 and XPD/ERCC2 were associated with altered risk for pancreatic cancer. PMID:18544627

  19. Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

    PubMed

    Nambiar, Dhanya K; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K; Agarwal, Rajesh; Singh, Rana P

    2015-12-01

    Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. PMID:26516160

  20. A chiroptical photoswitchable DNA complex.

    PubMed

    Mammana, Angela; Carroll, Gregory T; Areephong, Jetsuda; Feringa, Ben L

    2011-10-13

    The interesting structural, electronic, and optical properties of DNA provide fascinating opportunities for developing nanoscale smart materials by integrating DNA with opto-electronic components. In this article we demonstrate the electrostatic binding of an amine-terminated dithienylethene (DET) molecular switch to double-stranded synthetic polynucleotides. The DET switch can undergo photochemical ring-closure and opening reactions. Circular dichroism (CD) and UV-vis spectroscopy show that both the open, 1o, and the closed, 1c, forms of the switch bind to DNA. Upon addition of DNA to a solution of 1o or 1c, the UV-vis spectrum displays a hypochromic effect, indicative of an interaction between the switch and the DNA. The chirality of the DNA double-helix is transmitted to the switching unit which displays a well-defined CD signal upon supramolecular complexation to the DNA. Additionally, the CD signal of the DNA attenuates, demonstrating that both components of the complex mutually influence each other's structure; the DNA induces chirality in the switch, and the switch modifies the structure of the DNA. Modulation of the chiroptical properties of the complex is achieved by photochemically switching the DET between its ring open and closed isomers. A pH dependence study of the binding shows that when the pH is increased the switches lose their binding ability, indicating that electrostatic interactions between protonated amines and the negatively charged phosphate backbone are the dominant driving force for binding to the DNA. A comparison of poly(deoxyguanylic-deoxycytidylic) acid [poly(dGdC)(2)] polynucleotides with poly(deoxyadenylic-deoxythymidylic) acid [poly(dAdT)(2)] shows distinct differences in the CD spectra of the complexes. PMID:21879715

  1. Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity.

    PubMed

    Slyskova, Jana; Lorenzo, Yolanda; Karlsen, Anette; Carlsen, Monica H; Novosadova, Vendula; Blomhoff, Rune; Vodicka, Pavel; Collins, Andrew R

    2014-04-01

    The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity. Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways. Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p=0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by β-cryptoxanthin and β-tocopherol (p<0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p=0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p<0.05). We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity. PMID:24674629

  2. Dynamic control of strand excision during human DNA mismatch repair

    PubMed Central

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V.; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-01-01

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3′- or 5′-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2–HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1–HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2–HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1–HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR. PMID:26951673

  3. Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus

    SciTech Connect

    Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

    2005-01-01

    Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5α E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  4. Torin2 Suppresses Ionizing Radiation-Induced DNA Damage Repair.

    PubMed

    Udayakumar, Durga; Pandita, Raj K; Horikoshi, Nobuo; Liu, Yan; Liu, Qingsong; Wong, Kwok-Kin; Hunt, Clayton R; Gray, Nathanael S; Minna, John D; Pandita, Tej K; Westover, Kenneth D

    2016-05-01

    Several classes of inhibitors of the mammalian target of rapamycin (mTOR) have been developed based on its central role in sensing growth factor and nutrient levels to regulate cellular metabolism. However, its ATP-binding site closely resembles other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, resulting in reactivity with these targets that may also be therapeutically useful. The ATP-competitive mTOR inhibitor, Torin2, shows biochemical activity against the DNA repair-associated proteins ATM, ATR and DNA-PK, which raises the possibility that Torin2 and related compounds might radiosensitize cancerous tumors. In this study Torin2 was also found to enhance ionizing radiation-induced cell killing in conditions where ATM was dispensable, confirming the requirement for multiple PIKK targets. Moreover, Torin2 did not influence the initial appearance of γ-H2AX foci after irradiation but significantly delayed the disappearance of radiation-induced γ-H2AX foci, indicating a DNA repair defect. Torin2 increased the number of radiation-induced S-phase specific chromosome aberrations and reduced the frequency of radiation-induced CtIP and Rad51 foci formation, suggesting that Torin2 works by blocking homologous recombination (HR)-mediated DNA repair resulting in an S-phase specific DNA repair defect. Accordingly, Torin2 reduced HR-mediated repair of I-Sce1-induced DNA damage and contributed to replication fork stalling. We conclude that radiosensitization of tumor cells by Torin2 is associated with disrupting ATR- and ATM-dependent DNA damage responses. Our findings support the concept of developing combination cancer therapies that incorporate ionizing radiation therapy and Torin2 or compounds with similar properties. PMID:27135971

  5. Particulate matter inhibits DNA repair and enhances mutagenesis.

    PubMed

    Mehta, Manju; Chen, Lung-Chi; Gordon, Terry; Rom, William; Tang, Moon-Shong

    2008-12-01

    Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 microg/m3 elevation in long-term exposure to average PM2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors. PMID:18804180

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

    PubMed

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

    2016-01-01

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy. PMID:27589807

  7. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  8. Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

    PubMed

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  9. Chromosome Synapsis Alleviates Mek1-Dependent Suppression of Meiotic DNA Repair

    PubMed Central

    Subramanian, Vijayalakshmi V.; MacQueen, Amy J.; Vader, Gerben; Shinohara, Miki; Sanchez, Aurore; Borde, Valérie; Shinohara, Akira; Hochwagen, Andreas

    2016-01-01

    Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired (synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA+-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression. PMID:26870961

  10. Breaking bad: The mutagenic effect of DNA repair.

    PubMed

    Chen, Jia; Furano, Anthony V

    2015-08-01

    Species survival depends on the faithful replication of genetic information, which is continually monitored and maintained by DNA repair pathways that correct replication errors and the thousands of lesions that arise daily from the inherent chemical lability of DNA and the effects of genotoxic agents. Nonetheless, neutrally evolving DNA (not under purifying selection) accumulates base substitutions with time (the neutral mutation rate). Thus, repair processes are not 100% efficient. The neutral mutation rate varies both between and within chromosomes. For example it is 10-50 fold higher at CpGs than at non-CpG positions. Interestingly, the neutral mutation rate at non-CpG sites is positively correlated with CpG content. Although the basis of this correlation was not immediately apparent, some bioinformatic results were consistent with the induction of non-CpG mutations by DNA repair at flanking CpG sites. Recent studies with a model system showed that in vivo repair of preformed lesions (mismatches, abasic sites, single stranded nicks) can in fact induce mutations in flanking DNA. Mismatch repair (MMR) is an essential component for repair-induced mutations, which can occur as distant as 5 kb from the introduced lesions. Most, but not all, mutations involved the C of TpCpN (G of NpGpA) which is the target sequence of the C-preferring single-stranded DNA specific APOBEC deaminases. APOBEC-mediated mutations are not limited to our model system: Recent studies by others showed that some tumors harbor mutations with the same signature, as can intermediates in RNA-guided endonuclease-mediated genome editing. APOBEC deaminases participate in normal physiological functions such as generating mutations that inactivate viruses or endogenous retrotransposons, or that enhance immunoglobulin diversity in B cells. The recruitment of normally physiological error-prone processes during DNA repair would have important implications for disease, aging and evolution. This perspective

  11. 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

  12. Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.

    PubMed

    Bellacosa, A

    2001-11-01

    The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies. PMID:11687886

  13. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    SciTech Connect

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  14. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

    PubMed Central

    Mukherjee, Anirban; Vasquez, Karen M.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  15. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  16. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks

    PubMed Central

    Wyatt, Michael D.; Pittman, Douglas L.

    2008-01-01

    The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a ‘radiomimetic,’ i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired. PMID:17173371

  17. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    PubMed Central

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  18. Roles of PTEN with DNA Repair in Parkinson's Disease.

    PubMed

    Ogino, Mako; Ichimura, Mayuko; Nakano, Noriko; Minami, Akari; Kitagishi, Yasuko; Matsuda, Satoru

    2016-01-01

    Oxidative stress is considered to play key roles in aging and pathogenesis of many neurodegenerative diseases such as Parkinson's disease, which could bring DNA damage by cells. The DNA damage may lead to the cell apoptosis, which could contribute to the degeneration of neuronal tissues. Recent evidence suggests that PTEN (phosphatase and tensin homolog on chromosome 10) may be involved in the pathophysiology of the neurodegenerative disorders. Since PTEN expression appears to be one dominant determinant of the neuronal cell death, PTEN should be a potential molecular target of novel therapeutic strategies against Parkinson's disease. In addition, defects in DNA damage response and DNA repair are often associated with modulation of hormone signaling pathways. Especially, many observations imply a role for estrogen in a regulation of the DNA repair action. In the present review, we have attempted to summarize the function of DNA repair molecules at a viewpoint of the PTEN signaling pathway and the hormone related functional modulation of cells, providing a broad interpretation on the molecular mechanisms for treatment of Parkinson's disease. Particular attention will be paid to the mechanisms proposed to explain the health effects of food ingredients against Parkinson's disease related to reduce oxidative stress for an efficient therapeutic intervention. PMID:27314344

  19. Visualization of Eukaryotic DNA Mismatch Repair Reveals Distinct Recognition and Repair Intermediates

    PubMed Central

    Hombauer, Hans; Campbell, Christopher S.; Smith, Catherine E.; Desai, Arshad; Kolodner, Richard D.

    2011-01-01

    Summary DNA Mismatch Repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae mispairs are primarily detected by the Msh2-Msh6 complex and corrected following subsequent recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. Msh2-Msh6 formed foci in S-phase that colocalized with replication factories; this localized pool accounted for 10–15% of MMR in wild-type cells but was essential for MMR in the absence of the exonuclease Exo1. Mlh1-Pms1 also formed foci that, while requiring Msh2-Msh6 for their formation, rarely colocalized with Msh2-Msh6. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These results suggest that (I) mispair recognition can occur via either a replication factory-targeted or a second distinct pool of Msh2-Msh6, and (II) superstoichiometric Mlh1-Pms1 assembly triggered by mispair-bound Msh2-Msh6 defines sites of active MMR. PMID:22118461

  20. DNA repair variants, indoor tanning, and risk of melanoma.

    PubMed

    Torres, Salina M; Luo, Li; Lilyquist, Jenna; Stidley, Christine A; Flores, Kristina; White, Kirsten A M; Erdei, Esther; Gonzales, Melissa; Paine, Susan; Vogel, Rachel I; Lazovich, Deann; Berwick, Marianne

    2013-09-01

    Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 patients with melanoma and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cutoff of P < 0.05. ERCC6 and FBRSL1 gene variants and haplotypes interacted with indoor tanning. However, none of the 92 SNPs tested met the correction criteria for multiple comparisons. This study, based on an a priori interest in investigating the role of DNA repair capacity using variants in base excision and nucleotide excision repair, identified several genes that may play a role in resolving UV-induced DNA damage. PMID:23659246

  1. Prognostic value of DNA repair based stratification of hepatocellular carcinoma.

    PubMed

    Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

    2016-01-01

    Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

  2. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  3. Prognostic value of DNA repair based stratification of hepatocellular carcinoma

    PubMed Central

    Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

    2016-01-01

    Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

  4. p53 downregulates the Fanconi anaemia DNA repair pathway

    PubMed Central

    Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

    2016-01-01

    Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop. PMID:27033104

  5. Metal complex interactions with DNA.

    PubMed

    Pages, Benjamin J; Ang, Dale L; Wright, Elisé P; Aldrich-Wright, Janice R

    2015-02-28

    Increasing numbers of DNA structures are being revealed using biophysical, spectroscopic and genomic methods. The diversity of transition metal complexes is also growing, as the unique contributions that transition metals bring to the overall structure of metal complexes depend on the various coordination numbers, geometries, physiologically relevant redox potentials, as well as kinetic and thermodynamic characteristics. The vast range of ligands that can be utilised must also be considered. Given this diversity, a variety of biological interactions is not unexpected. Specifically, interactions with negatively-charged DNA can arise due to covalent/coordinate or subtle non-coordinate interactions such as electrostatic attraction, groove binding and intercalation as well as combinations of all of these modes. The potential of metal complexes as therapeutic agents is but one aspect of their utility. Complexes, both new and old, are currently being utilised in conjunction with spectroscopic and biological techniques to probe the interactions of DNA and its many structural forms. Here we present a review of metal complex-DNA interactions in which several binding modes and DNA structural forms are explored. PMID:25427534

  6. DNA Repair Pathway Gene Expression Score Correlates with Repair Proficiency and Tumor Sensitivity to Chemotherapy

    PubMed Central

    Pitroda, Sean. P.; Pashtan, Itai. M.; Logan, Hillary. L.; Budke, Brian; Darga, Thomas E.; Weichselbaum, Ralph. R.; Connell, Philip. P.

    2016-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The Recombination Proficiency Score (RPS) utilizes the expression levels for four genes involved in DNA repair pathway preference (RIF1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLC). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression plays a central role in promoting the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteracts the adverse prognosis associated with low RPS. Therefore, RPS may predict which therapies will be effective for individual patients, thereby enabling more personalized oncology care. PMID:24670686

  7. Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

    SciTech Connect

    Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

    2002-03-01

    Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.

  8. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  9. The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair.

    PubMed

    Ng, Hoi-Man; Wei, Leizhen; Lan, Li; Huen, Michael S Y

    2016-07-29

    Multisubunit protein assemblies offer integrated functionalities for efficient cell signal transduction control. One example of such protein assemblies, the BRCA1-A macromolecular complex, couples ubiquitin recognition and metabolism and promotes cellular responses to DNA damage. Specifically, the BRCA1-A complex not only recognizes Lys(63)-linked ubiquitin (K63-Ub) adducts at the damaged chromatin but is endowed with K63-Ub deubiquitylase (DUB) activity. To explore how the BRCA1-A DUB activity contributes to its function at DNA double strand breaks (DSBs), we used RNAi and genome editing approaches to target BRCC36, the protein subunit that confers the BRCA1-A complex its DUB activity. Intriguingly, we found that the K63-Ub DUB activity, although dispensable for maintaining the integrity of the macromolecular protein assembly, is important in enforcing the accumulation of the BRCA1-A complex onto DSBs. Inactivating BRCC36 DUB attenuated BRCA1-A functions at DSBs and led to unrestrained DSB end resection and hyperactive DNA repair. Together, our findings uncover a pivotal role of BRCC36 DUB in limiting DSB processing and repair and illustrate how cells may physically couple ubiquitin recognition and metabolizing activities for fine tuning of DNA repair processes. PMID:27288411

  10. Spatiotemporal analysis of DNA repair using charged particle radiation.

    PubMed

    Tobias, F; Durante, M; Taucher-Scholz, G; Jakob, B

    2010-01-01

    Approaches to visualise the dynamics of the DNA lesion processing substantially contributes to the understanding of the hierarchical organisation of the DNA damage response pathways. Charged particle irradiation has recently emerged as a tool to generate discrete sites of subnuclear damage by its means of extremely localised dose deposition at low energies, thus facilitating the spatiotemporal analysis of repair events. In addition, they are of high interest for risk estimations of human space exploration (e.g. mars mission) in the high energy regime (HZE). In this short review we will give examples for the application of charged particle irradiation to study spatiotemporal aspects of DNA damage recognition and repair in the context of recent achievements in this field. Beamline microscopy allows determining the exact kinetics of repair-related proteins after irradiation with different charged particles that induce different lesion densities. The classification into fast recruited proteins like DNA-PK or XRCC1 or slower recruited ones like 53BP1 or MDC1 helps to establish the hierarchical organisation of damage recognition and subsequent repair events. Additionally, motional analysis of DNA lesions induced by traversing particles proved information about the mobility of DSBs. Increased mobility or the absence of large scale motion has direct consequences on the formation of chromosomal translocations and, thus, on mechanisms of cancer formation. Charged particle microbeams offer the interesting perspective of precise nuclear or subnuclear targeting with a defined number of ions, avoiding the Poisson distribution of traversals inherent to broad beam experiments. With the help of the microbeam, geometrical patterns of traversing ions can be applied facilitating the analysis of spatial organisation of repair. PMID:19944777

  11. DNA repair in spermatocytes and spermatids of the mouse

    SciTech Connect

    Sega, G.A.

    1982-01-01

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced.

  12. Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms

    PubMed Central

    Kostyrko, Kaja; Mermod, Nicolas

    2016-01-01

    DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways. PMID:26657630

  13. Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

    NASA Technical Reports Server (NTRS)

    O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

    2007-01-01

    When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy

  14. When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions.

    PubMed

    Quiñones, Jason Luis; Demple, Bruce

    2016-08-01

    Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3'-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide ("long-patch") BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5'-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15-30min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60-70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5'-peptidyl-dL, and the

  15. Measurement of DNA repair deficiency in workers exposed to benzene

    SciTech Connect

    Hallberg, L.M.; Au, W.W.; El Zein, R.; Grossman, L.

    1996-05-01

    We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m{sup 2} UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m{sup 2} and 350 J/m{sup 2} were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs.

  16. Measurement of DNA repair deficiency in workers exposed to benzene.

    PubMed Central

    Hallberg, L M; el Zein, R; Grossman, L; Au, W W

    1996-01-01

    We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m2 UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m2 and 350 J/m2 were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (< 0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. PMID:8781377

  17. Complex DNA structures and structures of DNA complexes

    SciTech Connect

    Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

    1994-12-01

    Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

  18. Hypomorphic PCNA mutation underlies a human DNA repair disorder

    PubMed Central

    Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

    2014-01-01

    Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

  19. Chemical carcinogenesis — the price for DNA-repair?

    NASA Astrophysics Data System (ADS)

    Wintersberger, Ulrike

    1982-03-01

    This essay examines the possibility of merging the mutation theory of cancer with the hypothesis that cancer is a change in the state of the differentiation of cells. It is suggested that during normal development DNA rearrangements occur, concerning genes which code for differentiation specific cell communication proteins. These proteins are responsible for the proper functioning of growth control in a multicellular organism. DNA-damaging agents — mutagens — induce DNA repair enzymes, some of which may catalyse illegitimate genome rearrangements, thus leading to a change of the balance between growth and differentiation. A cell with a selective advantage may arise and become the origin of a tumor.

  20. Cycling with BRCA2 from DNA repair to mitosis

    SciTech Connect

    Lee, Hyunsook

    2014-11-15

    Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. - Highlights: • BRCA2 is a multifaceted tumor suppressor and is crucial in genetic integrity. • BRCA2 exerts distinct functions in cell cycle-specific manner. • Mitotic kinases regulate diverse functions of BRCA2 in mitosis and cytokinesis.

  1. Hybrid Approaches for Complex Parastomal Hernia Repair.

    PubMed

    Zhang, Heng; Xie, Jia-Ming; Miao, Jian-Qing; Wu, Hao-Rong

    2016-01-01

    Parastomal hernia is one of the major complications of colostomy with high occurrence. From October 2011 to November 2014, a retrospective study was conducted by analyzing and following up data of 16 patients suffering from parastomal hernia who underwent a hybrid technique repair. The safety and efficacy of the hybrid technique for parastomal hernia repair was investigated in terms of complications. All cases were operated successfully and had no major immediate postoperative complications other than mild abdominal pain in 5 cases. No long-term postoperative complications were reported in the follow-up. The authors found hybrid technique to be safe and effective for parastomal hernia repair with fewer complications. PMID:26787038

  2. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2012-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA with high efficiency, through a cylic light-driven electron transfer radical mechanism. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of five active-site residues. The significant loss of repair efficiency by the mutation indicates that those active-site residues play an important role in the DNA repair by photolyase. To understand how the active-site residues modulate the efficiency, we mapped out the entire evolution of each elementary step during the repair in those photolyase mutants with femtosecond resolution. We completely analyzed the electron transfer dynamics using the Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.

  3. Repair and recombination induced by triple helix DNA.

    PubMed

    Chin, Joanna Y; Schleifman, Erica B; Glazer, Peter M

    2007-01-01

    Triple-helix DNA structures can form endogenously at mirror repeat polypurine/polypyrimidine sequences or by introduction of triplex-forming oligonucleotides (TFOs). Recent evidence suggests that triple helices are sources of genetic instability, and are subject to increased rates of mutagenesis and recruitment of repair factors. Indeed, observations using TFOs suggest that triple helices provoke a variety of biological processes which can be harnessed to modulate gene expression and induce heritable changes in targeted genes. This review surveys the biological applications of TFOs, with particular attention to their recombinogenic and mutagenic potential, and summarizes available evidence for the mechanism of triplex and triplex-associated repair. PMID:17485375

  4. Effects of hyperthermia on DNA repair pathways: one treatment to inhibit them all.

    PubMed

    Oei, Arlene L; Vriend, Lianne E M; Crezee, Johannes; Franken, Nicolaas A P; Krawczyk, Przemek M

    2015-01-01

    The currently available arsenal of anticancer modalities includes many DNA damaging agents that can kill malignant cells. However, efficient DNA repair mechanisms protect both healthy and cancer cells against the effects of treatment and contribute to the development of drug resistance. Therefore, anti-cancer treatments based on inflicting DNA damage can benefit from inhibition of DNA repair. Hyperthermia - treatment at elevated temperature - considerably affects DNA repair, among other cellular processes, and can thus sensitize (cancer) cells to DNA damaging agents. This effect has been known and clinically applied for many decades, but how heat inhibits DNA repair and which pathways are targeted has not been fully elucidated. In this review we attempt to summarize the known effects of hyperthermia on DNA repair pathways relevant in clinical treatment of cancer. Furthermore, we outline the relationships between the effects of heat on DNA repair and sensitization of cells to various DNA damaging agents. PMID:26245485

  5. Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation.

    PubMed

    Mahaseth, Tulip; Kuzminov, Andrei

    2016-05-01

    Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H2O2, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN+HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN+HP-induced fragmentation only ∼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ∼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes. PMID:27078578

  6. Oxidative DNA damage background estimated by a system model of base excision repair

    SciTech Connect

    Sokhansanj, B A; Wilson, III, D M

    2004-05-13

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

  7. Electronic structure of (6-4) DNA photoproduct repair involving a non-oxetane pathway.

    PubMed

    Domratcheva, Tatiana; Schlichting, Ilme

    2009-12-16

    Mutagenic pyrimidine-pyrimidone (6-4) photoproducts are one of the main DNA lesions induced by solar UV radiation. These lesions can be photoreversed by (6-4) photolyases. The originally published repair mechanism involves rearrangement of the lesion into an oxetane intermediate upon binding to the (6-4) photolyase, followed by light-induced electron transfer from the reduced flavin cofactor. In a recent crystallographic study on a (6-4) photoproduct complexed with (6-4) photolyase from Drosophila melanogaster no oxetane was observed, raising the possibility of a non-oxetane repair mechanism. Using quantum-chemical calculations we find that in addition to repair via an oxetane, a direct transfer of the hydroxyl group results in reversal of the radical anion (6-4) photoproduct. In both mechanisms, the transition states have high energies and correspond to avoided crossings of the ground and excited electronic states. To study whether the repair can proceed via these state crossings, the excited-state potential energy curves were computed. The radical excitation energies and accessibility of the nonadiabatic repair path were found to depend on hydrogen bonds and the protonation state of the lesion. On the basis of the energy calculations, a nonadiabatic repair of the excited (6-4) lesion radical anion via hydroxyl transfer is probable. This repair mechanism is in line with the recent structural data on the (6-4) photolyase from D. melanogaster . PMID:19921821

  8. Sumoylation Influences DNA Break Repair Partly by Increasing the Solubility of a Conserved End Resection Protein

    PubMed Central

    Sarangi, Prabha; Steinacher, Roland; Altmannova, Veronika; Fu, Qiong; Paull, Tanya T.; Krejci, Lumir; Whitby, Matthew C.; Zhao, Xiaolan

    2015-01-01

    Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect. PMID:25569253

  9. BRCA Mutations, DNA Repair Deficiency, and Ovarian Aging1

    PubMed Central

    Oktay, Kutluk; Turan, Volkan; Titus, Shiny; Stobezki, Robert; Liu, Lin

    2015-01-01

    Oocyte aging has a significant impact on reproductive outcomes both quantitatively and qualitatively. However, the molecular mechanisms underlying the age-related decline in reproductive success have not been fully addressed. BRCA is known to be involved in homologous DNA recombination and plays an essential role in double-strand DNA break repair. Given the growing body of laboratory and clinical evidence, we performed a systematic review on the current understanding of the role of DNA repair in human reproduction. We find that BRCA mutations negatively affect ovarian reserve based on convincing evidence from in vitro and in vivo results and prospective studies. Because decline in the function of the intact gene occurs at an earlier age, women with BRCA1 mutations exhibit accelerated ovarian aging, unlike those with BRCA2 mutations. However, because of the still robust function of the intact allele in younger women and because of the masking of most severe cases by prophylactic oophorectomy or cancer, it is less likely one would see an effect of BRCA mutations on fertility until later in reproductive age. The impact of BRCA2 mutations on reproductive function may be less visible because of the delayed decline in the function of normal BRCA2 allele. BRCA1 function and ataxia-telangiectasia-mutated (ATM)-mediated DNA repair may also be important in the pathogenesis of age-induced increase in aneuploidy. BRCA1 is required for meiotic spindle assembly, and cohesion function between sister chromatids is also regulated by ATM family member proteins. Taken together, these findings strongly suggest the implication of BRCA and DNA repair malfunction in ovarian aging. PMID:26224004

  10. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    PubMed Central

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  11. A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

    PubMed Central

    Słabicki, Mikołaj; Theis, Mirko; Krastev, Dragomir B.; Samsonov, Sergey; Mundwiller, Emeline; Junqueira, Magno; Paszkowski-Rogacz, Maciej; Teyra, Joan; Heninger, Anne-Kristin; Poser, Ina; Prieur, Fabienne; Truchetto, Jérémy; Confavreux, Christian; Marelli, Cécilia; Durr, Alexandra; Camdessanche, Jean Philippe; Brice, Alexis; Shevchenko, Andrej; Pisabarro, M. Teresa; Stevanin, Giovanni; Buchholz, Frank

    2010-01-01

    DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair. PMID:20613862

  12. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    SciTech Connect

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo . E-mail: sueokae@post.saga-med.ac.jp

    2005-08-05

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

  13. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    PubMed

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  14. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

    PubMed Central

    Almeida, Karen H.; Sobol, Robert W.

    2007-01-01

    Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

  15. Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase

    SciTech Connect

    Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M.

    2010-08-12

    Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

  16. DNA damage by reactive species: Mechanisms, mutation and repair.

    PubMed

    Jena, N R

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA-protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA-protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented. PMID:22750987

  17. Targeting abnormal DNA repair in therapy-resistant breast cancers

    PubMed Central

    Tobin, Lisa A.; Robert, Carine; Nagaria, Pratik; Chumsri, Saranya; Twaddell, William; Ioffe, Olga B.; Greco, George E.; Brodie, Angela H.; Tomkinson, Alan E.; Rassool, Feyruz V.

    2012-01-01

    Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently we showed that, relative to non-tumorigenic breast epithelial MCF10A cells, estrogen receptor- and progesterone receptor-positive (ER/PR+) MCF7 breast cancer cells have reduced steady state levels of DNA ligase IV, a component of the major DNA-PK dependent non-homologous end-joining (NHEJ) pathway, whereas the steady state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER/PR- cells have even higher steady state levels of DNA ligase IIIα and increased levels of poly (ADP-ribose) polymerase (PARP1), another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double strand breaks (DSB)s and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER/PR- cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER/PR- tumors had elevated levels of ALT NHEJ and reduced levels of DNA-PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. PMID:22112941

  18. DNA repair activity in fish and interest in ecotoxicology: a review.

    PubMed

    Kienzler, Aude; Bony, Sylvie; Devaux, Alain

    2013-06-15

    The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O⁶-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities

  19. Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

    PubMed Central

    Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

    2015-01-01

    DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

  20. DNA mismatch repair: molecular mechanisms and biological function.

    PubMed

    Schofield, Mark J; Hsieh, Peggy

    2003-01-01

    DNA mismatch repair (MMR) guards the integrity of the genome in virtually all cells. It contributes about 1000-fold to the overall fidelity of replication and targets mispaired bases that arise through replication errors, during homologous recombination, and as a result of DNA damage. Cells deficient in MMR have a mutator phenotype in which the rate of spontaneous mutation is greatly elevated, and they frequently exhibit microsatellite instability at mono- and dinucleotide repeats. The importance of MMR in mutation avoidance is highlighted by the finding that defects in MMR predispose individuals to hereditary nonpolyposis colorectal cancer. In addition to its role in postreplication repair, the MMR machinery serves to police homologous recombination events and acts as a barrier to genetic exchange between species. PMID:14527292

  1. Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

    PubMed

    Wu, Yiping; Zhang, Bintian; Guo, Liang-Hong

    2013-07-16

    Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes. PMID:23777269

  2. A novel cell permeable DNA replication and repair marker

    PubMed Central

    Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25484186

  3. A novel cell permeable DNA replication and repair marker.

    PubMed

    Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25484186

  4. Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

    PubMed Central

    Peng, Guang; Lin, Curtis Chun-Jen; Mo, Wei; Dai, Hui; Park, Yun-Yong; Kim, Soo-Mi; Peng, Yang; Mo, Qianxing; Siwko, Stefan; Hu, Ruozhen; Lee, Ju-Seog; Hennessy, Bryan; Hanash, Samir; Mills, Gordon B.; Lin, Shiaw-Yih

    2014-01-01

    Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer. PMID:24553445

  5. Associations between polymorphisms in DNA repair genes and glioblastoma.

    PubMed

    McKean-Cowdin, Roberta; Barnholtz-Sloan, Jill; Inskip, Peter D; Ruder, Avima M; Butler, Maryann; Rajaraman, Preetha; Razavi, Pedram; Patoka, Joe; Wiencke, John K; Bondy, Melissa L; Wrensch, Margaret

    2009-04-01

    A pooled analysis was conducted to examine the association between select variants in DNA repair genes and glioblastoma multiforme, the most common and deadliest form of adult brain tumors. Genetic data for approximately 1,000 glioblastoma multiforme cases and 2,000 controls were combined from four centers in the United States that have conducted case-control studies on adult glioblastoma multiforme, including the National Cancer Institute, the National Institute for Occupational Safety and Health, the University of Texas M. D. Anderson Cancer Center, and the University of California at San Francisco. Twelve DNA repair single-nucleotide polymorphisms were selected for investigation in the pilot collaborative project. The C allele of the PARP1 rs1136410 variant was associated with a 20% reduction in risk for glioblastoma multiforme (odds ratio(CT or CC), 0.80; 95% confidence interval, 0.67-0.95). A 44% increase in risk for glioblastoma multiforme was found for individuals homozygous for the G allele of the PRKDC rs7003908 variant (odds ratio(GG), 1.44; 95% confidence interval, 1.13-1.84); there was a statistically significant trend (P = 0.009) with increasing number of G alleles. A significant, protective effect was found when three single-nucleotide polymorphisms (ERCC2 rs13181, ERCC1 rs3212986, and GLTSCR1 rs1035938) located near each other on chromosome 19 were modeled as a haplotype. The most common haplotype (AGC) was associated with a 23% reduction in risk (P = 0.03) compared with all other haplotypes combined. Few studies have reported on the associations between variants in DNA repair genes and brain tumors, and few specifically have examined their impact on glioblastoma multiforme. Our results suggest that common variation in DNA repair genes may be associated with risk for glioblastoma multiforme. PMID:19318434

  6. ASSOCIATIONS BETWEEN POLYMORPHISMS IN DNA REPAIR GENES AND GLIOBLASTOMA

    PubMed Central

    McKean-Cowdin, Roberta; Barnholtz-Sloan, Jill; Inskip, Peter; Ruder, Avima; Butler, MaryAnn; Rajaraman, Preetha; Razavi, Pedram; Patoka, Joe; Wiencke, John; Bondy, Melissa; Wrensch, Margaret

    2009-01-01

    A pooled analysis was conducted to examine the association between select variants in DNA repair genes and glioblastoma multiforme (GBM), the most common and deadliest form of adult brain tumors. Genetic data for approximately 1,000 GBM cases and 2,000 controls were combined from four centers in the United States that have conducted case-control studies of adult GBM including the National Cancer Institute, the National Institute for Occupational Safety and Health, the University of Texas M.D. Anderson Cancer Center, and the University of California at San Francisco. Twelve DNA repair SNPs were selected for investigation in the pilot collaborative project. The C allele of the PARP1 rs1136410 variant was associated with a 20% reduction in risk of GBM (ORCT or CC =0.80; 95%CI 0.67–0.95). A 44% increase in risk of GBM was found for individuals homozygous for the G allele of the PRKDC rs7003908 variant (ORGG 1.44; 95%CI 1.13–1.84); there was a statistically significant trend (p=0.009) with increasing number of G alleles. A significant, protective effect was found when 3 SNPs (ERCC2 rs13181, ERCC1 rs3212986, and GLTSCR1 rs1035938) located near each other on chromosome 19 were modeled as a haplotype. The most common haplotype (AGC) was associated with a 23% reduction in risk (p=0.03) compared to all other haplotypes combined. Few studies have reported on the associations between variants in DNA repair genes and brain tumors, and few specifically have examined their impact on GBMs. Our results suggest that common variation in DNA repair genes may be associated with risk of GBMs. PMID:19318434

  7. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

    2008-02-21

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  8. DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

    2007-12-01

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  9. DNA-repair capacity and lipid peroxidation in chronic alcoholics.

    PubMed

    Topinka, J; Binková, B; Srám, R J; Fojtíková, I

    1991-07-01

    The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p less than 0.01) and LPO in plasma was significantly higher (p less than 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = -0.078 x vit. C + 1.9; p less than 0.05) and between UDS and LPO values (UDS = -0.384 x LPO + 4.1; p less than 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol. PMID:2067553

  10. Clinical Radiation Sensitivity With DNA Repair Disorders: An Overview

    SciTech Connect

    Pollard, Julianne M.; Gatti, Richard A.

    2009-08-01

    Adverse reactions to radiotherapy represent a confounding phenomenon in radiation oncology. These reactions are rare, and many have been associated with individuals with DNA repair disorders such as ataxia-telangiectasia and Nijmegen Breakage syndrome. A paucity of published data is available detailing such circumstances. This overview describes four exemplary situations, a comprehensive list of 32 additional cases, and some insights gleaned from this overall experience. Fanconi anemia was associated with more than one-half of the reports. The lowest dose given to a patient that resulted in a reaction was 3 Gy, given to an ataxia-telangiectasia patient. Most patients died within months of exposure. It is clear that the patients discussed in this report had complicated illnesses, in addition to cancer, and the radiotherapy administered was most likely their best option. However, the underlying DNA repair defects make conventional radiation doses dangerous. Our findings support previous wisdom that radiotherapy should either be avoided or the doses should be selected with great care in the case of these radiosensitive genotypes, which must be recognized by their characteristic phenotypes, until more rapid, reliable, and functional assays of DNA repair become available.

  11. Clinical Radiation Sensitivity with DNA Repair Disorders: An Overview

    PubMed Central

    Pollard, Julianne M.; Gatti, Richard A.

    2009-01-01

    Adverse reactions to radiation therapy represent a confounding phenomenon in Radiation Oncology. These reactions are rare, and many have been associated with individuals with DNA repair disorders such as Ataxia-telangiectasia (A-T) and Nijmegen Breakage Syndrome (NBS). There is a paucity of literature detailing such circumstances. This overview describes four exemplary situations, a comprehensive list of 32 additional cases, and some insights gleaned from this overall experience. Fanconi Anemia was associated with over half of the reports. The lowest dose given to a patient that resulted in a reaction was 3 Gy, given to an A-T patient. Most patients died within months of exposure. It is clear that the patients discussed in this paper had complicated illnesses in addition to cancer, and the radiation therapy that administered was most likely their best option. However, the underlying DNA repair defects make conventional radiation therapy doses dangerous. Our review supports prior wisdom that radiation therapy should either be avoided, or doses should be selected with great care in the case of these radiosensitive genotypes which must be recognized with their characteristic phenotypes, until more rapid, reliable and functional assays of DNA repair become available. PMID:19616740

  12. Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

    PubMed

    Ricchetti, M; Fairhead, C; Dujon, B

    1999-11-01

    The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process. PMID:10573425

  13. Akt-mediated Phosphorylation of XLF Impairs Non-homologous End Joining DNA Repair

    PubMed Central

    Liu, Pengda; Gan, Wenjian; Guo, Chunguang; Xie, Anyong; Gao, Daming; Guo, Jianping; Zhang, Jinfang; Willis, Nicholas; Su, Arthur; Asara, John M.; Scully, Ralph; Wei, Wenyi

    2015-01-01

    SUMMARY Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3β leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCFβ-TRCP in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis. PMID:25661488

  14. Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

    PubMed Central

    Beishline, Kate; Kelly, Crystal M.; Olofsson, Beatrix A.; Koduri, Sravanthi; Emrich, Jacqueline; Greenberg, Roger A.

    2012-01-01

    Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H2O2. Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects. PMID:22826432

  15. Detection of the early stage of recombinational DNA repair by silicon nanowire transistors.

    PubMed

    Chiesa, Marco; Cardenas, Paula P; Otón, Francisco; Martinez, Javier; Mas-Torrent, Marta; Garcia, Fernando; Alonso, Juan C; Rovira, Concepció; Garcia, Ricardo

    2012-03-14

    A silicon nanowire-based biosensor has been designed and applied for label-free and ultrasensitive detection of the early stage of recombinational DNA repair by RecA protein. Silicon nanowires transistors were fabricated by atomic force microscopy nanolithography and integrated into a microfluidic environment. The sensor operates by measuring the changes in the resistance of the nanowire as the biomolecular reactions proceed. We show that the nanoelectronic sensor can detect and differentiate several steps in the binding of RecA to a single-stranded DNA filament taking place on the nanowire-aqueous interface. We report relative changes in the resistance of 3.5% which are related to the interaction of 250 RecA·single-stranded DNA complexes. Spectroscopy data confirm the presence of the protein-DNA complexes on the functionalized silicon surfaces. PMID:22364265

  16. Structural Complexity of DNA Sequence

    PubMed Central

    Liou, Cheng-Yuan; Cheng, Wei-Chen; Tsai, Huai-Ying

    2013-01-01

    In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

  17. Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms.

    PubMed

    Kuo, Ching-Ying; Chou, Wen-Cheng; Wu, Chin-Chung; Wong, Teng-Song; Kakadiya, Rajesh; Lee, Te-Chang; Su, Tsann-Long; Wang, Hui-Chun

    2015-09-22

    Alkylating agents are frequently used as first-line chemotherapeutics for various newly diagnosed cancers. Disruption of genome integrity by such agents can lead to cell lethality if DNA lesions are not removed. Several DNA repair mechanisms participate in the recovery of mono- or bi-functional DNA alkylation. Thus, DNA repair capacity is correlated with the therapeutic response. Here, we assessed the function of novel water-soluble N-mustard BO-1055 (ureidomustin) in DNA damage response and repair mechanisms. As expected, BO-1055 induces ATM and ATR-mediated DNA damage response cascades, including downstream Chk1/Chk2 phosphorylation, S/G2 cell-cycle arrest, and cell death. Further investigation revealed that cell survival sensitivity to BO-1055 is comparable to that of mitomycin C. Both compounds require nucleotide excision repair and homologous recombination, but not non-homologous end-joining, to repair conventional cross-linking DNA damage. Interestingly and unlike mitomycin C and melphalan, MGMT activity was also observed in BO-1055 damage repair systems, which reflects the occurrence of O-alkyl DNA lesions. Combined treatment with ATM/ATR kinase inhibitors significantly increases BO-1055 sensitivity. Our study pinpoints that BO-1055 can be used for treating tumors that with deficient NER, HR, and MGMT DNA repair genes, or for synergistic therapy in tumors that DNA damage response have been suppressed. PMID:26208482

  18. Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms

    PubMed Central

    Wu, Chin-Chung; Wong, Teng-Song; Kakadiya, Rajesh; Lee, Te-Chang; Su, Tsann-Long; Wang, Hui-Chun

    2015-01-01

    Alkylating agents are frequently used as first-line chemotherapeutics for various newly diagnosed cancers. Disruption of genome integrity by such agents can lead to cell lethality if DNA lesions are not removed. Several DNA repair mechanisms participate in the recovery of mono- or bi-functional DNA alkylation. Thus, DNA repair capacity is correlated with the therapeutic response. Here, we assessed the function of novel water-soluble N-mustard BO-1055 (ureidomustin) in DNA damage response and repair mechanisms. As expected, BO-1055 induces ATM and ATR-mediated DNA damage response cascades, including downstream Chk1/Chk2 phosphorylation, S/G2 cell-cycle arrest, and cell death. Further investigation revealed that cell survival sensitivity to BO-1055 is comparable to that of mitomycin C. Both compounds require nucleotide excision repair and homologous recombination, but not non-homologous end-joining, to repair conventional cross-linking DNA damage. Interestingly and unlike mitomycin C and melphalan, MGMT activity was also observed in BO-1055 damage repair systems, which reflects the occurrence of O-alkyl DNA lesions. Combined treatment with ATM/ATR kinase inhibitors significantly increases BO-1055 sensitivity. Our study pinpoints that BO-1055 can be used for treating tumors that with deficient NER, HR, and MGMT DNA repair genes, or for synergistic therapy in tumors that DNA damage response have been suppressed. PMID:26208482

  19. Chromatin dynamics during repair of chromosomal DNA double-strand breaks

    PubMed Central

    Sinha, Manisha; Peterson, Craig L

    2010-01-01

    The integrity of a eukaryotic genome is often challenged by DNA double-strand breaks (DSBs). Even a single, unrepaired DSB can be a lethal event, or such unrepaired damage can result in chromosomal instability and loss of genetic information. Furthermore, defects in the pathways that respond to and repair DSBs can lead to the onset of several human pathologic disorders with pleiotropic clinical features, including age-related diseases and cancer. For decades, studies have focused on elucidating the enzymatic mechanisms involved in recognizing, signaling and repairing DSBs within eukaryotic cells. The majority of biochemical and genetic studies have used simple, DNA substrates, whereas only recently efforts have been geared towards understanding how the repair machinery deals with DSBs within chromatin fibers, the nucleoprotein complex that packages DNA within the eukaryotic nucleus. The aim of this review is to discuss our recent understanding of the relationship between chromatin structure and the repair of DSBs by homologous recombination. In particular, we discuss recent studies implicating specialized roles for several, distinct ATP-dependent chromatin remodeling enzymes in facilitating multiple steps within the homologous recombination process. PMID:20495614

  20. Understanding the molecular mechanism of formaldehyde-induced DNA-protein crosslink repair

    EPA Science Inventory

    Formaldehyde induces DNA-protein crosslinks (DPCs) in several experimental in vitro and in vivo test systems, as well as in exposed human workers. DPCs are repaired by several DNA repair pathways in different species, but the molecular understanding of DPC repair in human tissues...

  1. Studying the organization of DNA repair by single-cell and single-molecule imaging

    PubMed Central

    Uphoff, Stephan; Kapanidis, Achillefs N.

    2014-01-01

    DNA repair safeguards the genome against a diversity of DNA damaging agents. Although the mechanisms of many repair proteins have been examined separately in vitro, far less is known about the coordinated function of the whole repair machinery in vivo. Furthermore, single-cell studies indicate that DNA damage responses generate substantial variation in repair activities across cells. This review focuses on fluorescence imaging methods that offer a quantitative description of DNA repair in single cells by measuring protein concentrations, diffusion characteristics, localizations, interactions, and enzymatic rates. Emerging single-molecule and super-resolution microscopy methods now permit direct visualization of individual proteins and DNA repair events in vivo. We expect much can be learned about the organization of DNA repair by linking cell heterogeneity to mechanistic observations at the molecular level. PMID:24629485

  2. DNA Repair Gene Polymorphisms and Their Relation With DNA Damage, DNA Repair, and Total Antioxidant Capacity in Childhood Acute Lymphoblastic Leukemia Survivors.

    PubMed

    Dincer, Yildiz; Yüksel, Selin; Batar, Bahadir; Güven, Mehmet; Onaran, Ilhan; Celkan, Tiraje

    2015-07-01

    Oxidative stress and defective DNA repair are major contributory factors in the initiation and progression of carcinogenesis. Chemotherapy and radiotherapy cause oxidative DNA damage, consume antioxidant capacity, and impair DNA repair activity. These effects of chemotherapy and radiotherapy may be contributory factors in the development of secondary malignancy in cancer survivors. Basal, H2O2-induced, and postrepair DNA damage; urinary 8-hydroxydeoxyguanosine level as a marker of oxidatively damaged DNA; and serum total antioxidant capacity were measured; XPD Lys751Gln, XRCC1 Arg399Gln, and XRCC1 Arg194Trp polymorphisms were analyzed in childhood acute lymphoblastic leukemia (ALL) survivors. Basal and H2O2-induced DNA damage were found to be higher in the ALL survivor group versus the control group, however, there was no significant difference between the other parameters. No association was found between the examined parameters and polymorphisms of XPD 751 and XRCC1 399 and both the groups. XRCC1 194Trp allele was found to be associated with a low level of postrepair DNA damage in the ALL survivors. In conclusion, basal DNA damage and susceptibility to oxidation are high in childhood ALL survivors. This situation which may easily lead to occurrence of a secondary cancer does not seem to be a result of deficient DNA repair. PMID:24577548

  3. DNA repair by the cryptic endonuclease activity of Mu transposase.

    PubMed

    Choi, Wonyoung; Harshey, Rasika M

    2010-06-01

    Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or "5'flap" after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway. PMID:20167799

  4. Resection is a major repair pathway of heavy ion-induced DNA lesions

    NASA Astrophysics Data System (ADS)

    Durante, Marco; Averbeck, Nicole; Taucher-Scholz, Gisela

    Space radiation include densely ionizing heavy ions, which can produce clustered DNA damage with high frequency in human cells. Repair of these complex lesions is generally assumed to be more difficult than for simple double-strand breaks. We show here that human cells use break resection with increasing frequency after exposure to heavy ions. Resection can lead to misrepair of the DNA lesion, via microhomology mediated end-joining. Resection can therefore be responsible for the increased effectiveness of heavy ions in the induction of mutations and genetic late effects.

  5. Inter-individual variation in DNA repair capacity: a need for multi-pathway functional assays to promote translational DNA repair research.

    PubMed Central

    Nagel, Zachary D.; Chaim, Isaac. A.; Samson, Leona D.

    2014-01-01

    Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA Repair Capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic. PMID:24780560

  6. Inter-individual variation in DNA repair capacity: a need for multi-pathway functional assays to promote translational DNA repair research.

    PubMed

    Nagel, Zachary D; Chaim, Isaac A; Samson, Leona D

    2014-07-01

    Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic. PMID:24780560

  7. DNA polymerase θ (POLQ), double-strand break repair, and cancer.

    PubMed

    Wood, Richard D; Doublié, Sylvie

    2016-08-01

    DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9. Pol θ participates in a route of DSB repair termed "alternative end-joining" (altEJ). AltEJ is independent of the DNA binding Ku protein complex and requires DNA end resection. Pol θ is able to mediate joining of two resected 3' ends harboring DNA sequence microhomology. "Signatures" of Pol θ action during altEJ are the frequent utilization of longer microhomologies, and the insertion of additional sequences at joining sites. The mechanism of end-joining employs the ability of Pol θ to tightly grasp a 3' terminus through unique contacts in the active site, allowing extension from minimally paired primers. Pol θ is involved in controlling the frequency of chromosome translocations and preserves genome integrity by limiting large deletions. It may also play a backup role in DNA base excision repair. POLQ is a member of a cluster of similarly upregulated genes that are strongly correlated with poor clinical outcome for breast cancer, ovarian cancer and other cancer types. Inhibition of pol θ is a compelling approach for combination therapy of radiosensitization. PMID:27264557

  8. Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair.

    PubMed

    Hermans, Nicolaas; Laffeber, Charlie; Cristovão, Michele; Artola-Borán, Mariela; Mardenborough, Yannicka; Ikpa, Pauline; Jaddoe, Aruna; Winterwerp, Herrie H K; Wyman, Claire; Jiricny, Josef; Kanaar, Roland; Friedhoff, Peter; Lebbink, Joyce H G

    2016-08-19

    DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks. PMID:27174933

  9. DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy.

    PubMed

    Fayzullina, Saniya; Martin, Lee J

    2016-09-01

    We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by γ-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After γ-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phospho-p53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA. PMID:27452406

  10. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  11. Replication Protein A: Single-stranded DNA's first responder : Dynamic DNA-interactions allow Replication Protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair

    PubMed Central

    Chen, Ran; Wold, Marc S.

    2015-01-01

    Summary Replication Protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. PMID:25171654

  12. Pol β associated complex and base excision repair factors in mouse fibroblasts.

    PubMed

    Prasad, Rajendra; Williams, Jason G; Hou, Esther W; Wilson, Samuel H

    2012-12-01

    During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate. PMID:23042675

  13. Mre11 Nuclease Activity has Essential Roles in DNA Repair and Genomic Stability Distinct from ATM Activation

    PubMed Central

    Buis, Jeffrey; Wu, Yipin; Deng, Yibin; Leddon, Jennifer; Westfield, Gerwin; Eckersdorff, Mark; Sekiguchi, JoAnn M.; Chang, Sandy; Ferguson, David O.

    2008-01-01

    Summary The Mre11/Rad50/NBS1 complex (MRN) maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution, but play unknown roles in mammals. To define functions of Mre11 we engineered targeted mouse alleles which either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology directed double strand break repair, and a contributing role in activating the ATR kinase. However, nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair distinct from MRN control of ATM signaling. PMID:18854157

  14. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  15. The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

    PubMed Central

    George, James W.; Stohr, Bradley A.; Tomso, Daniel J.; Kreuzer, Kenneth N.

    2001-01-01

    Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid. PMID:11459966

  16. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    SciTech Connect

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  17. Interplay between DNA repair and inflammation, and the link to cancer

    PubMed Central

    Kidane, Dawit; Chae, Wook Jin; Czochor, Jennifer; Eckert, Kristin A.; Glazer, Peter M.; Bothwell, Alfred L. M.; Sweasy, Joann B.

    2015-01-01

    DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer. PMID:24410153

  18. A moonlighting metabolic protein influences repair at DNA double-stranded breaks

    PubMed Central

    Torres-Machorro, Ana Lilia; Aris, John P.; Pillus, Lorraine

    2015-01-01

    Catalytically active proteins with divergent dual functions are often described as ‘moonlighting’. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair. PMID:25628362

  19. Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12

    SciTech Connect

    Strike, P.; Roberts, R.J.

    1982-04-01

    The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA/sup +/ and uvrB/sup +/ gene products, but not the host recA/sup +/ gene product. The requirement for both homologous DNA and the uvrA/sup +/ gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered.

  20. Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

    SciTech Connect

    Oliveri, D.R.

    1989-01-01

    The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens.

  1. MERIT40 facilitates BRCA1 localization and DNA damage repair

    PubMed Central

    Feng, Lin; Huang, Jun; Chen, Junjie

    2009-01-01

    The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization. PMID:19261748

  2. Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

    PubMed Central

    Mattarocci, Stefano; Hafner, Lukas; Lezaja, Aleksandra; Shyian, Maksym; Shore, David

    2016-01-01

    Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions. PMID:27066066

  3. Molecular mechanisms of DNA damage recognition for mammalian nucleotide excision repair.

    PubMed

    Sugasawa, Kaoru

    2016-08-01

    For faithful DNA repair, it is crucial for cells to locate lesions precisely within the vast genome. In the mammalian global genomic nucleotide excision repair (NER) pathway, this difficult task is accomplished through multiple steps, in which the xeroderma pigmentosum group C (XPC) protein complex plays a central role. XPC senses the presence of oscillating 'normal' bases in the DNA duplex, and its binding properties contribute to the extremely broad substrate specificity of NER. Unlike XPC, which acts as a versatile sensor of DNA helical distortion, the UV-damaged DNA-binding protein (UV-DDB) is more specialized, recognizing UV-induced photolesions and facilitating recruitment of XPC. Recent single-molecule analyses and structural studies have advanced our understanding of how UV-DDB finds its targets, particularly in the context of chromatin. After XPC binds DNA, it is necessary to verify the presence of damage in order to avoid potentially deleterious incisions at damage-free sites. Accumulating evidence suggests that XPA and the helicase activity of transcription factor IIH (TFIIH) cooperate to verify abnormalities in DNA chemistry. This chapter reviews recent findings about the mechanisms underlying the efficiency, versatility, and accuracy of NER. PMID:27264556

  4. DNA DSB repair pathway choice: an orchestrated handover mechanism

    PubMed Central

    Kakarougkas, A

    2014-01-01

    DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity. PMID:24363387

  5. Distinct effects of the UvrD helicase on topoisomerase-quinolone-DNA ternary complexes.

    PubMed

    Shea, M E; Hiasa, H

    2000-05-12

    Quinolone antibacterial drugs target both DNA gyrase (Gyr) and topoisomerase IV (Topo IV) and form topoisomerase-quinolone-DNA ternary complexes. The formation of ternary complexes results in the inhibition of DNA replication and leads to the generation of double-strand breaks and subsequent cell death. Here, we have studied the consequences of collisions between the UvrD helicase and the ternary complexes formed with either Gyr, Topo IV, or a mutant Gyr, Gyr (A59), which does not wrap the DNA strand around itself. We show (i) that Gyr-norfloxacin (Norf)-DNA and Topo IV-Norf-DNA, but not Gyr (A59)-Norf-DNA, ternary complexes inhibit the UvrD-catalyzed strand-displacement activity, (ii) that a single-strand break is generated at small portions of the ternary complexes upon their collisions with UvrD, and (iii) that the majority of Topo IV-Norf-DNA ternary complexes become nonreversible when UvrD collides with the Topo IV-Norf-DNA ternary complexes, whereas the majority of Gyr-Norf-DNA ternary complexes remain reversible after their collision with the UvrD helicase. These results indicated that different DNA repair mechanisms might be involved in the repair of Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes. PMID:10799552

  6. Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair

    PubMed Central

    Latimer, Jean J.; Kelly, Crystal M.

    2016-01-01

    The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. PMID:24623250

  7. Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene

    PubMed Central

    Morano, Annalisa; Angrisano, Tiziana; Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Bartollino, Silvia; Zuchegna, Candida; Babbio, Federica; Bonapace, Ian Marc; Allen, Brittany; Muller, Mark T.; Chiariotti, Lorenzo; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2014-01-01

    We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression. PMID:24137009

  8. Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

  9. Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling

    PubMed Central

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L.; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER byproduct 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, Z3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872

  10. RPA Antagonizes Microhomology-Mediated Repair of DNA Double-Strand Breaks

    PubMed Central

    Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino; Greene, Eric C; Symington, Lorraine S

    2014-01-01

    Microhomology-mediated end joining (MMEJ) is a Ku and Ligase IV independent mechanism for repair of DNA double-strand breaks, which contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the ssDNA overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity. PMID:24608368

  11. Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton

    SciTech Connect

    Malloy, K.D.; Holman, M.A.; Mitchell, D.

    1997-02-18

    The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlated with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. 54 refs., 4 figs., 2 tabs.

  12. Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton.

    PubMed

    Malloy, K D; Holman, M A; Mitchell, D; Detrich, H W

    1997-02-18

    The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlate with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. PMID:9037040

  13. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in primary melanocytes.

    PubMed

    Thompson, Benjamin C; Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2014-07-01

    Cutaneous melanoma is a significant cause of morbidity and mortality. Nicotinamide is a safe, widely available vitamin that reduces the immune suppressive effects of UV, enhances DNA repair in keratinocytes and has shown promise in the chemoprevention of non-melanoma skin cancer. Here, we report the effect of nicotinamide on DNA damage and repair in primary human melanocytes. Nicotinamide significantly enhanced the repair of oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine) and cyclobutane pyrimidine dimers induced by UV exposure. It also enhanced the repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine induced by the culture conditions in unirradiated melanocytes. A significant increase in the percentage of melanocytes undergoing unscheduled but not scheduled DNA synthesis was observed, confirming that nicotinamide enhances DNA repair in human melanocytes. In summary, nicotinamide, by enhancing DNA repair in melanocytes, is a potential agent for the chemoprevention of cutaneous melanoma. PMID:24798949

  14. An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

    PubMed Central

    Li, L; Lu, X; Peterson, C A; Legerski, R J

    1995-01-01

    Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential. PMID:7565690

  15. Analysis of DNA repair helicase UvrD from Arabidopsis thaliana and Oryza sativa.

    PubMed

    Tuteja, Renu; Tuteja, Narendra

    2013-10-01

    Mismatch repair (MMR) proteins play important roles in maintaining genome stability in all the organisms. Studies of MMR genes in plants have identified several homologs of the Escherichia coli genes. Crop yield is directly related to genome stability, which is crucially required for optimal plant growth and development. Numerous genotoxic stresses such as UV light, radiations, pollutants and heavy metals cause DNA damage leading to genome instability, which can interfere with the plant growth and crop productivity. But the efficient repair mechanisms can help to overcome the deleterious effects of the damage. Therefore it is important to study the genes involved in various repair pathways in the plants in greater detail. UvrD helicase is a component of MMR complex and plays an essential role in the DNA repair by providing the unwinding function. In the present manuscript we present an in silico analysis of UvrD helicase from two plant species (Arabidopsis and rice). The Arabidopsis thaliana and Oryza sativa UvrD are 1149 (~129 kDa) and 1165 amino-acids (~130 kDa) proteins, respectively. These proteins contain all the conserved domains and are larger than the E. coli UvrD because they contain a longer N-terminal extension. In order to decipher the role of plant UvrD in various stresses it will be important to study the biochemical and functional properties of this enzyme. PMID:23974358

  16. Chromosomal directionality of DNA mismatch repair in Escherichia coli

    PubMed Central

    Hasan, A. M. Mahedi; Leach, David R. F.

    2015-01-01

    Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5′ to 3′ and the 3′ to 5′ directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication. PMID:26170312

  17. SNF2H interacts with XRCC1 and is involved in repair of H2O2-induced DNA damage.

    PubMed

    Kubota, Yoshiko; Shimizu, Shinji; Yasuhira, Shinji; Horiuchi, Saburo

    2016-07-01

    The protein XRCC1 has no inherent enzymatic activity, and is believed to function in base excision repair as a dedicated scaffold component that coordinates other DNA repair factors. Repair foci clearly represent the recruitment and accumulation of DNA repair factors at sites of damage; however, uncertainties remain regarding their organization in the context of nuclear architecture and their biological significance. Here we identified the chromatin remodeling factor SNF2H/SMARCA5 as a novel binding partner of XRCC1, with their interaction dependent on the casein kinase 2-mediated constitutive phosphorylation of XRCC1. The proficiency of repairing H2O2-induced damage was strongly impaired by SNF2H knock-down, and similar impairment was observed with knock-down of both XRCC1 and SNF2H simultaneously, suggesting their role in a common repair pathway. Most SNF2H exists in the nuclear matrix fraction, forming salt extraction-resistant foci-like structures in unchallenged nuclei. Remarkably, damage-induced formation of both PAR and XRCC1 foci depended on SNF2H, and the PAR and XRCC1 foci co-localized with the SNF2H foci. We propose a model in which a base excision repair complex containing damaged chromatin is recruited to specific locations in the nuclear matrix for repair, with this recruitment mediated by XRCC1-SNF2H interaction. PMID:27268481

  18. Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

    PubMed

    Li, Qi; Huang, Yue; Liu, Xichun; Gan, Jianhua; Chen, Hao; Yang, Cai-Guang

    2016-05-20

    The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment. PMID:27015802

  19. DNA-PK is Involved in Repairing a Transient Surge of DNA BreaksInduced by Deceleration of DNA Replication.

    SciTech Connect

    Shimura, Tsutomu; Martin, Melvenia M.; Torres, Michael J.; Gu,Cory; Pluth, Janice M.; DiBernardi, Maria A.; McDonald, Jeffrey S.; Aladjem, Mirit I.

    2006-09-25

    ells that suffer substantial inhibition of DNA replication halt their cell cycle via a checkpoint response mediated by the PI3 kinases ATM and ATR. It is unclear how cells cope with milder replication insults, which are under the threshold for ATM and ATR activation. A third PI3 kinase, DNA-dependent protein kinase (DNA-PK), is also activated following replication inhibition, but the role DNA-PK might play in response to perturbed replication is unclear, since this kinase does not activate the signaling cascades involved in the S-phase checkpoint. Here we report that mild, transient drug-induced perturbation of DNA replication rapidly induced DNA breaks that promptly disappeared in cells that contained a functional DNA-PK whereas such breaks persisted in cells that were deficient in DNA-PK activity. After the initial transient burst of DNA breaks, cells with a functional DNA-PK did not halt replication and continued to synthesize DNA at a slow pace in the presence of replication inhibitors. In contrast, DNA-PK deficient cells subject to low levels of replication inhibition halted cell cycle progression via an ATR-mediated S-phase checkpoint. The ATM kinase was dispensable for the induction of the initial DNA breaks. These observations suggest that DNA-PK is involved in setting a high threshold for the ATR-Chkl-mediated S-phase checkpoint by promptly repairing DNA breaks that appear immediately following inhibition of DNA replication.

  20. Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy

    PubMed Central

    Xu, Yang; Her, Chengtao

    2015-01-01

    Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy. PMID:26287259

  1. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  2. Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

    PubMed

    Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

    2016-09-01

    The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance. PMID:27398742

  3. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

    PubMed

    Sijmons, Rolf H; Hofstra, Robert M W

    2016-02-01

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder. PMID:26746812

  4. Repeat instability during DNA repair: Insights from model systems

    PubMed Central

    Usdin, Karen; House, Nealia C. M.; Freudenreich, Catherine H.

    2015-01-01

    The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them. PMID:25608779

  5. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

    PubMed

    Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

    2014-04-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

  6. C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency

    PubMed Central

    Meier, Bettina; Cooke, Susanna L.; Weiss, Joerg; Bailly, Aymeric P.; Alexandrov, Ludmil B.; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R.; Campbell, Peter J.

    2014-01-01

    Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage–fusion–bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling “chromoanasynthesis,” a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. PMID:25030888

  7. Estrogen Receptor Expression Is Associated with DNA Repair Capacity in Breast Cancer

    PubMed Central

    Matta, Jaime; Morales, Luisa; Ortiz, Carmen; Adams, Damian; Vargas, Wanda; Casbas, Patricia; Dutil, Julie; Echenique, Miguel; Suárez, Erick

    2016-01-01

    Estrogen-receptor-positive (ER+) tumors employ complex signaling that engages in crosstalk with multiple pathways through genomic and non-genomic regulation. A greater understanding of these pathways is important for developing improved biomarkers that can better determine treatment choices, risk of recurrence and cancer progression. Deficiencies in DNA repair capacity (DRC) is a hallmark of breast cancer (BC); therefore, in this work we tested whether ER signaling influences DRC. We analyzed the association between ER positivity (% receptor activation) and DRC in 270 BC patients, then further stratified our analysis by HER2 receptor status. Our results show that among HER2 negative, the likelihood of having low DRC values among ER- women is 1.92 (95% CI: 1.03, 3.57) times the likelihood of having low DRC values among ER+ women, even adjusting for different potential confounders (p<0.05); however, a contrary pattern was observed among HER2 positives women. In conclusion, there is an association between DRC levels and ER status, and this association is modified by HER2 receptor status. Adding a DNA repair capacity test to hormone receptor testing may provide new information on defective DNA repair phenotypes, which could better stratify BC patients who have ER+ tumors. ER+/HER2- tumors are heterogeneous, incompletely defined, and clinically challenging to treat; the addition of a DRC test could better characterize and classify these patients as well as help clinicians select optimal therapies, which could improve outcomes and reduce recurrences. PMID:27032101

  8. In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

    PubMed Central

    Moné, Martijn J.; Bernas, Tytus; Dinant, Christoffel; Goedvree, Feliks A.; Manders, Erik M. M.; Volker, Marcel; Houtsmuller, Adriaan B.; Hoeijmakers, Jan H. J.; Vermeulen, Wim; van Driel, Roel

    2004-01-01

    Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of ≈30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells. PMID:15520397

  9. Visualization of mismatch repair complexes using fluorescence microscopy.

    PubMed

    Schmidt, Tobias T; Hombauer, Hans

    2016-02-01

    DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients. During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5' and 3' nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed. Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms. PMID:26725956

  10. Using the Bias from Flow to Elucidate Single DNA Repair Protein Sliding and Interactions with DNA

    PubMed Central

    Lin, Yihan; Zhao, Tong; Jian, Xing; Farooqui, Zishaan; Qu, Xiaohui; He, Chuan; Dinner, Aaron R.; Scherer, Norbert F.

    2009-01-01

    We perform single-molecule spatial tracking measurements of a DNA repair protein, the C-terminal domain of Ada (C-Ada) from Escherichia coli, moving on DNA extended by flow. The trajectories of single proteins labeled with a fluorophore are constructed. We analyze single-protein dwell times on DNA for different flow rates and conclude that sliding (with essentially no hopping) is the mechanism of C-Ada motion along stretched DNA. We also analyze the trajectory results with a drift-diffusion Langevin equation approach to elucidate the influence of flow on the protein motion; systematic variation of the flow enables one to estimate the microscopic friction. We integrate the step-size probability distribution to obtain a version of the fluctuation theorem that articulates the relation between the entropy production and consumption under the adjustable drag (i.e., bias) from the flow. This expression allows validation of the Langevin equation description of the motion. Comparison of the rate of sliding with recent computer simulations of DNA repair suggests that C-Ada could conduct its repair function while moving at near the one-dimensional diffusion limit. PMID:19254550

  11. Calculation of complex DNA damage induced by ions

    NASA Astrophysics Data System (ADS)

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-01

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  12. Calculation of complex DNA damage induced by ions

    SciTech Connect

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-15

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  13. DNA Repair in Despair-Vitamin D Is Not Fair.

    PubMed

    Gocek, Elżbieta; Studzinski, George P

    2016-08-01

    The role of vitamin D as a treatment option for neoplastic diseases, once considered to have a bright future, remains controversial. The preclinical studies discussed herein show compelling evidence that Vitamin D Derivatives (VDDs) can convert some cancer and leukemia cells to a benign phenotype, by differentiation/maturation, cell cycle arrest, or induction of apoptosis. Furthermore, there is considerable, though still evolving, knowledge of the molecular mechanisms underlying these changes. However, the attempts to clearly document that the treatment outcomes of human neoplastic diseases can be positively influenced by VDDs have been, so far, disappointing. The clinical trials to date of VDDs, alone or combined with other agents, have not shown consistent results. It is our contention, shared by others, that there were limitations in the design or execution of these trials which have not yet been fully addressed. Based on the connection between upregulation of JNK by VDDs and DNA repair, we propose a new avenue of attack on cancer cells by increasing the toxicity of the current, only partially effective, cancer chemotherapeutic drugs by combining them with VDDs. This can impair DNA repair and thus kill the malignant cells, warranting a comprehensive study of this novel concept. J. Cell. Biochem. 117: 1733-1744, 2016. © 2016 Wiley Periodicals, Inc. PMID:27122067

  14. Alterations in Chromosomal Synapses and DNA Repair in Apoptotic Spermatocytes of Mus m. Domesticus

    PubMed Central

    Ayarza, E.; González, M.; López, F.; Fernández-Donoso, R.; Page, J.; Berrios, S.

    2016-01-01

    We investigated whether apoptotic spermatocytes from the mouse Mus m. domesticus presented alterations in chromosomal synapses and DNA repair. To enrich for apoptotic spermatocytes, the scrotum’s temperature was raised by partially exposing animals for 15 min to a 42ºC water bath. Spermatocytes in initial apoptosis were identified in situ by detecting activated caspase-9. SYCP1 and SYCP3 were markers for evaluating synapses or the structure of synaptonemal complexes and Rad51 and γH2AX for detecting DNA repair and chromatin remodeling. Apoptotic spermatocytes were concentrated in spermatogenic cycle stages III-IV (50.3%), XI-XII (44.1%) and IX-X (4.2%). Among apoptotic spermatocytes, 48% were in middle pachytene, 44% in metaphase and 6% in diplotene. Moreover, apoptotic spermatocytes showed several structural anomalies in autosomal bivalents, including splitting of chromosomal axes and partial asynapses between homologous chromosomes. γH2AX and Rad51 were atypically distributed during pachytene and as late as diplotene and associated with asynaptic chromatin, single chromosome axes or discontinuous chromosome axes. Among apoptotic spermatocytes at pachytene, 70% showed changes in the structure of synapses, 67% showed changes in γH2AX and Rad51 distribution and 50% shared alterations in both synapses and DNA repair. Our results showed that apoptotic spermatocytes from Mus m. domesticus contain a high frequency of alterations in chromosomal synapses and in the recruitment and distribution of DNA repair proteins. Together, these observations suggest that these alterations may have been detected by meiotic checkpoints triggering apoptosis. PMID:27349323

  15. Alterations in chromosomal synapses and DNA repair in apoptotic spermatocytes of Mus m. domesticus.

    PubMed

    Ayarza, E; González, M; López, F; Fernández-Donoso, R; Page, J; Berrios, S

    2016-01-01

    We investigated whether apoptotic spermatocytes from the mouse Mus m. domesticus presented alterations in chromosomal synapses and DNA repair. To enrich for apoptotic spermatocytes, the scrotum's temperature was raised by partially exposing animals for 15 min to a 42ºC water bath. Spermatocytes in initial apoptosis were identified in situ by detecting activated Caspase-9.  SYCP1 and SYCP3 were markers for evaluating synapses or the structure of synaptonemal complexes and Rad51 and γH2AX for detecting DNA repair and chromatin remodeling. Apoptotic spermatocytes were concentrated in spermatogenic cycle stages III-IV (50.3%), XI-XII (44.1%) and IX-X (4.2%). Among apoptotic spermatocytes, 48% were in middle pachytene, 44% in metaphase and 6% in diplotene. Moreover, apoptotic spermatocytes showed several structural anomalies in autosomal bivalents, including splitting of chromosomal axes and partial asynapses between homologous chromosomes. gH2AX and Rad51 were atypically distributed during pachytene and as late as diplotene and associated with asynaptic chromatin, single chromosome axes or discontinuous chromosome axes. Among apoptotic spermatocytes at pachytene, 70% showed changes in the structure of synapses, 67% showed changes in gH2AX and Rad51 distribution and 50% shared alterations in both synapses and DNA repair. Our results showed that apoptotic spermatocytes from Mus m. domesticus contain a high frequency of alterations in chromosomal synapses and in the recruitment and distribution of DNA repair proteins. Together, these observations suggest that these alterations may have been detected by meiotic checkpoints triggering apoptosis. PMID:27349323

  16. Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes

    SciTech Connect

    Jones, I M; Thomas, C B; Xi, T; Mohrenweiser, H W; Nelson, D O

    2006-07-03

    Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by Random Forests Regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach 6 SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate

  17. Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes.

    PubMed

    Jones, Irene M; Thomas, Cynthia B; Xi, Tina; Mohrenweiser, Harvey W; Nelson, David O

    2007-03-01

    Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by random forests regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach six SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate

  18. Mismatch repair inhibits homeologous recombination via coordinated directional unwinding of trapped DNA structures.

    PubMed

    Tham, Khek-Chian; Hermans, Nicolaas; Winterwerp, Herrie H K; Cox, Michael M; Wyman, Claire; Kanaar, Roland; Lebbink, Joyce H G

    2013-08-01

    Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability. PMID:23932715

  19. Role of the translationally controlled tumor protein in DNA damage sensing and repair.

    PubMed

    Zhang, Jie; de Toledo, Sonia M; Pandey, Badri N; Guo, Guozheng; Pain, Debkumar; Li, Hong; Azzam, Edouard I

    2012-04-17

    The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G(1) delay but prolonged the G(2) delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents. PMID:22451927

  20. DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

    PubMed

    van Overbeek, Megan; Capurso, Daniel; Carter, Matthew M; Thompson, Matthew S; Frias, Elizabeth; Russ, Carsten; Reece-Hoyes, John S; Nye, Christopher; Gradia, Scott; Vidal, Bastien; Zheng, Jiashun; Hoffman, Gregory R; Fuller, Christopher K; May, Andrew P

    2016-08-18

    The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits. PMID:27499295

  1. A Human Orthologue of Archaeal DNA Repair Protein Hef is Defective in Fanconi Anemia Complementation Group M

    PubMed Central

    Meetei, Amom Ruhikanta; Medhurst, Annette L.; Ling, Chen; Xue, Yutong; Singh, Thiyam Ramsing; Bier, Patrick; Steltenpool, Jurgen; Stone, Stacie; Dokal, Inderjeet; Mathew, Christopher G.; Hoatlin, Maureen; Joenje, Hans; de Winter, Johan P.; Wang, Weidong

    2005-01-01

    Fanconi anemia (FA) is a genetic disease featuring genomic instability and cancer predisposition1. Nine FA genes have been identified, and their products participate in a DNA damage response network involving BRCA1 and BRCA22,3. We have previously purified a FA core complex containing the FANCL ubiquitin ligase and 6 other FA proteins4–6. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the FA DNA damage response pathway2,7. Here we show that another component of this complex, FAAP250, is mutated in FA patients of a new complementation group (FA-M). FAAP250, renamed FANCM, has sequence similarity to known DNA repair proteins, including archaeal Hef, yeast Mph1 and human ERCC4/XPF. FANCM can dissociate DNA triplex, possibly due to its ability to translocate on duplex DNA. FANCM is essential for FANCD2 monoubiquitination and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between FA proteins and DNA repair; FANCM may act as an engine that translocates the FA core complex along DNA. PMID:16116422

  2. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.

    PubMed

    Jia, Nan; Nakazawa, Yuka; Guo, Chaowan; Shimada, Mayuko; Sethi, Mieran; Takahashi, Yoshito; Ueda, Hiroshi; Nagayama, Yuji; Ogi, Tomoo

    2015-01-01

    DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete. PMID:25474029

  3. Sharpening the ends for repair: mechanisms and regulation of DNA resection.

    PubMed

    Paudyal, Sharad C; You, Zhongsheng

    2016-07-01

    DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival. Resection involves selective processing of 5' ends of broken DNA to generate ssDNA overhangs, which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability. Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment. PMID:27174871

  4. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging. PMID:26879629

  5. Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture).

    PubMed

    Sancar, Aziz

    2016-07-18

    Ultraviolet light damages DNA by converting two adjacent thymines into a thymine dimer which is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired by photolyase and the nucleotide excision repair system in E. coli by nucleotide excision repair in humans. The work leading to these results is presented by Aziz Sancar in his Nobel Lecture. PMID:27337655

  6. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  7. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    PubMed Central

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A.

    2015-01-01

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer. PMID:26197339

  8. Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme.

    PubMed

    Qi, Yan; Spong, Marie C; Nam, Kwangho; Banerjee, Anirban; Jiralerspong, Sao; Karplus, Martin; Verdine, Gregory L

    2009-12-10

    How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway. PMID:20010681

  9. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    SciTech Connect

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  10. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner.

    PubMed

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H F; Fousteri, Maria I

    2007-07-20

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase IIIalpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensable for ligation of NER-induced breaks and repair of UV lesions in quiescent cells. Furthermore, our results demonstrate that two distinct complexes differentially carry out gap filling in NER. XRCC1-Lig3 and DNA polymerase delta colocalize and interact with NER components in a UV- and incision-dependent manner throughout the cell cycle. In contrast, DNA ligase I and DNA polymerase epsilon are recruited to UV-damage sites only in proliferating cells. This study reveals an unexpected and key role for XRCC1-Lig3 in maintenance of genomic integrity by NER in both dividing and nondividing cells and provides evidence for cell-cycle regulation of NER-mediated repair synthesis in vivo. PMID:17643379

  11. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  12. Mechanism of Cluster DNA Damage Repair in Response to High-Atomic Number and Energy Particles Radiation

    PubMed Central

    Asaithamby, Aroumougame; Chen, David J.

    2012-01-01

    Low-linear energy transfer (LET) radiation (i.e., γ- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles are slowly repaired or are irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure. PMID:21126526

  13. DNA polymerase beta-catalyzed-PCNA independent long patch base excision repair synthesis: a mechanism for repair of oxidatively damaged DNA ends in post-mitotic brain.

    PubMed

    Wei, Wei; Englander, Ella W

    2008-11-01

    Oxidative DNA damage incidental to normal respiratory metabolism poses a particular threat to genomes of highly metabolic-long lived cells. We show that post-mitotic brain has capacity to repair oxidatively damaged DNA ends, which are targets of the long patch (LP) base excision repair (BER) subpathway. LP-BER relies, in part, on proteins associated with DNA replication, including proliferating cell nuclear antigen and is inherent to proliferating cells. Nonetheless, repair products are generated with brain extracts, albeit at slow rates, in the case of 5'-DNA ends modeled with tetrahydrofuran (THF). THF at this position is refractory to DNA polymerase beta 5'-deoxyribose 5-phosphate lyase activity and drives repair into the LP-BER subpathway. Comparison of repair of 5'-THF-blocked termini in the post-mitotic rat brain and proliferative intestinal mucosa, revealed that in mucosa, resolution of damaged 5'-termini is accompanied by formation of larger repair products. In contrast, adducts targeted by the single nucleotide BER are proficiently repaired with both extracts. Our findings reveal mechanistic differences in BER processes selective for the brain versus proliferative tissues. The differences highlight the physiological relevance of the recently proposed 'Hit and Run' mechanism of alternating cleavage/synthesis steps, in the proliferating cell nuclear antigen-independent LP-BER process. PMID:18752643

  14. Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

    PubMed Central

    Budd, Martin E.; Campbell, Judith L.

    2009-01-01

    The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway. PMID:19165339

  15. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

    NASA Astrophysics Data System (ADS)

    Vasquez, Karen M.; Christensen, Jesper; Li, Lei; Finch, Rick A.; Glazer, Peter M.

    2002-04-01

    Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

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

    PubMed

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  17. Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations

    NASA Astrophysics Data System (ADS)

    Okayasu, R.; Okabe, A.; Takakura, K.

    We irradiated plateau phase normal human fibroblasts with 2 Gy X-rays 70 keV um carbon 290MeV n and 200 keV um iron ions 500 MeV n and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break DSB repair GammaH2AX foci a marker for DSBs were detected immediately after irradiation and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci In contrast phosphorylation of an NHEJ protein DNA-PKcs threonine 2609 was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX Furthermore in the case of ATM protein phosphorylation serine 1981 iron irradiation alone caused a significant initial delay but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions These results suggest that 1 high LET irradiation induces complex and or severe DNA DSB damage which affects the function of DSB repair proteins 2 Both ATM and DNA-PKcs may recognize the complexity of DSBs but ATM may be more sensitive to detecting the complexity of DSB damage 3 gamma-H2AX may

  18. How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway

    PubMed Central

    Coleman, Kate E.; Huang, Tony T.

    2016-01-01

    Fanconi anemia (FA) is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs). ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination, nucleotide excision repair and translesion synthesis (TLS). SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligases (STUbLs) and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer. PMID:27148358

  19. Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

    NASA Technical Reports Server (NTRS)

    George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

  20. Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

    PubMed

    Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

    2015-10-23

    The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. PMID:26338705

  1. Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

    DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

  2. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes.

    PubMed

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  3. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  4. Rapid and Progressive Pulmonary Fibrosis in 2 Families with DNA Repair Deficiencies of Undetermined Etiology

    PubMed Central

    Vece, Timothy J.; Schecter, Marc G.; Gatti, Richard A.; Tunuguntla, Rashmi; Garcia, Christine Kim; Langston, Claire; Dishop, Megan K.; Moore, Robert H.; Fan, Leland L.

    2016-01-01

    Known genetic causes of pediatric interstitial lung disease include disorders of surfactant metabolism, telomerase, and DNA repair. We report 4 children from 2 families with rapidly progressive and fatal pulmonary fibrosis. A novel DNA repair defect unrelated to the ataxia-telangiectasia mutated gene was found in 1 child from each family. PMID:22240110

  5. Targeting the DNA repair pathway in Ewing sarcoma.

    PubMed

    Stewart, Elizabeth; Goshorn, Ross; Bradley, Cori; Griffiths, Lyra M; Benavente, Claudia; Twarog, Nathaniel R; Miller, Gregory M; Caufield, William; Freeman, Burgess B; Bahrami, Armita; Pappo, Alberto; Wu, Jianrong; Loh, Amos; Karlström, Åsa; Calabrese, Chris; Gordon, Brittney; Tsurkan, Lyudmila; Hatfield, M Jason; Potter, Philip M; Snyder, Scott E; Thiagarajan, Suresh; Shirinifard, Abbas; Sablauer, Andras; Shelat, Anang A; Dyer, Michael A

    2014-11-01

    Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice. PMID:25437539

  6. DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining

    PubMed Central

    Howard, Sean M.; Yanez, Diana A.; Stark, Jeremy M.

    2015-01-01

    Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection. PMID:25629353

  7. DNA-Damage Foci to Detect and Characterize DNA Repair Alterations in Children Treated for Pediatric Malignancies

    PubMed Central

    Kaiser, Mareike; Betten, Dominik; Furtwängler, Rhoikos; Rübe, Christian; Graf, Norbert; Rübe, Claudia E.

    2014-01-01

    Purpose In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA) suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. Methods and Materials In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. Results Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. Conclusions Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies. PMID:24637877

  8. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-12-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. PMID:1935910

  9. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed Central

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-01-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. Images PMID:1935910

  10. Effect of vanillin on toxicant-induced lethality in the Drosophila melanogaster DNA repair test.

    PubMed

    Furlanetto, Magda Patrícia; Sinigaglia, Marialva; Amaral, Viviane Souza do; Dihl, Rafael Rodrigues; de Andrade, Heloísa Helena Rodrigues

    2007-01-01

    Vanillin (VA) modulates the genotoxicity of chemical and physical agents in a complex manner. Previous studies indicate that VA inhibits the mutagenicity but increases the mitotic homologous recombination caused by at least some genotoxic agents. In the present study, we have evaluated the effects of VA on the repair of lethal damage produced by three genotoxins, N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and mitomycin C (MMC), using the DNA repair test (DRT) in Drosophila melanogaster. VA, 0.25% and 0.5% (w/v), increased the toxicity of MMC and EMS in repair-deficient flies, as measured by a decrease in the proportion of male to female progeny in the DRT; sex ratios decreased from 18-48% for MMC and 21-97% for EMS. These effects may be caused by the inhibition of nonhomologous DNA end joining caused by VA. In contrast to the results with MMC and EMS, VA protected against the lethality of ENU in repair-defective flies, as measured by a 43-207% increase in the survival of male flies in the DRT. It was inferred that the protective effect was due to VA modulating stages prior to the induction of ENU lesions in DNA, including modulating the antioxidant properties of VA and/or to its interference with the metabolic activation and/or detoxification of specific genotoxins. The results from this study indicate that the characterization of VA as a promising agent for preventing damage to genes and chromosomes should be tempered by observations that VA can increase the toxicity of chemical agents. PMID:17177210

  11. A novel small molecule inhibitor of the DNA repair protein Ku70/80.

    PubMed

    Weterings, Eric; Gallegos, Alfred C; Dominick, Lauren N; Cooke, Laurence S; Bartels, Trace N; Vagner, Josef; Matsunaga, Terry O; Mahadevan, Daruka

    2016-07-01

    Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents. PMID:27130816

  12. Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation

    PubMed Central

    Jiang, Yuhui; Qian, Xu; Shen, Jianfeng; Wang, Yugang; Li, Xinjian; Liu, Rui; Xia, Yan; Chen, Qianming; Peng, Guang; Lin, Shiaw-Yih; Lu, Zhimin

    2016-01-01

    Histone methylation regulates DNA repair. However, the mechanisms that underlie the regulation of histone methylation during this repair remain to be further defined. Here, we show that ionizing radiation (IR) induces DNA-PK-dependent phosphorylation of nuclear fumarase at T236, which leads to an interaction between fumarase and the histone variant H2A.Z at DNA double-strand break (DSB) regions. Locally generated fumarate inhibits KDM2B histone demethylase activity, resulting in enhanced dimethylation of histone H3 K36; in turn, this increases the accumulation of the Ku70-containing DNA-PK at DSB regions for non-homologous end joining (NHEJ) DNA repair and cell survival. These findings reveal a feedback mechanism that underlies DNA-PK regulation by chromatin-associated fumarase and an instrumental function of fumarase in regulating histone H3 methylation and DNA repair. PMID:26237645

  13. UV-Induced Charge Transfer States in DNA Promote Sequence Selective Self-Repair.

    PubMed

    Bucher, Dominik Benjamin; Kufner, Corinna Lucia; Schlueter, Alexander; Carell, Thomas; Zinth, Wolfgang

    2016-01-13

    Absorption of UV-radiation in nucleotides initiates a number of photophysical and photochemical processes, which may finally cause DNA damage. One major decay channel of photoexcited DNA leads to reactive charge transfer states. This study shows that these states trigger self-repair of DNA photolesions. The experiments were performed by UV spectroscopy and HPLC on different single and double stranded oligonucleotides containing a cyclobutane pyrimidine dimer (CPD) lesion. In a first experiment we show that photoexcitation of adenine adjacent to a CPD has no influence on this lesion. However, excitation of a guanine (G) adenine (A) sequence leads to reformation of the intact thymine (T) bases. The involvement of two bases for the repair points to a long-living charge transfer state between G and A to be responsible for the repair. The negatively charged A radical anion donates an electron to the CPD, inducing ring splitting and repair. In contrast, a TA sequence, having an inverted charge distribution (T radical anion, A radical cation), is not able to repair the CPD lesion. The investigations show that the presence of an adjacent radical ion is not sufficient for repair. More likely it is the driving power represented by the oxidation potential of the radical ion, which controls the repair. Thus, repair capacities are strongly sequence-dependent, creating DNA regions with different tendencies of self-repair. This self-healing activity represents the simplest sequence-dependent DNA repair system. PMID:26651219

  14. Laser microbeam - kinetic studies combined with molecule - structures reveal mechanisms of DNA repair

    NASA Astrophysics Data System (ADS)

    Altenberg, B.; Greulich, K. O.

    2011-10-01

    Kinetic studies on double strand DNA damages induced by a laser microbeam have allowed a precise definition of the temporal order of recruitment of repair molecules. The order is KU70 / KU80 - XRCC4 --NBS1 -- RAD51. These kinetic studies are now complemented by studies on molecular structures of the repair proteins, using the program YASARA which does not only give molecular structures but also physicochemical details on forces involved in binding processes. It turns out that the earliest of these repair proteins, the KU70 / KU80 heterodimer, has a hole with high DNA affinity. The next molecule, XRCC4, has a body with two arms, the latter with extremely high DNA affinity at their ends and a binding site for Ligase 4. Together with the laser microbeam results this provides a detailed view on the early steps of DNA double strand break repair. The sequence of DNA repair events is presented as a movie.

  15. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.

    PubMed

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  16. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    PubMed Central

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  17. Genotoxicity of dichlorvos in strains of Drosophila melanogaster defective in DNA repair.

    PubMed

    Mishra, Manish; Sharma, A; Shukla, A K; Kumar, R; Dwivedi, U N; Kar Chowdhuri, D

    2014-05-15

    Dichlorvos (DDVP), an organophosphate pesticide, is reported to be genotoxic at high concentrations. However, the roles of DNA damage repair pathways in DDVP genotoxicity are not well characterized. To test whether pre- and post-replication pathways are involved, we measured changes in DNA migration (Comet assay) in the midgut cells of Drosophila melanogaster Oregon R+ larvae and in some mutants of pre- (mei-9, mus201, and mus207) and post- (mei-41 and mus209)replication DNA repair pathways. Insects were exposed to environmentally relevant concentrations of DDVP (up to 15ng/ml) for 48h. After insect exposure to 0.15ng/ml DDVP, we observed greater DNA damage in pre-replication repair mutants; effects on Oregon R+ and post-replication repair mutants were insignificant. In contrast, significant DNA damage was observed in the post-replication repair mutants after their exposure to 1.5 and 15ng/ml DDVP. The pre-replication repair mutant mus207 showed maximum sensitivity to DDVP, suggestive of alkylation damage to DNA. We also examined mutants (SOD- and urate-null) that are sensitive to oxidative stress and the results indicate that significant oxidative DNA damage occurs in DDVP-exposed mutants. This study suggests involvement of both pre- and post-replication repair pathways against DDVP-induced DNA damage in Drosophila, with oxidative DNA damage leading to genotoxicity. PMID:24614193

  18. Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes

    PubMed Central

    Powley, Ian R.; Kondrashov, Alexander; Young, Lucy A.; Dobbyn, Helen C.; Hill, Kirsti; Cannell, Ian G.; Stoneley, Mark; Kong, Yi-Wen; Cotes, Julia A.; Smith, Graeme C.M.; Wek, Ron; Hayes, Christopher; Gant, Timothy W.; Spriggs, Keith A.; Bushell, Martin; Willis, Anne E.

    2009-01-01

    UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5′ untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis. PMID:19451221

  19. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  20. Approaches to diagnose DNA mismatch repair gene defects in cancer.

    PubMed

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-02-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance

  1. Essential roles of Jab1 in cell survival, spontaneous DNA damage and DNA repair.

    PubMed

    Tian, L; Peng, G; Parant, J M; Leventaki, V; Drakos, E; Zhang, Q; Parker-Thornburg, J; Shackleford, T J; Dai, H; Lin, S-Y; Lozano, G; Rassidakis, G Z; Claret, F X

    2010-11-18

    Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to γ-radiation-induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibits its activity and represents a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival. PMID:20802511

  2. Temperature effects on survival and DNA repair in four freshwater cladoceran Daphnia species exposed to UV radiation.

    PubMed

    Connelly, Sandra J; Moeller, Robert E; Sanchez, Guillermo; Mitchell, David L

    2009-01-01

    fact suffered the highest damage rates. Melanin content in this species was not temperature dependent under the experimental growth conditions, and so did not contribute to temperature-dependent responses. It is evident that different species within the same genus have developed diverse biological responses to UVB. Our data strongly suggest that DNA damage is lethal to Daphnia and that photoenzymatic repair is the primary mechanism for removing these lesions. In the absence of light, few species are capable of removing any DNA damage. Surprisingly, the single species in which significant excision repair was detected did so only at reduced temperature. This temperature-dependence of excision repair is striking and may reflect adaptations of certain organisms to stress in a complex and changing environment. PMID:18700864

  3. Brain capacity for repair of oxidatively damaged DNA and preservation of neuronal function.

    PubMed

    Englander, Ella W

    2008-01-01

    Accumulation of oxidative DNA damage in the human brain has been implicated in etiologies of post-traumatic and age-associated declines in neuronal function. In neurons, because of high metabolic rates and prolonged life span, exposure to free radicals is intense and risk for accumulation of damaged DNA is amplified. While data indicate that the brain is equipped to repair nuclear and mitochondrial DNA, it is unclear whether repair is executed by distinct subsets of the DNA-repair machinery. Likewise, there are no firm assessments of brain capacity for accurate DNA repair under normal and more so compromised conditions. Consequently, the scope of DNA repair in the brain and the impact of resolution of oxidative lesions on neuronal survival and function remain largely unknown. This review considers evidences for brain levels and activities of the base excision repair (BER) pathway in the context of newly available, comprehensive in situ hybridization analyses of genes encoding repair enzymes. These analyses suggest that not all subsets of BER are equally represented in the brain. Because BER is the major repair process for oxidatively damaged DNA, to what extent parsimonious BER may contribute to development of neuronal dysfunction and brain injury under compromised conditions, is discussed. PMID:18374390

  4. Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2008-03-01

    Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

  5. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  6. Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

    PubMed

    Thielmann, H W; Vosberg, H P; Reygers, U

    1975-08-15

    Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds. PMID:170107

  7. TDP1 promotes assembly of non-homologous end joining protein complexes on DNA.

    PubMed

    Heo, Jinho; Li, Jing; Summerlin, Matthew; Hays, Annette; Katyal, Sachin; McKinnon, Peter J; Nitiss, Karin C; Nitiss, John L; Hanakahi, Leslyn A

    2015-06-01

    The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. PMID:25841101

  8. DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

    PubMed

    Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

    2016-03-01

    Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV. PMID:26573366

  9. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  10. DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks.

    PubMed

    Yu, Yaping; Mahaney, Brandi L; Yano, Ken-Ichi; Ye, Ruiqiong; Fang, Shujuan; Douglas, Pauline; Chen, David J; Lees-Miller, Susan P

    2008-10-01

    Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo. PMID:18644470

  11. GENETIC AND MOLECULAR ANALYSIS OF DNA DAMAGE REPAIR AND TOLERANCE PATHWAYS.

    SciTech Connect

    SUTHERLAND, B.M.

    2001-07-26

    Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts in a specific type (e.g., DNA) and conformation (double-stranded B conformation) of nucleic acid. Others, for example, nucleotide excision repair, deal with larger classes of damages, in this case bulky adducts in DNA. A detailed discussion of DNA repair mechanisms is beyond the scope of this article, but one can be found in the excellent book of Friedberg et al. [1] for further detail. However, some DNA damages and paths for repair of those damages important for photobiology will be outlined below as a basis for the specific examples of genetic and molecular analysis that will be presented below.

  12. Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation

    PubMed Central

    Al-Khalaf, Mohammad H; Blake, Leanne E; Larsen, Brian D; Bell, Ryan A; Brunette, Steve; Parks, Robin J; Rudnicki, Michael A; McKinnon, Peter J; Jeffrey Dilworth, F; Megeney, Lynn A

    2016-01-01

    Transient DNA strand break formation has been identified as an effective means to enhance gene expression in living cells. In the muscle lineage, cell differentiation is contingent upon the induction of caspase-mediated DNA strand breaks, which act to establish the terminal gene expression program. This coordinated DNA nicking is rapidly resolved, suggesting that myoblasts may deploy DNA repair machinery to stabilize the genome and entrench the differentiated phenotype. Here, we identify the base excision repair pathway component XRCC1 as an indispensable mediator of muscle differentiation. Caspase-triggered XRCC1 repair foci form rapidly within differentiating myonuclei, and then dissipate as the maturation program proceeds. Skeletal myoblast deletion of Xrcc1 does not have an impact on cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. Together, these results demonstrate that XRCC1 manages a temporally responsive DNA repair process to advance the muscle differentiation program. PMID:27462438

  13. Epigenetic regulation of DNA repair machinery in Helicobacter pylori-induced gastric carcinogenesis

    PubMed Central

    Santos, Juliana Carvalho; Ribeiro, Marcelo Lima

    2015-01-01

    Although thousands of DNA damaging events occur in each cell every day, efficient DNA repair pathways have evolved to counteract them. The DNA repair machinery plays a key role in maintaining genomic stability by avoiding the maintenance of mutations. The DNA repair enzymes continuously monitor the chromosomes to correct any damage that is caused by exogenous and endogenous mutagens. If DNA damage in proliferating cells is not repaired because of an inadequate expression of DNA repair genes, it might increase the risk of cancer. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes has been associated with carcinogenesis. Gastric cancer represents the second highest cause of cancer mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes during the lifetime. Among the risk factors, Helicobacter pylori (H. pylori) infection is considered the main driving factor to gastric cancer development. Thus, in this review, we summarize the current knowledge of the role of H. pylori infection on the epigenetic regulation of DNA repair machinery in gastric carcinogenesis. PMID:26290630

  14. Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans

    SciTech Connect

    Hartman, P.S.; Hevelone, J.; Dwarakanath, V.; Mitchell, D.L. )

    1989-06-01

    Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.

  15. Different repair kinetics for short and long DNA double-strand gaps in Saccharomyces cervisiae.

    PubMed

    Glasunov, A V; Frankenberg-Schwager, M; Frankenberg, D

    1995-10-01

    The kinetics of recombinational repair of plasmid DNA double-strand breaks (dsb) and gaps (dsg) of different sizes and ends were studied. For this purpose we used the mutant rad54-3 of the yeast Saccharomyces cerevisiae, which is temperature dependent with respect to genetic recombination and rejoining of dsb/dsg, allowing us to stop these processes by shifting cells to the restrictive temperature. We found that the kinetics of repair of cohesive-ended dsb and small gaps (up to 400 bp) are similar and characterized by two phases separated by a plateau. In contrast, large gap (1.4 kbp) repair proceeds with different kinetics exhibiting only the second phase. We also investigated the repair kinetics of 400 bp gaps introduced into plasmid DNA with and without homology to chromosomal DNA allowing recombinational repair and non-recombinational repair (ligation), respectively. We found that gaps introduced in plasmid sequences homologous to chromosomal DNA are rapidly repaired by recombination. In contrast, recircularization of the gapped plasmid by ligation is as slow and inefficient as ligation of a cohesive-ended dsb. The kinetics of repair of gapped plasmids may be explained by assuming a constitutive level of enzymes responsible for the first phase of recombinational repair, while inducible enzymes, which become available at the end of the plateau, carry out the second phase of repair. PMID:7594968

  16. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  17. Impeded repair of abasic site damaged lesions in DNA adsorbed over functionalized multiwalled carbon nanotube and graphene oxide.

    PubMed

    Kumari, Rina; Mondal, Titash; Bhowmick, Anil K; Das, Prolay

    2016-06-01

    The processing of abasic site DNA damage lesions in extracellular DNA in the presence of engineered carbon nanomaterials (CNMs) is demonstrated. The efficacy of the apurinic-apyrimidinic endonuclease 1 (APE1) in the cleavage of abasic site lesions in the presence of carboxylated multi-walled carbon nanotubes (MWCNT-COOH) and graphene oxide (GO) are compared. The CNMs were found to perturb the incision activity of APE1. The reason for such perturbation process was anticipated to take place either by the non-specific adsorption of APE1 over the free surface of the CNMs or steric hindrance offered by the CNM-DNA complex. Accordingly, bovine serum albumin (BSA) was selectively utilized to block the free surface of the CNM-DNA hybrid material. Further treatment of the CNM-DNA-BSA complex with APE1 resulted in a marginal increase in APE1 efficiency. This indicates that APE1 in solution is unable to process the abasic sites on DNA adsorbed over the CNMs. However, the cleavage activity of APE1 was restored in the presence of non-ionic surfactant (Tween 20) that inhibits adsorption of the DNA on the surface of the CNMs. The conformational deformation of the DNA, along with steric hindrance induced by the CNMs resulted in the inhibition of abasic site DNA repair by APE1. Moreover, appreciable changes in the secondary structure of APE1 adsorbed over the CNMs were observed that contribute further to the repair refractivity of the abasic sites. From a toxicological viewpoint, these findings can be extended to the study of the effect of engineered nanoparticles in the intracellular DNA repair process. PMID:27265379

  18. Statistical mechanics of topologically constrained DNA and nucleoprotein complexes

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan Michael

    A complex connection exists between the 3 dimensional topological state of DNA in living organisms and biological processes including gene expression, DNA replication, recombination and repair. A significant limitation in developing a detailed, quantitative understanding of this connection is due to a lack of rigorous methods to calculate statistical mechanical properties of DNA molecules with complex topologies, including supercoiling, looping and knotting. This dissertation's main focus is on developing such methods and applying them to realistic DNA and nucleoprotein models. In chapter 2, a method is presented to calculate free energies and J factors of protein mediated DNA loops by normal mode analysis (NMA). This method is similar to calculations performed previously but with several significant advances. We apply the method to the specific case of DNA looping mediated by Cre recombinase protein. J factors calculated by our method are compared to experimental measurements to extract geometric and elastic properties of the Cre-DNA synaptic complex. In particular, the results suggest the existence of a synaptic complex that is more flexible than previously expected and may be explained by a stable intermediate in the reaction pathway that deviates significantly from the planar crystal structure. Calculating free energies of DNA looping is difficult in general, especially when considering intermediate length scales such as plasmid sized DNA which may readily adopt multiple topological states. In chapter 3, a novel method is presented to obtain free energies of semiflexible biopolymers with fixed topologies and arbitrary ratios of contour length L to persistence length P. High accuracy is demonstrated by calculating free energies of specific DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex

  19. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

    PubMed

    Li, Hui; Swiercz, Rafal; Englander, Ella W

    2009-09-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136

  20. Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery

    PubMed Central

    Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W; Palle, Komaraiah

    2016-01-01

    Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB. PMID:26871286

  1. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes.

    PubMed

    Bekker-Jensen, Simon; Rendtlew Danielsen, Jannie; Fugger, Kasper; Gromova, Irina; Nerstedt, Annika; Lukas, Claudia; Bartek, Jiri; Lukas, Jiri; Mailand, Niels

    2010-01-01

    Regulatory ubiquitylation is emerging as an important mechanism to protect genome integrity in cells exposed to DNA damage. However, the spectrum of known ubiquitin regulators of the DNA damage response (DDR) is limited and their functional interplay is poorly understood. Here, we identify HERC2 as a factor that regulates ubiquitin-dependent retention of repair proteins on damaged chromosomes. In response to ionising radiation (IR), HERC2 forms a complex with RNF8, a ubiquitin ligase involved in the DDR. The HERC2-RNF8 interaction requires IR-inducible phosphorylation of HERC2 at Thr 4827, which in turn binds to the forkhead-associated (FHA) domain of RNF8. Mechanistically, we provide evidence that HERC2 facilitates assembly of the ubiquitin-conjugating enzyme Ubc13 with RNF8, thereby promoting DNA damage-induced formation of Lys 63-linked ubiquitin chains. We also show that HERC2 interacts with, and maintains the levels of, RNF168, another ubiquitin ligase operating downstream of RNF8 (Refs 7, 8). Consequently, knockdown of HERC2 abrogates ubiquitin-dependent retention of repair factors such as 53BP1, RAP80 and BRCA1. Together with the increased radiosensitivity of HERC2-depleted cells, these results uncover a regulatory layer in the orchestration of protein interactions on damaged chromosomes and they underscore the role of ubiquitin-mediated signalling in genome maintenance. PMID:20023648

  2. Spatio-temporal analysis of DNA damage repair using the X-ray microbeam

    NASA Astrophysics Data System (ADS)

    Schettino, G.; Ghita, M.; Prise, K. M.

    2010-10-01

    Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

  3. RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

    PubMed Central

    Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M.; Shih, Hsiu-Ming; Ann, David K.

    2016-01-01

    Abstract Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 re