The Experts below are selected from a list of 156 Experts worldwide ranked by ideXlab platform
Wei Xiao - One of the best experts on this subject based on the ideXlab platform.
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The Pol30-K196 residue plays a critical role in budding yeast DNA Postreplication Repair through interaction with Rad18.
DNA repair, 2016Co-Authors: Li Fan, Wei XiaoAbstract:PCNA plays critical roles in DNA replication and various DNA Repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA Postreplication Repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.
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The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair
PloS one, 2014Co-Authors: Lindsay G. Ball, Michelle Hanna, Amanda D. Lambrecht, Bryan A. Mitchell, Barry Ziola, Jennifer A. Cobb, Wei XiaoAbstract:Yeast DNA Postreplication Repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.
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DNA damage checkpoints are involved in Postreplication Repair
Genetics, 2006Co-Authors: Leslie Barbour, Lindsay G. Ball, Ke Zhang, Wei XiaoAbstract:Saccharomyces cerevisiae MMS2 encodes a ubiquitin-conjugating enzyme variant, belongs to the error-free branch of the RAD6 Postreplication Repair (PRR) pathway, and is parallel to the REV3-mediated mutagenesis branch. A mutation in genes of either the MMS2 or the REV3 branch does not result in extreme sensitivity to DNA-damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. Nevertheless, the double mutant is not as sensitive to DNA-damaging agents as a rad6 or rad18 mutant defective in the entire PRR pathway, suggesting the presence of an additional subpathway within PRR. A synthetic lethal screen was employed in the presence of a sublethal dose of a DNA-damaging agent to identify novel genes involved in PRR, which resulted in the isolation of RAD9 as a candidate PRR gene. Epistatic analysis showed that rad9 is synergistic to both mms2 and rev3 with respect to killing by methyl methanesulfonate (MMS), and the triple mutant is nearly as sensitive as the rad18 single mutant. In addition, rad9 rad18 is no more sensitive to MMS than the rad18 single mutant, suggesting that rad9 plays a role within the PRR pathway. Moreover, deletion of RAD9 reduces damage-induced mutagenesis and the mms2 spontaneous and induced mutagenesis is partially dependent on the RAD9 gene. We further demonstrated that the observed synergistic interactions apply to any two members between different branches of PRR and G1/S and G2/M checkpoint genes. These results suggest that a damage checkpoint is essential for tolerance mediated by both the error-free and error-prone branches of PRR.
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DNA Postreplication Repair modulated by ubiquitination and sumoylation.
Advances in protein chemistry, 2004Co-Authors: Landon Pastushok, Wei XiaoAbstract:Publisher Summary The living cell has developed ways to reduce or avoid detrimental changes to its genetic material. Numerous external and internal agents act and modify DNA. The incredible task of ensuring DNA fidelity and the survival of individual organisms is made possible by a variety of DNA Repair and replication processes. It is conceivable that both prokaryotic and eukaryotic organisms employ a centrally controlled postreplicative survival mechanism to deal with ssDNA gaps left by replication blocks during DNA synthesis. The discovery of novel signal transduction mechanisms in Postreplication Repair (PRR) through the sequential modification of proliferating cell-nuclear antigen (PCNA) by two E2–E3 ubiquitination complexes sets an important milestone in the research of eukaryotic DNA Repair. The Rad6–Rad18 complex bridges monoubiquitination of PCNA with error-prone Postreplication Repair (PRR), whereas Ubc13-Mms2-Rad5 ties novel Lys-63 polyubiquitination of PCNA with error-free PRR.
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Structural and functional conservation of error-free DNA Postreplication Repair in Schizosaccharomyces pombe.
DNA repair, 2002Co-Authors: Morgan L. Brown, Yu Zhu, Sean M. Hemmingsen, Wei XiaoAbstract:DNA Postreplication Repair (PRR) is a cellular process by which cells survive replication-blocking lesions without removing the lesion. In the budding yeast Saccharomyces cerevisiae , MMS2 plays a key role in the error-free PRR pathway: the mms2 null mutant displays an increased spontaneous mutation rate and sensitivity to a variety of DNA damaging agents. In contrast, its human homologs appear to play a different role. In order to address whether the MMS2 -mediated PRR pathway is conserved in eukaryotes, we isolated a Schizosaccharomyces pombe cDNA homologous to MMS2 , which we named spm2 + . Using spm2 + as a bait in a yeast two-hybrid screen, we identified a fission yeast cDNA homologous to UBC13 from various species and named it spu13 + . Two-hybrid analysis confirmed physical interaction between Spm2 and Spu13, and between Spm2 and budding yeast Ubc13. Genetic analysis shows that both spm2 + and spu13 + are able to functionally complement the corresponding budding yeast mutants. Furthermore, deletion of either spm2 + , spu13 + or both genes from fission yeast results in an increased sensitivity to DNA damaging agents, suggesting that spm2 + and spu13 + indeed function in PRR. The fact that the spm2 − spu13 − double mutant showed sensitivity similar to that of the single mutant indicates that these two gene products act at the same step. Hence, our data strongly support the hypothesis that the PRR function mediated by UBC13 – MMS2 is conserved throughout eukaryotes.
Louise Prakash - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast
2013Co-Authors: Venkateswarlu Gangavarapu, Satya Prakash, Sergio Santa R. Maria, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases � and � or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpointmediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DN
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requirement of replication checkpoint protein kinases mec1 rad53 for Postreplication Repair in yeast
Mbio, 2011Co-Authors: Venkateswarlu Gangavarapu, Satya Prakash, Sergio Santa R. Maria, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.
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Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast
mBio, 2011Co-Authors: Venkateswarlu Gangavarapu, Sergio R. Santa Maria, Satya Prakash, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.
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Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent Postreplication Repair of UV-damaged DNA in Saccharomyces cerevisiae.
Molecular and cellular biology, 2007Co-Authors: Sergio R. Santa Maria, Louise Prakash, Venkateswarlu Gangavarapu, Robert E. Johnson, Satya PrakashAbstract:Received 23 August 2007/Returned for modification 14 September 2007/Accepted 20 September 2007 In Saccharomyces cerevisiae, Postreplication Repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this Repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.
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Requirement of RAD5 and MMS2 for Postreplication Repair of UV-damaged DNA in Saccharomyces cerevisiae.
Molecular and cellular biology, 2002Co-Authors: Carlos A. Torres-ramos, Satya Prakash, Louise PrakashAbstract:UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the Postreplication Repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not Repaired in the rad5Δ and mms2Δ mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this Repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase η (Polη) but not in the absence of REV3-encoded DNA Polζ. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polζ, error-free translesion synthesis by Polη, and Postreplication Repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a “copy choice” type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Polδ. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.
Hong Lin - One of the best experts on this subject based on the ideXlab platform.
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Parathyroid Hormone-Like Hormone ( PTHLH ) Feedback Mitosis to Downstream DNA Replication Coupling Postreplication Repair-Induced Cell Proliferation Network in No-Tumor Hepatitis/Cirrhotic Tissues (HBV or HCV Infection) by Systems-Theoretical Analysi
Molecular Biology, 2012Co-Authors: Juxiang Huang, Lin Wang, Minghu Jiang, Hong Lin, Haizhen DiaoAbstract:Based on analysis of biological processes in the same low expression Parathyroid Hormone-Like Hormone (PTHLH) activated feedback mitosis and downstream DNA replication-mediated cell proliferation Gene Ontology (GO) network of no-tumor Hepatitis/Cirrhotic Tissues (HBV or HCV infection) compared with the corresponding high expression (fold change ≥2) activated (Gene Ontology) GO network of human Hepatocellular Carcinoma (HCC), we proposed PTHLH activated network that upstream consisted of cell division, cell proliferation, mitosis, mitotic checkpoint, nucleosome assembly, spindle organization and biogenesis; Downstream network cell cycle, cell cycle arrest, centrosome cycle, chromosome segregation, DNA replication, DNA replication checkpoint, G1/S transition of mitotic cell cycle, mitotic chromosome condensation, mitotic G2 checkpoint, mitotic spindle checkpoint, positive regulation of cell proliferation, regulation of cell proliferation, traversing start control point of mitotic cell cycle, positive regulation of DNA Repair, Postreplication Repair, DNA damage response, response to DNA damage stimulus, cell division, cell proliferation, mitosis, mitotic checkpoint, nucleosome assembly, spindle organization and biogenesis, as a result of feedback mitosis to downstream DNA replication coupling Postreplication Repair-induced cell proliferation in no-tumor hepatitis/cirrhotic tissues. Our hypothesis was verified by the different PTHLH activated feedback mitosis and downstream DNA replication-mediated cell proliferation GO network of no-tumor hepatitis/cirrhotic tissues compared with the corresponding inhibited GO network of HCC, or the same compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues. We constructed PTHLH feedback mitosis to downstream DNA replication coupling Postreplication Repair-induced cell proliferation network that upstream BUB1B activated PTHLH, and downstream PTHLH-activated BUB1B, CCNA2, CDC6, BRCA1 in no-tumor hepatitis/cirrhotic tissues from (Gene Expression Omnibus) GEO data set using gene regulatory network inference method and our programming.
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Tissue-specific transplantation antigen P35B (TSTA3) immune response-mediated metabolism coupling cell cycle to Postreplication Repair network in no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) by biocomputation.
Immunologic research, 2012Co-Authors: Lin Wang, Juxiang Huang, Minghu Jiang, Hong LinAbstract:We constructed the low-expression tissue-specific transplantation antigen P35B (TSTA3) immune response–mediated metabolism coupling cell cycle to Postreplication Repair network in no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) compared with high-expression (fold change ≥ 2) human hepatocellular carcinoma in GEO data set, by using integration of gene regulatory network inference method with gene ontology analysis of TSTA3-activated up- and downstream networks. Our results showed TSTA3 upstream–activated CCNB2, CKS1B, ELAVL3, GAS7, NQO1, NTN1, OCRL, PLA2G1B, REG3A, SSTR5, etc. and TSTA3 downstream–activated BAP1, BRCA1, CCL20, MCM2, MS4A2, NTN1, REG1A, TP53I11,VCAN, SLC16A3, etc. in no-tumor hepatitis/cirrhotic tissues. TSTA3-activated network enhanced the regulation of apoptosis, cyclin-dependent protein kinase activity, cell migration, insulin secretion, transcription, cell division, cell proliferation, DNA replication, Postreplication Repair, cell differentiation, T-cell homeostasis, neutrophil-mediated immunity, neutrophil chemotaxis, interleukin-8 production, inflammatory response, immune response, B-cell activation, humoral immune response, actin filament organization, xenobiotic metabolism, lipid metabolism, phospholipid metabolism, leukotriene biosynthesis, organismal lipid catabolism, phosphatidylcholine metabolism, arachidonic acid secretion, activation of phospholipase A2, deoxyribonucleotide biosynthesis, heterophilic cell adhesion, activation of MAPK activity, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, G-protein-coupled receptor protein signaling pathway, response to toxin, acute-phase response, DNA damage response, intercellular junction assembly, cell communication, and cell recognition, as a result of inducing immune response–mediated metabolism coupling cell cycle to Postreplication Repair in no-tumor hepatitis/cirrhotic tissues.
Stacey Broomfield - One of the best experts on this subject based on the ideXlab platform.
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DNA Postreplication Repair and mutagenesis in Saccharomyces cerevisiae.
Mutation research, 2001Co-Authors: Stacey Broomfield, Todd Hryciw, Wei XiaoAbstract:DNA Postreplication Repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.
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The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free Postreplication Repair pathways.
Genetics, 2000Co-Authors: Wei Xiao, Stacey Broomfield, Barbara L. Chow, Michelle HannaAbstract:The RAD6 Postreplication Repair and mutagenesis pathway is the only major radiation Repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 Postreplication Repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free Postreplication Repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of Postreplication Repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.
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Genetic interactions between error-prone and error-free Postreplication Repair pathways in Saccharomyces cerevisiae.
Mutation research, 1999Co-Authors: Wei Xiao, Barbara L. Chow, Todd Hryciw, Treena Fontanie, Silvia Bacchetti, Stacey BroomfieldAbstract:Evidence obtained from recent studies supports the existence of an error-free Postreplication Repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA Repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA Repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free Repair and mutagenesis, respectively.
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MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free Postreplication Repair pathway
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Stacey Broomfield, Barbara L. Chow, Wei XiaoAbstract:Among the three Saccharomyces cerevisiae DNA Repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate Repair pathways: an error-free Postreplication Repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free Postreplication Repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. & Prakash, S. (1977) Genetics 86, 33–55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in Postreplication Repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free Postreplication Repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6Δ1–9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6Δ1–9 and pol30-46, define the error-free mode of DNA Postreplication Repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.
Satya Prakash - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast
2013Co-Authors: Venkateswarlu Gangavarapu, Satya Prakash, Sergio Santa R. Maria, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases � and � or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpointmediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DN
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requirement of replication checkpoint protein kinases mec1 rad53 for Postreplication Repair in yeast
Mbio, 2011Co-Authors: Venkateswarlu Gangavarapu, Satya Prakash, Sergio Santa R. Maria, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.
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Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast
mBio, 2011Co-Authors: Venkateswarlu Gangavarapu, Sergio R. Santa Maria, Satya Prakash, Louise PrakashAbstract:ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that Postreplication Repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.
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Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent Postreplication Repair of UV-damaged DNA in Saccharomyces cerevisiae.
Molecular and cellular biology, 2007Co-Authors: Sergio R. Santa Maria, Louise Prakash, Venkateswarlu Gangavarapu, Robert E. Johnson, Satya PrakashAbstract:Received 23 August 2007/Returned for modification 14 September 2007/Accepted 20 September 2007 In Saccharomyces cerevisiae, Postreplication Repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this Repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.
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Requirement of RAD5 and MMS2 for Postreplication Repair of UV-damaged DNA in Saccharomyces cerevisiae.
Molecular and cellular biology, 2002Co-Authors: Carlos A. Torres-ramos, Satya Prakash, Louise PrakashAbstract:UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the Postreplication Repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not Repaired in the rad5Δ and mms2Δ mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this Repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase η (Polη) but not in the absence of REV3-encoded DNA Polζ. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polζ, error-free translesion synthesis by Polη, and Postreplication Repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a “copy choice” type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Polδ. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.
