The Experts below are selected from a list of 490659 Experts worldwide ranked by ideXlab platform
Peter M J Burgers - One of the best experts on this subject based on the ideXlab platform.
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lagging strand maturation factor dna2 is a component of the replication checkpoint initiation machinery
Genes & Development, 2013Co-Authors: Sandeep Kumar, Peter M J BurgersAbstract:Initiation of the DNA replication checkpoint in yeast is mainly mediated by Mec1 protein kinase, the ortholog of human ATR, while its homolog Tel1, the ortholog of human ATM, has a minor replication checkpoint function. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. Saccharomyces cerevisiae Dna2, a nuclease-helicase that is essential for Okazaki fragment maturation, is employed specifically during S phase to stimulate Mec1 kinase and initiate the replication checkpoint. Mutations (W128A and Y130A) in the unstructured N terminus of Dna2 abrogate its checkpoint function in vitro and in vivo. Dna2 shows partial redundancy for the replication checkpoint with checkpoint initiators 9-1-1 (S. cerevisiae Ddc1–Mec3–RAD17 and human Rad9–Rad1–Hus1) and Dpb11, the ortholog of human TopBP1. A triple mutant that eliminates the checkpoint functions of all three initiators abrogates the Mec1-dependent checkpoint.
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yeast dna replication protein dpb11 activates the mec1 atr checkpoint kinase
Journal of Biological Chemistry, 2008Co-Authors: Vasundhara M Navadgipatil, Peter M J BurgersAbstract:Abstract The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase (human ATR-ATRIP) initiates a signal transduction pathway in response to DNA damage and replication stress to mediate cell cycle arrest. The yeast DNA damage checkpoint clamp Ddc1-Mec3-RAD17 (human Rad9-Hus1-Rad1: 9-1-1) is loaded around effector DNA and thereby activates Mec1 kinase. Dpb11 (Schizosaccharomyces pombe Cut5/Rad4 or human TopBP1) is an essential protein required for the initiation of DNA replication and has a role in checkpoint activation. In this study, we demonstrate that Dpb11 directly activates the Mec1 kinase in phosphorylating the downstream effector kinase Rad53 (human Chk1/2) and DNA bound RPA. However, DNA was not required for Dpb11 to function as an activator. Dpb11 and yeast 9-1-1 independently activate Mec1, but substantial synergism in activation was observed when both activators were present. Our studies suggest that Dpb11 and 9-1-1 may partially compensate for each other during yeast checkpoint function.
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function of RAD17 mec3 ddc1 and its partial complexes in the dna damage checkpoint
DNA Repair, 2005Co-Authors: Jerzy Majka, Peter M J BurgersAbstract:The Saccharomyces cerevisiaeheterotrimeric checkpoint clamp consisting of the RAD17, Mec3, and Ddc1 subunits (RAD17/3/1, the 9-1-1 complex in humans) is an early response factor to DNA damage in a signal transduction pathway leading to the activation of the checkpoint system and eventually to cell cycle arrest. These subunits show structural similarities with the replication clamp PCNA and indeed, it was demonstrated in vitro that RAD17/3/1 could be loaded onto DNA by checkpoint specific clamp loader Rad24-RFC, analogous to the PCNARFC clamp–clamp loader system. We have studied the interactions between the checkpoint clamp subunits and the activity of partial clamp complexes. We find that none of the possible partial complexes makes up a clamp that can be loaded onto DNA by Rad24-RFC. In agreement, overexpression of DDC1 or RAD17 in a MEC3� strain, or of MEC3 or RAD17 in a DDC1∆ strain shows no rescue of damage sensitivity. © 2005 Elsevier B.V. All rights reserved.
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yeast RAD17 mec3 ddc1 a sliding clamp for the dna damage checkpoint
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Jerzy Majka, Peter M J BurgersAbstract:The Saccharomyces cerevisiae Rad24 and RAD17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. RAD17 forms a complex with Mec3 and Ddc1 (RAD17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp RAD17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the RAD17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the RAD17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. RAD17/3/1 showed no detectable exonuclease activity.
Naoto Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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nuclear translocation promotes proteasomal degradation of human RAD17 protein through the n terminal destruction boxes
Journal of Biological Chemistry, 2021Co-Authors: Yasunori Fukumoto, Masayoshi Ikeuchi, Yuji Nakayama, Naoto Yamaguchi, Tyuji Hoshino, Yasumitsu OgraAbstract:The ATR pathway is one of the major DNA damage checkpoints, and RAD17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. RAD17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of RAD17 is important for recovery from the ATR pathway. Here, we identified several RAD17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of RAD17. RAD17 Δ230–270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of RAD17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of RAD17 is functionally linked to the proteasomal degradation. The ATP-binding activity of RAD17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The RAD17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of RAD17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of RAD17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of RAD17. We propose a model in which RAD17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.
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human RAD17 c terminal tail is phosphorylated by concerted action of ck1δ e and ck2 to promote interaction with the 9 1 1 complex
Biochemical and Biophysical Research Communications, 2019Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the RAD17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate RAD17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between RAD17 and the 9‒1‒1 complex. In human RAD17, the Y665 and S667 residues are conserved in iVERGE. The RAD17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of RAD17, T670 in human RAD17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. RAD17-T670 is phosphorylated by casein kinase 1δ/e. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.
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casein kinase 2 promotes interaction between RAD17 and the 9 1 1 complex through constitutive phosphorylation of the c terminal tail of human RAD17
Biochemical and Biophysical Research Communications, 2018Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Yasumitsu Ogra, Kazuaki Takahashi, Noriyuki Suzuki, Naoto YamaguchiAbstract:Abstract An interaction between the RAD17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human RAD17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that RAD17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of RAD17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. RAD17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between RAD17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the RAD17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate RAD17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE.
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the polyanionic c terminal tail of human RAD17 regulates interaction with the 9 1 1 complex
The Japanese Biochemical Society The Molecular Biology Society of Japan, 2017Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract In the activation and maintenance of ATR-dependent DNA damage checkpoint, the interaction between the RAD17–RFC2-5 and 9–1–1 complexes is essential, however, the regulatory mechanism of the interaction is not known. Here we show that vertebrate RAD17 proteins contain a polyanionic 12-amino acid sequence in the C-terminal ends that is important for the 9–1–1 interaction. We demonstrate that the C-terminal tail contains a conserved sequence designated iVERGE that must be intact for the 9–1–1 interaction and contains potential posttranslational modification sites. Our data raise a possibility that the RAD17 C-terminal tail is a molecular switch that regulates the 9–1–1 interaction and the ATR pathway.
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the kyxxl motif in RAD17 protein is essential for the interaction with the 9 1 1 complex
Biochemical and Biophysical Research Communications, 2016Co-Authors: Yasunori Fukumoto, Masayoshi Ikeuchi, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The RAD17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of RAD17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced RAD17 phosphorylation is increased in the RAD17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required for RAD17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the RAD17 phosphorylation. We also show that the nucleotide-binding activity of RAD17 is required for its nuclear localization.
William G Dunphy - One of the best experts on this subject based on the ideXlab platform.
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The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks
Molecular biology of the cell, 2013Co-Authors: Joon Lee, William G DunphyAbstract:The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), RAD17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both RAD17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, RAD17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, RAD17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure–function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.
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RAD17 plays a central role in establishment of the interaction between topbp1 and the rad9 hus1 rad1 complex at stalled replication forks
Molecular Biology of the Cell, 2010Co-Authors: Joon Lee, William G DunphyAbstract:RAD17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that RAD17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that RAD17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with RAD17 mutants indicate that binding of ATP to RAD17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by RAD17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, RAD17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both RAD17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a RAD17-dependent manner before activation of Chk1.
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claspin a chk1 regulatory protein monitors dna replication on chromatin independently of rpa atr and RAD17
Molecular Cell, 2003Co-Authors: Joon Lee, Akiko Kumagai, William G DunphyAbstract:Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and RAD17 require RPA for association with DNA. Claspin, ATR, and RAD17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and RAD17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.
Yasunori Fukumoto - One of the best experts on this subject based on the ideXlab platform.
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nuclear translocation promotes proteasomal degradation of human RAD17 protein through the n terminal destruction boxes
Journal of Biological Chemistry, 2021Co-Authors: Yasunori Fukumoto, Masayoshi Ikeuchi, Yuji Nakayama, Naoto Yamaguchi, Tyuji Hoshino, Yasumitsu OgraAbstract:The ATR pathway is one of the major DNA damage checkpoints, and RAD17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. RAD17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of RAD17 is important for recovery from the ATR pathway. Here, we identified several RAD17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of RAD17. RAD17 Δ230–270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of RAD17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of RAD17 is functionally linked to the proteasomal degradation. The ATP-binding activity of RAD17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The RAD17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of RAD17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of RAD17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of RAD17. We propose a model in which RAD17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.
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human RAD17 c terminal tail is phosphorylated by concerted action of ck1δ e and ck2 to promote interaction with the 9 1 1 complex
Biochemical and Biophysical Research Communications, 2019Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the RAD17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate RAD17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between RAD17 and the 9‒1‒1 complex. In human RAD17, the Y665 and S667 residues are conserved in iVERGE. The RAD17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of RAD17, T670 in human RAD17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. RAD17-T670 is phosphorylated by casein kinase 1δ/e. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.
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casein kinase 2 promotes interaction between RAD17 and the 9 1 1 complex through constitutive phosphorylation of the c terminal tail of human RAD17
Biochemical and Biophysical Research Communications, 2018Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Yasumitsu Ogra, Kazuaki Takahashi, Noriyuki Suzuki, Naoto YamaguchiAbstract:Abstract An interaction between the RAD17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human RAD17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that RAD17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of RAD17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. RAD17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between RAD17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the RAD17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate RAD17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE.
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the polyanionic c terminal tail of human RAD17 regulates interaction with the 9 1 1 complex
The Japanese Biochemical Society The Molecular Biology Society of Japan, 2017Co-Authors: Yasunori Fukumoto, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract In the activation and maintenance of ATR-dependent DNA damage checkpoint, the interaction between the RAD17–RFC2-5 and 9–1–1 complexes is essential, however, the regulatory mechanism of the interaction is not known. Here we show that vertebrate RAD17 proteins contain a polyanionic 12-amino acid sequence in the C-terminal ends that is important for the 9–1–1 interaction. We demonstrate that the C-terminal tail contains a conserved sequence designated iVERGE that must be intact for the 9–1–1 interaction and contains potential posttranslational modification sites. Our data raise a possibility that the RAD17 C-terminal tail is a molecular switch that regulates the 9–1–1 interaction and the ATR pathway.
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the kyxxl motif in RAD17 protein is essential for the interaction with the 9 1 1 complex
Biochemical and Biophysical Research Communications, 2016Co-Authors: Yasunori Fukumoto, Masayoshi Ikeuchi, Yuji Nakayama, Naoto YamaguchiAbstract:Abstract ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The RAD17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of RAD17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced RAD17 phosphorylation is increased in the RAD17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required for RAD17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the RAD17 phosphorylation. We also show that the nucleotide-binding activity of RAD17 is required for its nuclear localization.
Christopher W Lawrence - One of the best experts on this subject based on the ideXlab platform.
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the error free component of the rad6 rad18 dna damage tolerance pathway of budding yeast employs sister strand recombination
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Hengshan Zhang, Christopher W LawrenceAbstract:Evidence for an error-free DNA damage tolerance process in eukaryotes (also called postreplication repair) has existed for more than two decades, but its underlying mechanism, although known to be different from that in prokaryotes, has remained elusive. We have investigated this mechanism in Saccharomyces cerevisiae, in which it is the major component of the RAD6/RAD18 pathway, by transforming an isogenic set of rad1Δ excision-defective strains with plasmids that carry a single thymine-thymine pyrimidine (6-4) pyrimidinone photoadduct in each strand at staggered positions 28 base pairs apart. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the events that can lead to the completion of replication: sister-strand recombination or translesion replication on one or the other strand. Despite the severe block to replication that these lesions impose, we find that more than half of the plasmids were fully replicated in a rad1Δ strain and that >90% of them achieved this end by recombination between partially replicated sister strands within the interlesion region. Approximately 60-70% of these events depended on the error-free component of the RAD6/RAD18 pathway, with the remaining events depended on RAD52; these two processes account for almost all of the recombination, which depended neither on DNA polymerase ζ nor on mismatch repair. We conclude that the error-free component of the RAD6/RAD18 pathway completes replication by a mechanism employing recombination between partially replicated sister strands, possibly by means of transient template strand switching or copy choice.
