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• W2069894032 abstract "DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. Rad5 is required to resolve forks stalled by DNA damage and to restart replication Homologous recombination factors function along with Rad5 in the same pathway Rad5 enables a Holliday junction to form between sister chromatids at a stalled fork Replication fork recombination requires Rad5 ATPase and ubiquitin ligase activities The capability to replicate damaged DNA templates is crucial for living cells, because prolonged stalling or collapse of replication forks at DNA lesions can lead to cell death or genomic instability and cancer (Kolodner et al., 2002Kolodner R.D. Putnam C.D. Myung K. Maintenance of genome stability in Saccharomyces cerevisiae.Science. 2002; 297: 552-557Crossref PubMed Scopus (385) Google Scholar). In Saccharomyces cerevisiae, replication of damaged DNA requires the RAD6 epistasis group of genes (Lawrence, 1994Lawrence C. The RAD6 DNA repair pathway in Saccharomyces cerevisiae: what does it do, and how does it do it?.Bioessays. 1994; 16: 253-258Crossref PubMed Scopus (143) Google Scholar). Genetic analysis identified two distinct DNA damage bypass pathways: translesion synthesis, error prone for most types of damage, but operating with high fidelity for UV lesions (Prakash et al., 2005Prakash S. Johnson R.E. Prakash L. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (791) Google Scholar), and the alternative error-free bypass, hypothesized to involve a template switch in which the blocked nascent strand uses the undamaged sister chromatid as a temporary replication template (Xiao et al., 2000Xiao W. Chow B.L. Broomfield S. Hanna M. The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways.Genetics. 2000; 155: 1633-1641Crossref PubMed Google Scholar). DNA damage bypass is associated with posttranslational modifications of proliferating cell nuclear antigen (PCNA) (Ulrich, 2009Ulrich H.D. Regulating post-translational modifications of the eukaryotic replication clamp PCNA.DNA Repair (Amst.). 2009; 8: 461-469Crossref PubMed Scopus (126) Google Scholar). In the error-prone pathway, PCNA monoubiquitination on lysine-164 by the Rad6/Rad18 E2/E3 ubiquitin-conjugating complex activates translesion synthesis by damage-tolerant DNA polymerases (Waters et al., 2009Waters L.S. Minesinger B.K. Wiltrout M.E. D'Souza S. Woodruff R.V. Walker G.C. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.Microbiol. Mol. Biol. Rev. 2009; 73: 134-154Crossref PubMed Scopus (419) Google Scholar). The alternative, error-free, template-switch bypass is controlled by Rad5, Mms2, and Ubc13 that form a second ubiquitin-conjugating complex (Chang and Cimprich, 2009Chang D.J. Cimprich K.A. DNA damage tolerance: when it's OK to make mistakes.Nat. Chem. Biol. 2009; 5: 82-90Crossref PubMed Scopus (138) Google Scholar). Rad5, through its RING-domain E3 activity, stimulates the Mms2/Ubc13-dependent synthesis of lysine-63-linked polyubiquitin chains onto monoubiquitinated PCNA (Hoege et al., 2002Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1657) Google Scholar, Ulrich, 2009Ulrich H.D. Regulating post-translational modifications of the eukaryotic replication clamp PCNA.DNA Repair (Amst.). 2009; 8: 461-469Crossref PubMed Scopus (126) Google Scholar). Rad5 exhibits also a DNA-dependent ATPase activity through its helicase domain, which has homology to those of chromatin remodeling factors in the Snf2 family (Johnson et al., 1992Johnson R.E. Henderson S.T. Petes T.D. Prakash S. Bankmann M. Prakash L. Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome.Mol. Cell. Biol. 1992; 12: 3807-3818Crossref PubMed Scopus (187) Google Scholar). With limited physical data, it is not understood how the template switch bypass is induced and executed, and what roles the Rad5 activities play at stalled replication forks in vivo. One model of template switch involves the regression of a blocked replication fork into a four-way “chicken foot” structure, possibly without any requirement for recombination proteins. Regressed forks have been visualized in vivo in mammalian cells in certain conditions (Higgins et al., 1976Higgins N.P. Kato K. Strauss B. A model for replication repair in mammalian cells.J. Mol. Biol. 1976; 101: 417-425Crossref PubMed Scopus (353) Google Scholar) and synthetic forked DNA structures can be reversed in vitro by Rad5 exclusively through its ATPase activity (Blastyak et al., 2007Blastyak A. Pinter L. Unk I. Prakash L. Prakash S. Haracska L. Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression.Mol. Cell. 2007; 28: 167-175Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). However, there is no direct support for this model in budding yeast cells, in which regressed replication forks have been observed only in pathologic conditions (Sogo et al., 2002Sogo J.M. Lopes M. Foiani M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.Science. 2002; 297: 599-602Crossref PubMed Scopus (650) Google Scholar). An alternative template-switch model entails a recombination-like invasion of the opposite undamaged sister chromatid by the blocked nascent strand either at a replication fork upon leading-strand stalling or behind the fork in the case of postreplicative filling of gaps formed opposite DNA lesions. Recombination would generate sister chromatid exchanges (SCE) and branched DNA structures with Holliday junctions between sister chromatids (Krogh and Symington, 2004Krogh B.O. Symington L.S. Recombination proteins in yeast.Annu. Rev. Genet. 2004; 38: 233-271Crossref PubMed Scopus (605) Google Scholar, San Filippo et al., 2008San Filippo J. Sung P. Klein H. Mechanism of eukaryotic homologous recombination.Annu. Rev. Biochem. 2008; 77: 229-257Crossref PubMed Scopus (1055) Google Scholar). Recombination factors are required for gap-filling repair and for the formation of SCE and X-shaped DNA structures (X-DNA) in UV-treated nucleotide excision repair (NER) mutant cells, but their role is independent of Rad5 damage-bypass, and they are thought to contribute to lagging-strand lesion bypass (Kadyk and Hartwell, 1993Kadyk L.C. Hartwell L.H. Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae.Genetics. 1993; 133: 469-487Crossref PubMed Google Scholar, Neecke et al., 1999Neecke H. Lucchini G. Longhese M.P. Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.EMBO J. 1999; 18: 4485-4497Crossref PubMed Scopus (82) Google Scholar, Zhang and Lawrence, 2005Zhang H. Lawrence C.W. The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination.Proc. Natl. Acad. Sci. USA. 2005; 102: 15954-15959Crossref PubMed Scopus (151) Google Scholar, Gangavarapu et al., 2007Gangavarapu V. Prakash S. Prakash L. Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.Mol. Cell. Biol. 2007; 27: 7758-7764Crossref PubMed Scopus (79) Google Scholar). The X-DNA induced by methyl-methanesulfonate (MMS) exposure in sgs1 or top3 mutants has hemicatenane properties with no canonical Holliday junctions but rather ssDNA-containing sister chromatid junctions (Liberi et al., 2005Liberi G. Maffioletti G. Lucca C. Chiolo I. Baryshnikova A. Cotta-Ramusino C. Lopes M. Pellicioli A. Haber J.E. Foiani M. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.Genes Dev. 2005; 19: 339-350Crossref PubMed Scopus (264) Google Scholar). Hemicatenane formation is Rad18-dependent and thought to promote gap filling in the lagging strand behind the replication fork (Branzei et al., 2008Branzei D. Vanoli F. Foiani M. SUMOylation regulates Rad18-mediated template switch.Nature. 2008; 456: 915-920Crossref PubMed Scopus (203) Google Scholar). A template switch mechanism at stalled replication forks in the leading strand has not been identified, and possible roles for Rad5 and recombination factors in restarting replication at stalled forks are not known. DNA damage bypass has been studied using mainly UV irradiation or MMS as DNA-damaging agents. Although the corresponding lesions appear to slow the rate of replication-fork progression (Tercero and Diffley, 2001Tercero J.A. Diffley J.F. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint.Nature. 2001; 412: 553-557Crossref PubMed Scopus (541) Google Scholar, Lopes et al., 2006Lopes M. Foiani M. Sogo J.M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.Mol. Cell. 2006; 21: 15-27Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), fork stalling has not been directly observed and linked to recombination-mediated restart of replication. Also, the natural occurrence of UV lesions has resulted in evolutionary selection for efficient NER and error-free translesion synthesis pathways (Prakash et al., 2005Prakash S. Johnson R.E. Prakash L. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (791) Google Scholar), with Rad5-dependent damage bypass playing only a lesser role in UV-damage survival. However, the intricate relationship between these repair and damage bypass pathways, and their coordination with DNA replication and recombination, may be different for other types of DNA lesions. Earlier we found that DNA alkylation by adozelesin induces site-specific fork stalling at a replication origin analyzed by two-dimensional (2D) gel electrophoresis (Wang et al., 2001Wang Y. Beerman T.A. Kowalski D. Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants.Cancer Res. 2001; 61: 3787-3794PubMed Google Scholar). Adozelesin, a DNA alkylating drug, forms bulky adducts, which, unlike UV lesions, target the DNA minor groove (Swenson et al., 1982Swenson D.H. Li L.H. Hurley L.H. Rokem J.S. Petzold G.L. Dayton B.D. Wallace T.L. Lin A.H. Krueger W.C. Mechanism of interaction of CC-1065 (NSC 298223) with DNA.Cancer Res. 1982; 42: 2821-2828PubMed Google Scholar). This, together with its specificity for AT-rich DNA sequences (Weiland and Dooley, 1991Weiland K.L. Dooley T.P. In vitro and in vivo DNA bonding by the CC-1065 analogue U-73975.Biochemistry. 1991; 30: 7559-7565Crossref PubMed Scopus (46) Google Scholar), contributes to the site-specific fork stalling. The ability to directly detect stalled forks and other damage-induced DNA structures in replication intermediates through 2D-gel analysis can provide molecular evidence for DNA damage bypass mechanisms in vivo and help identify the roles of specific proteins. In the present work, we took advantage of this approach and used adozelesin, as well as MMS, to examine the role of Rad5-dependent damage bypass in DNA replication and the interplay with homologous recombination. We show that Rad5 and recombination factors are required for complete replication of damaged chromosomal DNA and for the formation of X-DNA structures with Holliday junctions at forks stalled at replication origins. In the absence of Rad5 or recombination factors, stalled forks were defective in restarting replication and formed abnormal structures. The ATPase and ubiquitin ligase activities of Rad5 were both required for full damage bypass function. Our findings indicate that multiple activities of Rad5 coordinate replication template switch events at stalled forks through homologous recombination between sister chromatids. An investigation of cell survival in a panel of yeast deletion strains defective in various DNA repair pathways revealed that inactivation of Rad5-dependent DNA damage bypass or homologous recombination conferred extremely high sensitivity to adozelesin when compared with inactivation of NER or translesion synthesis (data not shown). To determine whether the Rad5-dependent tolerance is related to replication over adozelesin-induced DNA lesions, we restricted DNA damage to S phase in cells progressing synchronously from a G1-block. Following a 1 hr drug exposure in S phase, cells were allowed to recover in drug-free medium for up to 18 hr (Figure 1A ). We analyzed the dynamics of damaged chromosomal DNA replication in wild-type (WT) and rad5Δ cells by pulsed-field gel electrophoresis. The assay resolves linear chromosomal DNA from agarose-embedded cells, but not DNA containing replication bubbles, that is trapped inside the agarose plugs in the loading wells (Mesner et al., 2006Mesner L.D. Crawford E.L. Hamlin J.L. Isolating apparently pure libraries of replication origins from complex genomes.Mol. Cell. 2006; 21: 719-726Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Accordingly, intact chromosomal DNA from G1-blocked cells was separated as individual bands, whereas, after 60 min of drug exposure in S phase, most of the chromosomal DNA from WT and rad5Δ cells was retained in the loading wells (Figure 1B), indicating ongoing DNA replication. This is consistent with the DNA content detected by flow cytometry, intermediate between 1C and 2C (Figure 1C). Chromosomal DNA from WT cells reentered the gel at 3 hr of recovery in drug-free medium, with progressively increasing amounts at later times (6 and 18 hr) due to subsequent duplications. This correlates with the flow-cytometry profiles as well as the microscopy and cell counting (Figures 1C, 1D, and 1E) showing nuclear division and replicated 2C DNA at 3 hr, actively dividing cells at 6 hr, and stationary cells with 1C DNA content at 18 hr. In marked contrast to WT cells, most of the chromosomal DNA from rad5Δ cells remained in the loading well throughout the recovery period (Figure 1B), indicating that, in the absence of Rad5, damaged DNA replication was not completed. This in turn blocked nuclear division and cells failed to undergo mitosis, becoming progressively larger with time (Figures 1D and 1E). The apparently higher than 2C DNA content (Figure 1C) at 6 and 18 hr is due partly to the dramatic increase in cell volume, which alters the optical transmission of the fluorescent signal in flow cytometry (Haase and Reed, 2002Haase S.B. Reed S.I. Improved flow cytometric analysis of the budding yeast cell cycle.Cell Cycle. 2002; 1: 132-136Crossref PubMed Scopus (56) Google Scholar), and to the increasing mitochondrial DNA content, because we did not observe higher-than-2C signals in similar experiments performed with rho0 rad5Δ mutants devoid of mitochondrial DNA (data not shown). Similar to adozelesin, MMS also led to incomplete chromosomal DNA replication in rad5Δ cells after S-phase exposure (see Figure S1 available online). These results demonstrate that Rad5 is required for the completion of chromosome replication in the presence of alkylation DNA damage. We previously showed that, in asynchronous WT cells exposed to adozelesin, replication-fork progression stalls at a specific site within a replication origin, ORI305 (Wang et al., 2001Wang Y. Beerman T.A. Kowalski D. Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants.Cancer Res. 2001; 61: 3787-3794PubMed Google Scholar). The inability of rad5Δ cells to complete chromosomal DNA replication in the presence of adozelesin could be caused by prolonged fork stalling or fork collapse at such a site. Alternatively, Rad5 inactivation in the presence of DNA damage could inhibit replication origin activity. To determine how adozelesin affects DNA replication at a molecular level in WT and rad5Δ cells progressing synchronously through S phase, we employed 2D-gel analysis of replication intermediates in a fragment containing early-firing origin ORI305 (Huang and Kowalski, 1993Huang R.Y. Kowalski D. A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome.EMBO J. 1993; 12: 4521-4531Crossref PubMed Scopus (90) Google Scholar) (Figure 2A ). Schematics of the signals resolved by this method in WT cells are illustrated in Figures 2B and 2C (top right panels). Both WT and rad5Δ cells showed normal replication intermediates during the release from the G1 block in medium without drug (Figure 2B). ORI305 had a maximum of activity at 30 min postrelease as indicated by strong bubble and late-Y signals, which decreased progressively at 45 and 60 min. A faint X signal was visible at 30 min in both WT and rad5Δ cells, representing hemicatenanes that form during normal replication (Lopes et al., 2003Lopes M. Cotta-Ramusino C. Liberi G. Foiani M. Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms.Mol. Cell. 2003; 12: 1499-1510Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Upon release into S phase in the presence of adozelesin, the ORI305 firing was the same in WT and rad5Δ cells and similar to untreated controls in this time frame, indicating that the absence of Rad5 does not influence the origin activity. However, the presence of the drug induced a strong fork-stalling signal at ORI305 at the peak of the Y arc and an intensified the late-Y arc in both WT and rad5Δ cells (Figure 2C). The fork stalling, first seen at 30 min after the G1-block release, persisted at 45 and 60 min. The lack of an early-Y arc at 30 min indicates that the forks involved in stalling were generated locally and not at a distal origin. In WT cells, the drug also induced a strong vertical spike signal containing X-DNA. The X signal migrated similarly to the 2N linear DNA spot in the first dimension of electrophoresis, indicating fully replicated DNA structures containing joined sister chromatids. The X spike was most intense at its upper margin, which contains branched DNA structures with centered junctions, corresponding to the position of the fork-stalling signal in the middle of the fragment and proximal to the replication origin. Less intense signals on the X spike were associated with minor fork-stalling signals on the late Y arc. The X-spike intensity increased from 30 to 45 min, along with a decrease in the bubble-arc signal, and remained strong also at 60 min. A Y to X arc signal, connecting the fork-stalling signal to the upper margin of the X spike, was observed most strongly at 60 min. The convex shape of the high-rising Y to X arc resembles that of a bubble arc, as opposed to the linear-shaped signal generated by an incoming fork approaching a centrally located stalled fork (Martín-Parras et al., 1991Martín-Parras L. Hernández P. Martínez-Robles M.L. Schvartzman J.B. Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis.J. Mol. Biol. 1991; 220: 843-853Crossref PubMed Scopus (68) Google Scholar). In rad5Δ cells, the X-DNA was greatly reduced when compared with WT cells (Figure 2C), despite the presence of a similar fork-stalling signal. Also, the Y to X arc was eliminated, but not the faint early-Y arc due to passive replication of ORI305 by minimal incoming forks (60 min), implying that the Y to X arc contains Rad5-dependent replication intermediates elongating away from the origin following damage bypass and stalled fork restart to form fully duplicated X-DNA. Finally, the late-Y arc in rad5Δ cells showed an atypical concave shape (compared with WT), indicating the formation of abnormal replication fork structures. We observed adozelesin-induced fork stalling also at another replication origin, ORI508 (Figure S2A). In rad5Δ cells, the fork stalling was again accompanied by deficient X-DNA formation, loss of the Y to X arc, and an abnormal late-Y arc (Figure S2A). These results imply that, during adozelesin exposure, Rad5 promotes damage bypass through the formation of junctions between sister chromatids at stalled forks, giving rise to replication intermediates that form X-DNA molecules when fully duplicated. Damage bypass failure in rad5Δ cells leads to abnormal replication fork structures, possibly due to prolonged fork stalling. The inability to complete the replication of damaged DNA and the reduction in X-DNA at fork-stalling sites in rad5Δ cells, suggest that a Rad5-mediated template switch that joins sister chromatids is required to restart replication at stalled forks. To determine the fate of stalled replication forks following adozelesin exposure in S phase in WT and rad5Δ cells, we analyzed the replication intermediates during a subsequent recovery period in the absence of drug. The experimental outline was similar to that described in Figure 1A. In WT cells, the replication intermediates at ORI305, including the fork-stalling signal and the X-DNA, still visible at 30 min of recovery, were reduced to a minimum level at 1 and 2 hr, indicating the completion of DNA replication in this region (Figure 3A ), which preceded the completion of chromosomal DNA replication observed by PFGE at 3 hr (Figure 1B). As most WT cells entered a new S phase between 3 and 6 hr of recovery (Figures 1B, 1C and 1D), strong bubble and late-Y arcs reappeared at ORI305 at 4 and 6 hr with no fork stalling and a faint X signal (Figure 3A), a profile similar to that observed during an unperturbed S phase (Figure 2B). This suggests that adozelesin adducts were removed from DNA, and thus caused no more fork stalling. At 18 hr, the replication intermediates were again at minimum levels (Figure 3A), as cells accumulated in stationary phase with 1C DNA content (Figure 1C). 2D-gel analysis at early firing origin ORI508 revealed similar structures and kinetics (Figure S2A). In contrast to WT cells, in rad5Δ cells, the fork-stalling signal at ORI305 was still present at 1 hr of recovery, along with the abnormal late-Y arc. Both the fork-stalling and the late-Y signals became progressively wider and less defined at 30 min to 1 hr of recovery (Figure 3A, brackets), indicating further alterations in the DNA structure at stalled forks in the absence of proper replication restart. At 2 hr of recovery, the replication intermediates were reduced to a low background level, which, in contrast with WT cells, was detected for up to 18 hr with no subsequent origin firing (Figure 3A). Again, a similar phenotype was observed at ORI508 (Figure S2A). These results indicate that replication forks stalled by DNA damage at early-firing origins cannot properly restart replication in the absence of Rad5 and the associated X-DNA, and progressively change into abnormal DNA structures. Late-firing origins are repressed by the intra-S-phase checkpoint in the presence of DNA damage (Shirahige et al., 1998Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Regulation of DNA-replication origins during cell-cycle progression.Nature. 1998; 395: 618-621Crossref PubMed Scopus (350) Google Scholar, Santocanale and Diffley, 1998Santocanale C. Diffley J.F. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication.Nature. 1998; 395: 615-618Crossref PubMed Scopus (514) Google Scholar). Accordingly, we detected no bubble arcs at late-firing origins ORI1412 and ORI501 in WT or rad5Δ cells after 1 hr of adozelesin exposure in S phase (Figure 3B and Figure S2B). After the drug removal, in WT cells, the late origins showed activity at 30 min, 1 and 2 hr of recovery as cells completed the first S phase, as well as at 4 and 6 hr, during the subsequent S phase (Figure 3B and Figure S2B). Interestingly, in rad5Δ cells, late origins also showed activity at 2, 4 and 6 hr of recovery (Figure 3B and Figure S2B). Unlike early origin firing, the late origin activity was not accompanied by fork stalling in WT or rad5Δ cells, and it did not result in abundant X-DNA formation in WT cells. Taken together, these results show that during recovery from DNA damage, Rad5 is required to form X-DNA structures and restart replication forks at early-firing origins but not at late origins, whose activity was repressed during the prior exposure to DNA damage, possibly allowing lesion repair. The requirement of Rad5 for X-DNA formation during replication under genotoxic stress is not limited to adozelesin-induced damage. We observed a similar requirement during MMS exposure in S phase (Figure 4A ). However, in contrast to adozelesin, MMS did not induce visible fork stalling at discrete sites within the ORI305-containing DNA fragment. This could be due to wide variations in size and DNA sequence specificity between MMS lesions and bulky adozelesin adducts leading to a different degree or distribution of DNA polymerase stalling and/or to different repair pathways. To gain insight into the structure of the Rad5-mediated X-DNA molecules, we assayed whether they can be resolved under conditions that promote spontaneous branch migration of synthetic Holliday junctions in vitro (Panyutin and Hsieh, 1994Panyutin I.G. Hsieh P. The kinetics of spontaneous DNA branch migration.Proc. Natl. Acad. Sci. USA. 1994; 91: 2021-2025Crossref PubMed Scopus (226) Google Scholar). After incubation of DNA preparations between the first and second dimension of electrophoresis in branch-migrating conditions, the X-DNA structures induced by MMS and adozelesin in WT cells at 45 min after the G1-block release resolved as linear molecules (Figures 4A and 4B, arrows), indicating that they branch migrate. To further test the Holliday junction nature of these structures and to distinguish them from other types of branched DNA molecules described at replication origins like hemicatenanes (Lopes et al., 2003Lopes M. Cotta-Ramusino C. Liberi G. Foiani M. Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms.Mol. Cell. 2003; 12: 1499-1510Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we subjected the DNA intermediates to an in-gel digestion with RuvC Holliday junction resolvase (Dunderdale et al., 1994Dunderdale H.J. Sharples G.J. Lloyd R.G. West S.C. Cloning, overexpression, purification, and characterization of the Escherichia coli RuvC Holliday junction resolvase.J. Biol. Chem. 1994; 269: 5187-5194Abstract Full Text PDF PubMed Google Scholar), prior to the second dimension of electrophoresis (Zou and Rothstein, 1997Zou H. Rothstein R. Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism.Cell. 1997; 90: 87-96Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The enzymatic treatment resulted in the formation of linear molecules indicating that the damage-induced X-DNA contains Holliday junctions (Figure 4B). Inefficient RuvC activity during in-gel conditions and on DNA with bulky adozelesin lesions that also appear to hinder branch migration may account for partial linearization of X-DNA as could presence of RuvC-insensitive hemicatenanes, in addition to Holliday junctions. Finally, we also tested whether the X-DNA contains ssDNA, by incubation prior to the first-dimension electropho" @default.
• W2069894032 created "2016-06-24" @default.
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• W2069894032 date "2010-06-01" @default.
• W2069894032 modified "2023-10-14" @default.
• W2069894032 title "Multiple Rad5 Activities Mediate Sister Chromatid Recombination to Bypass DNA Damage at Stalled Replication Forks" @default.
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• W2069894032 doi "https://doi.org/10.1016/j.molcel.2010.03.020" @default.
• W2069894032 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2887677" @default.
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