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Post-replication repair suppresses duplication-mediated genome instability.

Putnam CD, Hayes TK, Kolodner RD - PLoS Genet. (2010)

Bottom Line: The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164.The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5.In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
RAD6 is known to suppress duplication-mediated gross chromosomal rearrangements (GCRs) but not single-copy sequence mediated GCRs. Here, we found that the RAD6- and RAD18-dependent post-replication repair (PRR) and the RAD5-, MMS2-, UBC13-dependent error-free PRR branch acted in concert with the replication stress checkpoint to suppress duplication-mediated GCRs formed by homologous recombination (HR). The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164. The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5. In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5. Our analysis is consistent with models in which PRR prevents replication damage from becoming double strand breaks (DSBs) and/or regulates the activity of HR on DSBs.

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Related in: MedlinePlus

Models for the suppression of duplication-mediated GCRs by PRR.A. The most important RAD6-dependent pathway that suppresses duplication-mediated GCRs (thick lines) corresponds to the “error-free” PRR branch, which is downstream of Srs2. Other Rad6- and Rad18-dependent branches are less important (thin lines). The presence of specific PCNA and DNA states are inferred based on the genes involved in the pathway. Sgs1 appears to act downstream of the Rad5-dependent branches. The existence of Rad5 branches that are independent of Ubc13 and Rev3 that could be dependent upon Rad6 and Rad18 or independent of Rad6 is inferred by the observation of synergistic interactions between mutations in RAD5 and mutations in RAD6, UBC13 and REV3. Our data do not directly address the previously identified Rad5- and Rev3-dependent branch [46]. B. PRR could potentially suppress duplication-mediated GCRs by preventing replication damage from being converted into DSBs and other HR substrates. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. C. PRR could potentially suppress duplication-mediated GCRs by functioning as an alternative to HR. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. The red arrows and Xs in B and C indicate the consequences of PRR defects.
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pgen-1000933-g002: Models for the suppression of duplication-mediated GCRs by PRR.A. The most important RAD6-dependent pathway that suppresses duplication-mediated GCRs (thick lines) corresponds to the “error-free” PRR branch, which is downstream of Srs2. Other Rad6- and Rad18-dependent branches are less important (thin lines). The presence of specific PCNA and DNA states are inferred based on the genes involved in the pathway. Sgs1 appears to act downstream of the Rad5-dependent branches. The existence of Rad5 branches that are independent of Ubc13 and Rev3 that could be dependent upon Rad6 and Rad18 or independent of Rad6 is inferred by the observation of synergistic interactions between mutations in RAD5 and mutations in RAD6, UBC13 and REV3. Our data do not directly address the previously identified Rad5- and Rev3-dependent branch [46]. B. PRR could potentially suppress duplication-mediated GCRs by preventing replication damage from being converted into DSBs and other HR substrates. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. C. PRR could potentially suppress duplication-mediated GCRs by functioning as an alternative to HR. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. The red arrows and Xs in B and C indicate the consequences of PRR defects.

Mentions: In the present study, we have demonstrated that suppression of duplication-mediated GCRs by RAD6 is epistatic to the replication stress checkpoint and that the RAD18-, RAD5-, UBC13-, and MMS2-dependent error-free PRR pathway is the RAD6-dependent pathway that is primarily responsible for suppressing duplication-mediated GCRs. The translesion polymerase-dependent pathways for PRR and the BRE1- and UBR1-dependent RAD6 pathways played small roles in suppressing duplication-mediated GCRs. In addition, genes that are not typically considered as encoding components of the PRR pathways, but which have been implicated in PRR by a few genetic studies, including RAD9 [53] and TAF14 (ANC1) [41], as well as the Shu complex genes, PSY3 and CSM2, implicated as acting downstream of RAD5 [54], did not appear to play significant roles in suppressing duplication-mediated GCRs (Tables 1 & 2, and not shown). The suppression of duplication-mediated GCRs exhibited remarkably complex genetic interactions between downstream PRR components (Figure 2a), involved the helicase and not the RING-finger functions of Rad5, and required Sgs1 for processing of repair intermediates. Our analysis using the sensitive duplication-mediated GCR assay revealed a number of surprising results that appear paradoxical in the context of commonly accepted models for PRR [4], but fit with a growing body of evidence that indicate that the in vivo pathways are more complicated than can be accounted for by present models [26], [45]–[47], [55]–[57].


Post-replication repair suppresses duplication-mediated genome instability.

Putnam CD, Hayes TK, Kolodner RD - PLoS Genet. (2010)

Models for the suppression of duplication-mediated GCRs by PRR.A. The most important RAD6-dependent pathway that suppresses duplication-mediated GCRs (thick lines) corresponds to the “error-free” PRR branch, which is downstream of Srs2. Other Rad6- and Rad18-dependent branches are less important (thin lines). The presence of specific PCNA and DNA states are inferred based on the genes involved in the pathway. Sgs1 appears to act downstream of the Rad5-dependent branches. The existence of Rad5 branches that are independent of Ubc13 and Rev3 that could be dependent upon Rad6 and Rad18 or independent of Rad6 is inferred by the observation of synergistic interactions between mutations in RAD5 and mutations in RAD6, UBC13 and REV3. Our data do not directly address the previously identified Rad5- and Rev3-dependent branch [46]. B. PRR could potentially suppress duplication-mediated GCRs by preventing replication damage from being converted into DSBs and other HR substrates. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. C. PRR could potentially suppress duplication-mediated GCRs by functioning as an alternative to HR. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. The red arrows and Xs in B and C indicate the consequences of PRR defects.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865514&req=5

pgen-1000933-g002: Models for the suppression of duplication-mediated GCRs by PRR.A. The most important RAD6-dependent pathway that suppresses duplication-mediated GCRs (thick lines) corresponds to the “error-free” PRR branch, which is downstream of Srs2. Other Rad6- and Rad18-dependent branches are less important (thin lines). The presence of specific PCNA and DNA states are inferred based on the genes involved in the pathway. Sgs1 appears to act downstream of the Rad5-dependent branches. The existence of Rad5 branches that are independent of Ubc13 and Rev3 that could be dependent upon Rad6 and Rad18 or independent of Rad6 is inferred by the observation of synergistic interactions between mutations in RAD5 and mutations in RAD6, UBC13 and REV3. Our data do not directly address the previously identified Rad5- and Rev3-dependent branch [46]. B. PRR could potentially suppress duplication-mediated GCRs by preventing replication damage from being converted into DSBs and other HR substrates. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. C. PRR could potentially suppress duplication-mediated GCRs by functioning as an alternative to HR. Suppression of single-copy GCRs also requires that PRR promotes other GCR forming pathways (such as NHEJ and de novo telomere addition) or requires PRR-dependent suppression of HR. The red arrows and Xs in B and C indicate the consequences of PRR defects.
Mentions: In the present study, we have demonstrated that suppression of duplication-mediated GCRs by RAD6 is epistatic to the replication stress checkpoint and that the RAD18-, RAD5-, UBC13-, and MMS2-dependent error-free PRR pathway is the RAD6-dependent pathway that is primarily responsible for suppressing duplication-mediated GCRs. The translesion polymerase-dependent pathways for PRR and the BRE1- and UBR1-dependent RAD6 pathways played small roles in suppressing duplication-mediated GCRs. In addition, genes that are not typically considered as encoding components of the PRR pathways, but which have been implicated in PRR by a few genetic studies, including RAD9 [53] and TAF14 (ANC1) [41], as well as the Shu complex genes, PSY3 and CSM2, implicated as acting downstream of RAD5 [54], did not appear to play significant roles in suppressing duplication-mediated GCRs (Tables 1 & 2, and not shown). The suppression of duplication-mediated GCRs exhibited remarkably complex genetic interactions between downstream PRR components (Figure 2a), involved the helicase and not the RING-finger functions of Rad5, and required Sgs1 for processing of repair intermediates. Our analysis using the sensitive duplication-mediated GCR assay revealed a number of surprising results that appear paradoxical in the context of commonly accepted models for PRR [4], but fit with a growing body of evidence that indicate that the in vivo pathways are more complicated than can be accounted for by present models [26], [45]–[47], [55]–[57].

Bottom Line: The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164.The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5.In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
RAD6 is known to suppress duplication-mediated gross chromosomal rearrangements (GCRs) but not single-copy sequence mediated GCRs. Here, we found that the RAD6- and RAD18-dependent post-replication repair (PRR) and the RAD5-, MMS2-, UBC13-dependent error-free PRR branch acted in concert with the replication stress checkpoint to suppress duplication-mediated GCRs formed by homologous recombination (HR). The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164. The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5. In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5. Our analysis is consistent with models in which PRR prevents replication damage from becoming double strand breaks (DSBs) and/or regulates the activity of HR on DSBs.

Show MeSH
Related in: MedlinePlus