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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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PRR defects result in increased rates of duplication-mediated translocations.A. The pre-duplication (yel068c::CAN1/URA3) and post-duplication (yel072w::CAN1/URA3) assays differ by whether or not they include the DSF1-HXT13 homology in the breakpoint region (the left arm of chromosome V between the CAN1/URA3 cassette and the most telomeric essential gene, PCM1). The hygromycin resistance marker is indicated by hph. Grey boxes indicate regions of homologies between the chromosomes. B. The rates of the total CanR 5FOAR product and the rates of t(V;XIV) and t(V;IV or X) translocations, and non-duplication-mediated GCR products in the yel072w::CAN1/URA3 assay are depicted in a bar graph. Error bars indicate 95% confidence intervals and the fold increase for each rate is displayed in parentheses, (<) indicates that no isolates of that class were identified. The number of isolates analyzed is shown in parentheses after the genotype. The numerical GCR rates are presented in Tables 1, 2, 4 and 5.
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pgen-1000933-g001: PRR defects result in increased rates of duplication-mediated translocations.A. The pre-duplication (yel068c::CAN1/URA3) and post-duplication (yel072w::CAN1/URA3) assays differ by whether or not they include the DSF1-HXT13 homology in the breakpoint region (the left arm of chromosome V between the CAN1/URA3 cassette and the most telomeric essential gene, PCM1). The hygromycin resistance marker is indicated by hph. Grey boxes indicate regions of homologies between the chromosomes. B. The rates of the total CanR 5FOAR product and the rates of t(V;XIV) and t(V;IV or X) translocations, and non-duplication-mediated GCR products in the yel072w::CAN1/URA3 assay are depicted in a bar graph. Error bars indicate 95% confidence intervals and the fold increase for each rate is displayed in parentheses, (<) indicates that no isolates of that class were identified. The number of isolates analyzed is shown in parentheses after the genotype. The numerical GCR rates are presented in Tables 1, 2, 4 and 5.

Mentions: Deletion of RAD6 was previously found to specifically increase the spontaneous rate of duplication-mediated GCRs by comparing the rates of loss of a CAN1/URA3 cassette on chromosome V in the yel068c::CAN1/URA3 GCR assay, which lacks a duplication in the breakpoint region, with the yel072w::CAN1/URA3 GCR assay, which contains the DSF1-HXT13 duplicated region in the breakpoint region (Table 1; Figure 1a)[27]. The DSF1-HXT13 region shares ∼4.2 kb of homology with chromosome XIV and ∼1.7 kb of homology with highly similar regions of chromosomes IV and X, and consequently most of the duplication-mediated GCRs are translocations between the DSF1 HXT13 region on chromosome V and the homology regions on chromosomes XIV, IV and X. We analyzed the GCRs obtained in the yel072w::CAN1/URA3 assay in the rad6Δ background and observed that the increased rates of forming homology-mediated t(V;XIV) and t(V;IV or X) translocations were responsible for most of the rate increases (Figure 1b). The majority of both homology and non-homology-mediated GCRs lost the telomeric end of chromosome V as determined by the loss of the telomeric hygromycin resistance marker (Table S1).


Post-replication repair suppresses duplication-mediated genome instability.

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

PRR defects result in increased rates of duplication-mediated translocations.A. The pre-duplication (yel068c::CAN1/URA3) and post-duplication (yel072w::CAN1/URA3) assays differ by whether or not they include the DSF1-HXT13 homology in the breakpoint region (the left arm of chromosome V between the CAN1/URA3 cassette and the most telomeric essential gene, PCM1). The hygromycin resistance marker is indicated by hph. Grey boxes indicate regions of homologies between the chromosomes. B. The rates of the total CanR 5FOAR product and the rates of t(V;XIV) and t(V;IV or X) translocations, and non-duplication-mediated GCR products in the yel072w::CAN1/URA3 assay are depicted in a bar graph. Error bars indicate 95% confidence intervals and the fold increase for each rate is displayed in parentheses, (<) indicates that no isolates of that class were identified. The number of isolates analyzed is shown in parentheses after the genotype. The numerical GCR rates are presented in Tables 1, 2, 4 and 5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865514&req=5

pgen-1000933-g001: PRR defects result in increased rates of duplication-mediated translocations.A. The pre-duplication (yel068c::CAN1/URA3) and post-duplication (yel072w::CAN1/URA3) assays differ by whether or not they include the DSF1-HXT13 homology in the breakpoint region (the left arm of chromosome V between the CAN1/URA3 cassette and the most telomeric essential gene, PCM1). The hygromycin resistance marker is indicated by hph. Grey boxes indicate regions of homologies between the chromosomes. B. The rates of the total CanR 5FOAR product and the rates of t(V;XIV) and t(V;IV or X) translocations, and non-duplication-mediated GCR products in the yel072w::CAN1/URA3 assay are depicted in a bar graph. Error bars indicate 95% confidence intervals and the fold increase for each rate is displayed in parentheses, (<) indicates that no isolates of that class were identified. The number of isolates analyzed is shown in parentheses after the genotype. The numerical GCR rates are presented in Tables 1, 2, 4 and 5.
Mentions: Deletion of RAD6 was previously found to specifically increase the spontaneous rate of duplication-mediated GCRs by comparing the rates of loss of a CAN1/URA3 cassette on chromosome V in the yel068c::CAN1/URA3 GCR assay, which lacks a duplication in the breakpoint region, with the yel072w::CAN1/URA3 GCR assay, which contains the DSF1-HXT13 duplicated region in the breakpoint region (Table 1; Figure 1a)[27]. The DSF1-HXT13 region shares ∼4.2 kb of homology with chromosome XIV and ∼1.7 kb of homology with highly similar regions of chromosomes IV and X, and consequently most of the duplication-mediated GCRs are translocations between the DSF1 HXT13 region on chromosome V and the homology regions on chromosomes XIV, IV and X. We analyzed the GCRs obtained in the yel072w::CAN1/URA3 assay in the rad6Δ background and observed that the increased rates of forming homology-mediated t(V;XIV) and t(V;IV or X) translocations were responsible for most of the rate increases (Figure 1b). The majority of both homology and non-homology-mediated GCRs lost the telomeric end of chromosome V as determined by the loss of the telomeric hygromycin resistance marker (Table S1).

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