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New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion.

Gellon L, Razidlo DF, Gleeson O, Verra L, Schulz D, Lahue RS, Freudenreich CH - PLoS Genet. (2011)

Bottom Line: Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats.Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC.Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Tufts University, Medford, Massachusetts, United States of America.

ABSTRACT
Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.

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Analysis of rad52 effects on triplet repeat instability phenotype of dcc1.Contractions, expansions, and fragility were measured as described in Table S1. (A) Contraction and expansion phenotypes normalized to wild type. *, p<0.05, **, p<0.01 compared to wild type; Δ, p<0.05 compared to dcc1. (B) Fragility measurements as in Figure 1; the dcc1 rad52 mutant had a (CAG)65 repeat. Error bars, ±1 SEM.
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pgen-1001298-g003: Analysis of rad52 effects on triplet repeat instability phenotype of dcc1.Contractions, expansions, and fragility were measured as described in Table S1. (A) Contraction and expansion phenotypes normalized to wild type. *, p<0.05, **, p<0.01 compared to wild type; Δ, p<0.05 compared to dcc1. (B) Fragility measurements as in Figure 1; the dcc1 rad52 mutant had a (CAG)65 repeat. Error bars, ±1 SEM.

Mentions: Since SCC defects did not account for instability of triplet repeats, we tested the idea that strains deficient for Ctf18-RFC suffer enhanced DNA damage at the trinucleotide repeat, as suggested by the increased repeat fragility in mutants of the complex (Figure 1). If so, this damage might be susceptible to RAD52-dependent recombinational repair and therefore a rad52 background should alter the mutational spectrum in the absence of Ctf18-RFC. The results show that mutation of RAD52 suppressed, partially or completely, every dcc1 mutability phenotype–contractions of both short and medium CAG/CTG tracts and expansion of medium tracts (Figure 3A). We conclude that Rad52-dependent repair in the absence of Ctf18-RFC does not proceed with fidelity in the context of a CAG repeat, since it results in expansions and contractions. A similar result was also observed in srs2 and mre11 mutants, where increased levels of medium- and long-tract repeat expansions and contractions were dependent on Rad52 [28], [29]. Some medium tract contractions were Rad52-independent (Figure 3A); previous data indicated that an additional source of contractions could be processing of DSBs within the repeat tract followed by microhomology-mediated end joining [29]. Fragility was not suppressed or significantly increased in a dcc1 rad52 double mutant compared to the dcc1 single mutant (Figure 3B), indicating that Rad52 does not contribute to fragility resulting from DNA damage in Ctf18-RFC deficient cells. In summary, in the absence of Ctf18-RFC, a Rad52-dependent pathway is operative that is responsible for the majority of the observed contractions and expansions.


New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion.

Gellon L, Razidlo DF, Gleeson O, Verra L, Schulz D, Lahue RS, Freudenreich CH - PLoS Genet. (2011)

Analysis of rad52 effects on triplet repeat instability phenotype of dcc1.Contractions, expansions, and fragility were measured as described in Table S1. (A) Contraction and expansion phenotypes normalized to wild type. *, p<0.05, **, p<0.01 compared to wild type; Δ, p<0.05 compared to dcc1. (B) Fragility measurements as in Figure 1; the dcc1 rad52 mutant had a (CAG)65 repeat. Error bars, ±1 SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037408&req=5

pgen-1001298-g003: Analysis of rad52 effects on triplet repeat instability phenotype of dcc1.Contractions, expansions, and fragility were measured as described in Table S1. (A) Contraction and expansion phenotypes normalized to wild type. *, p<0.05, **, p<0.01 compared to wild type; Δ, p<0.05 compared to dcc1. (B) Fragility measurements as in Figure 1; the dcc1 rad52 mutant had a (CAG)65 repeat. Error bars, ±1 SEM.
Mentions: Since SCC defects did not account for instability of triplet repeats, we tested the idea that strains deficient for Ctf18-RFC suffer enhanced DNA damage at the trinucleotide repeat, as suggested by the increased repeat fragility in mutants of the complex (Figure 1). If so, this damage might be susceptible to RAD52-dependent recombinational repair and therefore a rad52 background should alter the mutational spectrum in the absence of Ctf18-RFC. The results show that mutation of RAD52 suppressed, partially or completely, every dcc1 mutability phenotype–contractions of both short and medium CAG/CTG tracts and expansion of medium tracts (Figure 3A). We conclude that Rad52-dependent repair in the absence of Ctf18-RFC does not proceed with fidelity in the context of a CAG repeat, since it results in expansions and contractions. A similar result was also observed in srs2 and mre11 mutants, where increased levels of medium- and long-tract repeat expansions and contractions were dependent on Rad52 [28], [29]. Some medium tract contractions were Rad52-independent (Figure 3A); previous data indicated that an additional source of contractions could be processing of DSBs within the repeat tract followed by microhomology-mediated end joining [29]. Fragility was not suppressed or significantly increased in a dcc1 rad52 double mutant compared to the dcc1 single mutant (Figure 3B), indicating that Rad52 does not contribute to fragility resulting from DNA damage in Ctf18-RFC deficient cells. In summary, in the absence of Ctf18-RFC, a Rad52-dependent pathway is operative that is responsible for the majority of the observed contractions and expansions.

Bottom Line: Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats.Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC.Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Tufts University, Medford, Massachusetts, United States of America.

ABSTRACT
Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.

Show MeSH
Related in: MedlinePlus