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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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Cell cycle dependency of Rad52 focus formation in ctf18 cells.(A) Cultures of wild type and ctf18 cells were grown to mid-log phase before mounting on a microscope slide. The panel shows differential interference contrast (DIC), DAPI-stained DNA, and Rad52-yellow fluorescent protein (Rad52-YFP) images of selected cells among WT and ctf18 cells with no tract (−) or with a medium (CAG)70 tract (M). Scale bar is 10 µm. (B) Quantification of Rad52 foci formation in WT or ctf18 cells with no tract (−), medium (CAG)70 tract (M) or long (CAG)155 tract (L). *, p<0.05, **, p<0.01 compared to wild type of same tract length; ∧, p<0.05, ∧∧, p<0.01 compared to no tract of the same strain. (C) Cell cycle distribution of Rad52 foci. The occurrence of Rad52 foci in G1, S or G2 cells was determined after incubation with α-factor, 40 min after release from G1 or nocodazole, respectively (see Methods for details). Representative examples of ctf18 cells with medium (CAG)70 tract in S or G2 are shown as DIC images (top) or Rad52-YFP foci (bottom). (D) Quantification of Rad52 foci in G1, S, or G2 cell cycle stage. Labels and statistical analysis are as in (B). Percentages obtained for WT cells treated with α-factor are indicated. See Table S2 for complete set of data.
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pgen-1001298-g006: Cell cycle dependency of Rad52 focus formation in ctf18 cells.(A) Cultures of wild type and ctf18 cells were grown to mid-log phase before mounting on a microscope slide. The panel shows differential interference contrast (DIC), DAPI-stained DNA, and Rad52-yellow fluorescent protein (Rad52-YFP) images of selected cells among WT and ctf18 cells with no tract (−) or with a medium (CAG)70 tract (M). Scale bar is 10 µm. (B) Quantification of Rad52 foci formation in WT or ctf18 cells with no tract (−), medium (CAG)70 tract (M) or long (CAG)155 tract (L). *, p<0.05, **, p<0.01 compared to wild type of same tract length; ∧, p<0.05, ∧∧, p<0.01 compared to no tract of the same strain. (C) Cell cycle distribution of Rad52 foci. The occurrence of Rad52 foci in G1, S or G2 cells was determined after incubation with α-factor, 40 min after release from G1 or nocodazole, respectively (see Methods for details). Representative examples of ctf18 cells with medium (CAG)70 tract in S or G2 are shown as DIC images (top) or Rad52-YFP foci (bottom). (D) Quantification of Rad52 foci in G1, S, or G2 cell cycle stage. Labels and statistical analysis are as in (B). Percentages obtained for WT cells treated with α-factor are indicated. See Table S2 for complete set of data.

Mentions: The results above suggested that Ctf18-RFC helps cope with triplet repeat-associated damage and in stabilizing replication forks, so we tested directly whether the Ctf18-RFC complex has a role in progression through the cell cycle. Cells from a log phase liquid culture were plated on solid media, and microscopic analysis was used to monitor the proportion of cells in each phase of the cell cycle: unbudded (G1), small budded (bud size one-third or less the size of the mother cell (S), and large budded (G2/M). The results are quantified in Figure 4A, and representative micrographs are shown in Figure 4B. In wild-type cells with no CAG/CTG tract, there was a distribution of 30% unbudded, 12% small budded, and 55% large budded (Figure 4A). The presence of a CAG/CTG tract changed this distribution in two ways. First, there were more small budded (S phase) cells, consistent with replication stress. Second, a new category of cells was observed that were either swollen with large buds or contained multiple buds (Figure 4B), a phenotype that is indicative of unresolved damage in G2/M [37]. The proportion of the multi-budded/swollen cells rose with increasing repeat tract length to as much as 20% of the wild type population (Figure 4A). In general the swelling was modest and most multi-budded clusters contained only one extra bud in wild-type cells (Figure 4B). In dcc1 and ctf18 cells, even without a repeat, multi-budded/swollen cells comprised 18–30% of the population, a level significantly greater than wild-type cells with no tract (Figure 4A). This indicates that the absence of the Ctf18-RFC complex leads to some level of repeat-independent damage that causes accumulation of cells in G2/M. Even more strikingly, the combination of an expanded repeat plus the lack of a functional Ctf18-RFC led to an increase in the multi-budded category to 42–54% of cells (Figure 4B). In addition, the morphological defects in dcc1 and ctf18 mutants with a repeat tract often showed a more severe phenotype, with extreme swelling and many connected buds (Figure 4B). Staining of nuclei revealed that some of the cells within multibudded clusters, often the more swollen ones, had fragmented or missing DNA (example in Figure 6A). We conclude that Ctf18-RFC has an important function in helping resolve repeat-independent DNA damage, and that damage is persisting into the G2 or M phase. Since this phenotype is enhanced by an expanded triplet repeat, and since the expanded repeat causes replication stress, we also infer that Ctf18-RFC helps cope with repeat-induced replication stress during S phase.


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)

Cell cycle dependency of Rad52 focus formation in ctf18 cells.(A) Cultures of wild type and ctf18 cells were grown to mid-log phase before mounting on a microscope slide. The panel shows differential interference contrast (DIC), DAPI-stained DNA, and Rad52-yellow fluorescent protein (Rad52-YFP) images of selected cells among WT and ctf18 cells with no tract (−) or with a medium (CAG)70 tract (M). Scale bar is 10 µm. (B) Quantification of Rad52 foci formation in WT or ctf18 cells with no tract (−), medium (CAG)70 tract (M) or long (CAG)155 tract (L). *, p<0.05, **, p<0.01 compared to wild type of same tract length; ∧, p<0.05, ∧∧, p<0.01 compared to no tract of the same strain. (C) Cell cycle distribution of Rad52 foci. The occurrence of Rad52 foci in G1, S or G2 cells was determined after incubation with α-factor, 40 min after release from G1 or nocodazole, respectively (see Methods for details). Representative examples of ctf18 cells with medium (CAG)70 tract in S or G2 are shown as DIC images (top) or Rad52-YFP foci (bottom). (D) Quantification of Rad52 foci in G1, S, or G2 cell cycle stage. Labels and statistical analysis are as in (B). Percentages obtained for WT cells treated with α-factor are indicated. See Table S2 for complete set of data.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037408&req=5

pgen-1001298-g006: Cell cycle dependency of Rad52 focus formation in ctf18 cells.(A) Cultures of wild type and ctf18 cells were grown to mid-log phase before mounting on a microscope slide. The panel shows differential interference contrast (DIC), DAPI-stained DNA, and Rad52-yellow fluorescent protein (Rad52-YFP) images of selected cells among WT and ctf18 cells with no tract (−) or with a medium (CAG)70 tract (M). Scale bar is 10 µm. (B) Quantification of Rad52 foci formation in WT or ctf18 cells with no tract (−), medium (CAG)70 tract (M) or long (CAG)155 tract (L). *, p<0.05, **, p<0.01 compared to wild type of same tract length; ∧, p<0.05, ∧∧, p<0.01 compared to no tract of the same strain. (C) Cell cycle distribution of Rad52 foci. The occurrence of Rad52 foci in G1, S or G2 cells was determined after incubation with α-factor, 40 min after release from G1 or nocodazole, respectively (see Methods for details). Representative examples of ctf18 cells with medium (CAG)70 tract in S or G2 are shown as DIC images (top) or Rad52-YFP foci (bottom). (D) Quantification of Rad52 foci in G1, S, or G2 cell cycle stage. Labels and statistical analysis are as in (B). Percentages obtained for WT cells treated with α-factor are indicated. See Table S2 for complete set of data.
Mentions: The results above suggested that Ctf18-RFC helps cope with triplet repeat-associated damage and in stabilizing replication forks, so we tested directly whether the Ctf18-RFC complex has a role in progression through the cell cycle. Cells from a log phase liquid culture were plated on solid media, and microscopic analysis was used to monitor the proportion of cells in each phase of the cell cycle: unbudded (G1), small budded (bud size one-third or less the size of the mother cell (S), and large budded (G2/M). The results are quantified in Figure 4A, and representative micrographs are shown in Figure 4B. In wild-type cells with no CAG/CTG tract, there was a distribution of 30% unbudded, 12% small budded, and 55% large budded (Figure 4A). The presence of a CAG/CTG tract changed this distribution in two ways. First, there were more small budded (S phase) cells, consistent with replication stress. Second, a new category of cells was observed that were either swollen with large buds or contained multiple buds (Figure 4B), a phenotype that is indicative of unresolved damage in G2/M [37]. The proportion of the multi-budded/swollen cells rose with increasing repeat tract length to as much as 20% of the wild type population (Figure 4A). In general the swelling was modest and most multi-budded clusters contained only one extra bud in wild-type cells (Figure 4B). In dcc1 and ctf18 cells, even without a repeat, multi-budded/swollen cells comprised 18–30% of the population, a level significantly greater than wild-type cells with no tract (Figure 4A). This indicates that the absence of the Ctf18-RFC complex leads to some level of repeat-independent damage that causes accumulation of cells in G2/M. Even more strikingly, the combination of an expanded repeat plus the lack of a functional Ctf18-RFC led to an increase in the multi-budded category to 42–54% of cells (Figure 4B). In addition, the morphological defects in dcc1 and ctf18 mutants with a repeat tract often showed a more severe phenotype, with extreme swelling and many connected buds (Figure 4B). Staining of nuclei revealed that some of the cells within multibudded clusters, often the more swollen ones, had fragmented or missing DNA (example in Figure 6A). We conclude that Ctf18-RFC has an important function in helping resolve repeat-independent DNA damage, and that damage is persisting into the G2 or M phase. Since this phenotype is enhanced by an expanded triplet repeat, and since the expanded repeat causes replication stress, we also infer that Ctf18-RFC helps cope with repeat-induced replication stress during S phase.

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