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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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Related in: MedlinePlus

Single cell analysis of cell cycle dynamics.Aliquots from mid-logarithmic phase liquid cultures were plated onto solid media. Single unbudded cells were isolated by micromanipulation, and their progression was monitored by microscopy every 30 min for 6.0–8.5 h (1–4 cell divisions). (A) Examples of how cells were followed and scored. (B) Time spent in each phase of the cell cycle, as scored by budding index (see Materials and Methods). Red bars, S phase; green bars, G2 phase; blue bars, G2+G1 phases.
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pgen-1001298-g005: Single cell analysis of cell cycle dynamics.Aliquots from mid-logarithmic phase liquid cultures were plated onto solid media. Single unbudded cells were isolated by micromanipulation, and their progression was monitored by microscopy every 30 min for 6.0–8.5 h (1–4 cell divisions). (A) Examples of how cells were followed and scored. (B) Time spent in each phase of the cell cycle, as scored by budding index (see Materials and Methods). Red bars, S phase; green bars, G2 phase; blue bars, G2+G1 phases.

Mentions: To measure cell cycle dynamics with more precision, we isolated unbudded G1 cells by micromanipulation and followed their progression through 2–3 cell cycles by microscopy. This single-cell approach measures the time spent in each phase of the cell cycle, and therefore it allows assignment of the cell cycle stage in which defects can first be detected. A schematic example of the approach and some representative data are shown in Figure 5A. The majority of wild type cells with no repeat spent ∼30 min in S phase, with a slight shift to longer S phases when a medium-length (CAG)70 repeat was present (Figure 5B). Cells containing a (CAG)70 tract and lacking DCC1 or CTF18 exhibited several cell cycle phenotypes. First, they divided much more slowly. Average division time was 5.8 h for dcc1 (range 2.5–8.5 h) and 3.5 h for ctf18 (range 2.0–6.0 h), compared to 2.0 h for wild type (CAG)70 strain. The presence of the repeats enhanced the delay as the dcc1 and ctf18 mutants with no repeat averaged 2.5 h and 2.0 h per division, respectively. Second, some ctf18 and dcc1 cells stayed small budded 1–2 h, consistent with an S-phase delay, a phenotype that was exacerbated by the presence of the repeat (Figure 5B). In contrast, all wild type cells completed S phase in 1 h or less, regardless of whether the repeat tract was present. Thus, single-cell analysis provides additional evidence for a role of Ctf18-RFC during S phase, as its absence leads to an extended S phase in some cells.


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)

Single cell analysis of cell cycle dynamics.Aliquots from mid-logarithmic phase liquid cultures were plated onto solid media. Single unbudded cells were isolated by micromanipulation, and their progression was monitored by microscopy every 30 min for 6.0–8.5 h (1–4 cell divisions). (A) Examples of how cells were followed and scored. (B) Time spent in each phase of the cell cycle, as scored by budding index (see Materials and Methods). Red bars, S phase; green bars, G2 phase; blue bars, G2+G1 phases.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001298-g005: Single cell analysis of cell cycle dynamics.Aliquots from mid-logarithmic phase liquid cultures were plated onto solid media. Single unbudded cells were isolated by micromanipulation, and their progression was monitored by microscopy every 30 min for 6.0–8.5 h (1–4 cell divisions). (A) Examples of how cells were followed and scored. (B) Time spent in each phase of the cell cycle, as scored by budding index (see Materials and Methods). Red bars, S phase; green bars, G2 phase; blue bars, G2+G1 phases.
Mentions: To measure cell cycle dynamics with more precision, we isolated unbudded G1 cells by micromanipulation and followed their progression through 2–3 cell cycles by microscopy. This single-cell approach measures the time spent in each phase of the cell cycle, and therefore it allows assignment of the cell cycle stage in which defects can first be detected. A schematic example of the approach and some representative data are shown in Figure 5A. The majority of wild type cells with no repeat spent ∼30 min in S phase, with a slight shift to longer S phases when a medium-length (CAG)70 repeat was present (Figure 5B). Cells containing a (CAG)70 tract and lacking DCC1 or CTF18 exhibited several cell cycle phenotypes. First, they divided much more slowly. Average division time was 5.8 h for dcc1 (range 2.5–8.5 h) and 3.5 h for ctf18 (range 2.0–6.0 h), compared to 2.0 h for wild type (CAG)70 strain. The presence of the repeats enhanced the delay as the dcc1 and ctf18 mutants with no repeat averaged 2.5 h and 2.0 h per division, respectively. Second, some ctf18 and dcc1 cells stayed small budded 1–2 h, consistent with an S-phase delay, a phenotype that was exacerbated by the presence of the repeat (Figure 5B). In contrast, all wild type cells completed S phase in 1 h or less, regardless of whether the repeat tract was present. Thus, single-cell analysis provides additional evidence for a role of Ctf18-RFC during S phase, as its absence leads to an extended S phase in some cells.

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