The Elg1 Clamp Loader Plays a Role in Sister Chromatid Cohesion

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring

Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability

Elg1 and Ctf18 are present both at the chromatin and soluble fractions.

<p>Whole cell extracts from cells at mid-logarithmic growth phase were fractionated into a chromatin and a soluble fraction, and the presence of Myc- tagged Elg1 was followed with anti-Myc specific antibodies. As controls, anti-Carboxypeptidase Y and anti- Acetylated histone H4 antibodies were used. (A) Elg1 is present in both fractions, but its abundance decreases with DNA damage. Cells were untreated, or incubated in the presence of methyl methanesulfonate (MMS) at two different concentrations (0.02% and 0.3%). (B) Ctf18 is present in both fractions, and its localization in G2 depends on Elg1. Wild type or <i>elg1</i> cells were arrested with either alpha factor (G1), hydroxyurea (early S) or nocodazole (G2) before being fractionated.</p

Schematic explanation of the genetic screen carried out.

<p>A double mutant <i>elg1 ctf4</i> strain is kept alive by the presence of a plasmid carrying the <i>ELG1</i> gene. The <i>ADE3</i> marker on the plasmid confers a red pigment to the cells carrying it. As these cells are unable to lose the plasmid during colony formation, all colonies are uniformly red. This strain was transformed with a high copy number library carrying random fragments of the yeast genome. Cells that received a plasmid that suppresses the synthetic lethality phenotype can now lose the <i>ELG1</i>-containing plasmid, becoming white. These cells create white or red/white sectored colonies.</p

Synthetic genetic interactions between <i>elg1</i> and cohesin subunits/accessory factors.

<p>Synthetic genetic interactions between <i>elg1</i> and cohesin subunits/accessory factors.</p

List of strains used in this study.

<p>List of strains used in this study.</p

Overexpression of <i>SCC1</i> or <i>SCC2</i> suppress the synthetic lethality between <i>elg1</i> and <i>ctf4</i>.

<p>A <i>elg1 ctf4</i> double mutant maintained alive by the presence of a <i>URA3</i>-marked plasmid carrying the <i>ELG1</i> gene was transformed with <i>LEU2</i>-marked plasmids overexpressing <i>SCC1, SCC2, SMC1</i> or <i>SMC3,</i> or, as controls, with an empty vector or a plasmid carrying <i>ELG1</i>. Cells were successively diluted ten-fold and plated on plates lacking Uracil (SD-Ura), Leucine (SD-Leu), or on plates containing 5-fluoroorotic acid (5-FOA), which select for cells that became Ura- (i.e., lost the <i>URA3</i>-marked, <i>ELG1</i>-containing plasmid). Only plasmids overexpressing <i>SCC1</i> and <i>SCC2</i> allowed cells to grow on 5-FOA plates.</p

Overexpression of Scc1 and Scc2 does not affect the hyper-recombination phenotype of <i>ctf4</i> or <i>elg1</i>.

<p>Overexpression of Scc1 and Scc2 does not affect the hyper-recombination phenotype of <i>ctf4</i> or <i>elg1</i>.</p

Overexpression of <i>SCC1</i> suppresses the sensitivity of both <i>elg1</i> and <i>ctf4</i> to DNA damaging agents.

<p>Isogenic wild type, <i>elg1</i> or <i>ctf4</i> strains were serially diluted and plated on plates without, and with methyl methanesulfonate (MMS) at two different concentrations (0.001% and 0.02%). Although <i>ctf4</i> strains are more sensitive than <i>elg1</i> strains, <i>SCC1</i> overexpression suppresses the sensitivity of both to MMS.</p

Elg1 and Ctf18, but not Rad24, play a role in sister chromatid cohesion.

<p>(A) Cohesion test. Isogenic strains carrying TetO tandem repeats on chromosome <i>V</i> were arrested in G2 with nocodazole and the percent of cells exhibiting two GFP dots (indicating separated sister chromatids) was counted. (B) Percentage of cells exhibiting precocious sister chromatid separation. At lest 400 cells were counted for each strain.</p