ELG1, a yeast gene required for genome stability, forms a complex related to replication factor C

Abstract

Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. We have screened to isolate yeast mutants exhibiting increased levels of recombination between repeated sequences. Here we characterize one of these mutants, elg1. Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats. These strains also exhibit increased levels of chromosome loss. The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18. Elg1p forms a complex with the Rfc2-5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18. Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses.

Fig. 1.

(A) Genetic system used to isolate ELG1. The strain SBA1 carries several substrates for recombination. The presence of the SUP4 suppressor tRNA within a Ty element renders the cells white (Ade⁺) and sensitive to CAN (Can^S) by suppressing the mutations can1-100 and ade2-1. Recombination events (gene conversion) between Ty1^Sup and other Ty elements in the genome, or interactions between the LTRs of the Ty (triangles), yield Can^R Ade^– cells, which can be selected on CAN plates. In addition, SBA1 carries a duplication of part of the HIS4 gene, created by the insertion of a TRP1-marked plasmid; DRR between the duplicated segments (black rectangles) gives rise to His⁺ Trp^– cells. (B) Schematic representation of the genetic system used to measure crossing over and chromosome loss. Crossing over between CAN1 and URA3 on chromosome V gives rise to Can^R Ura⁺ His⁺ colonies, whereas chromosome loss generates Can^R Ura^– His^– colonies. These events can be selected on appropriate plates. Rates of crossing over and chromosome loss in elg1/elg1 and in WT controls are shown. (C) Schematic representation of the genetic system used to measure USCE. Two nonfunctional fragments of the ADE3 gene overlap in a small region (black square). Only unequal sister chromatid recombination after DNA replication generates a functional ADE3 gene. Rates of USCE in elg1 and WT strains are shown. Fold induction appears in parentheses.

Fig. 2.

Elg1p shares similarities with Rfc1p, Rad24p, and Ctf18p. (A) Schematic representation of the four proteins. Boxes I–VIII represent motives conserved in RFC proteins (23). The overall identity/similarity between Elg1p and the other three proteins are as follows: Rfc1p, 20/13.3%; Ctf18p, 15.4/15.4%; and Rad24p, 17.2/12.8%. (B) Detailed comparison between the four proteins. Boxes II–VIII are underscored. Black squares represent identical residues and gray squares mark conserved amino acids. The comparison was generated with clustalw and matchbox programs.

Fig. 3.

elg1, ctf18, and rad24 are additive in their sensitivity to MMS and HU. (A) Midlogarithmic cells were treated for 20 min at 30°C with various concentrations of MMS, washed, and plated on YPD plates. (B) Mid-logarithmic cells were plated on plates containing different concentrations of HU.

Fig. 4.

Elg1p coimmunoprecipitates with Rfc4p and Rfc5p, but not with Ctf18p, Rfc1p, or Rad24p. (A) Extracts were prepared from strains carrying combinations of the following alleles: ELG1::3myc, CTF18::9myc, RFC4::6HA, and RFC5::6HA. After IP with anti-myc antibody, immunocomplexes were separated by SDS/PAGE and immunoblotted with anti-myc and anti-HA antibodies. Rfc4p and Rfc5p coimmunoprecipitated with both Elg1p and Ctf18p. MK244, carrying no tags, served as a negative control. (B) Proteins were extracted from strains in which the genomic ELG1 gene was HA-tagged and carried myc-tagged versions of CTF18, RFC1, RAD24, or, as a positive control, RFC4. Anti-HA antibody was used to precipitate Elg1-3HA. As expected, Rfc4-myc was detected in the immunocomplexes, whereas no coimmunoprecipitation of Elg1p with Rfc1p, Ctf18p, or Rad24p could be detected. As additional controls, we show that Rfc4-HA is easily detected after IP of Elg1-myc or Ctf18-myc. (C) Samples treated similarly, but without IP, are shown to indicate the expected electrophoretic mobility of the various tagged proteins. MK244, carrying no tags, served as a negative control.