Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2

Abstract

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5'-3' exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.

Figure 1

Interaction mating test showing target specificity of yMSH2 interactor C39. Also shown is the apparently nonspecific weak yMSH2 interactor 1-27, which expresses an activation-tagged C-terminal region of MOT1 beginning at amino acid 1579. The interaction mating test was performed as described (18). Briefly, haploid MATa strains that contain a lacZ reporter plasmid (URA3 vector) and different bait plasmids (HIS3 vector) were streaked in horizontal rows on a Ura⁻His⁻ plate, and MATα strains that contain galactose-induced yMSH2 interactor plasmids (TRP1 vector) C39 and 1-27 were streaked in vertical columns on a Trp⁻ plate. Both plates were replica plated onto a single yeast extract/peptone/dextrose (YPD) plate to allow mating, replica plated to a Ura⁻His⁻Trp⁻ glucose plate to select diploids, then replica plated to Ura⁻His⁻Trp⁻ 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) glucose and galactose indicator plates and incubated for 2 days at 30°C (shown). The plasmids contained in each strain tested are indicated at the top of each column and at the right of each row.

Figure 2

Homology among the XPG/RAD2/EXO1 family of endo- and exonucleases. (A) Alignment of the N-terminal conserved regions of S. pombe EXO1, S. cerevisiae EXO1 and DIN7, and D. melanogaster Tosca. (B) Illustration of the sequence relatedness of the XPG/RAD2/EXO1 family of endo- and exonucleases adapted from figure 1 of Szankasi and Smith (7). The highly conserved N and I regions are represented by boxes filled with forward and backward diagonal lines, respectively. (C) Phylogenetic tree of the XPG/RAD2/EXO1 family of endo- and exonucleases generated from alignment of the first 240 amino acids of these proteins. YEN1, a related but divergent protein sequence found during database searches, is also included for clarity.

Figure 3

Immunoprecipitation of proteins expressed in two-hybrid strains. (A) Cell extracts (200 μg) were prepared from the two-hybrid strains expressing LexA-tagged baits and HA-tagged preys. The extracts were precleared with protein-G Sepharose, immunoprecipitated with monoclonal 12CA5 anti-HA antibody and protein-G Sepharose, fractionated by SDS/PAGE, and analyzed by Western blotting with rabbit anti-LexA antiserum. The two bands seen in every IP and PC lane are proteins nonspecifically precipitated by protein-G Sepharose. (B) To ensure appropriate expression and immunoprecipitation of HA-tagged preys, the immunoblot was stripped and reprobed with 12CA5 antibody. E, extract; IP, immunoprecipitation; PC, proteins eluted from protein G-Sepharose used to preclear.

Figure 4

Expression of EXO1 in E. coli results in induction of an exonuclease activity. Extracts from E. coli expressing either pET28b vector alone (A) or RDK480 (B) were chromatographed on a PBE94 column. Fractions were analyzed for protein (•), exonuclease activity (○), and conductivity (NaCl, top scale). Activity is pmol acid-soluble nucleotides released per 5 μl of each fraction.

Figure 5

Overexpression of EXO1 suppresses both the temperature sensitive and mutator phenotypes of rad27(rth1) mutants. Isogenic wild-type (wt) (RKY2672), exo1 (RKY3044), or rad27 (RKY2608) strains were transformed with either YEp213 (a 2μ LEU2-marked plasmid) or pRDK480 (YEp213 with a yeast genomic insert containing the EXO1 gene) and the resulting strains streaked out on SD Leu⁻ plates and grown for 48 h at either 30°C (A) or 37°C (B), or they were patched onto a SD Leu⁻ plate, grown at 30°C for 2 days, then replica plated to a SD Leu⁻ canavanine plate and grown at 30°C for 3 days to detect the presence of Can^r mutations, which appear as papillae (C).