Evidence for short-patch mismatch repair in Saccharomyces cerevisiae

Abstract

Recombination events between non-identical sequences most often involve heteroduplex DNA intermediates that are subjected to mismatch repair. The well-characterized long-patch mismatch repair process, controlled in eukaryotes by bacterial MutS and MutL orthologs, is the major system involved in repair of mispaired bases. Here we present evidence for an alternative short-patch mismatch repair pathway that operates on a broad spectrum of mismatches. In msh2 mutants lacking the long-patch repair system, sequence analysis of recombination tracts resulting from exchanges between similar but non-identical (homeologous) parental DNAs showed the occurrence of short-patch repair events that can involve <12 nucleotides. Such events were detected both in mitotic and in meiotic recombinants. Confirming the existence of a distinct short-patch repair activity, we found in a recombination assay involving homologous alleles that closely spaced mismatches are repaired independently with high efficiency in cells lacking MSH2 or PMS1. We show that this activity does not depend on genes required for nucleotide excision repair and thus differs from the short-patch mismatch repair described in Schizosaccharomyces pombe.

Fig. 1. Structure of the ARG4 chromosomal region and schematic representation of the different genetic systems used. (A) The organization of this region is similar in S.cerevisiae and S.douglasii. The ARG4 ORFs differ by 8% of base substitutions, and the intergenic regions by ∼20% of substitutions and small insertions/deletions. The HpaI segment, shown in black, is the region substituted in S.cerevisiae by the corresponding fragment of S.douglasii. Frameshift mutations were introduced at the EcoRV (RV, –2 bp), the AgeI (Ag, +4 bp) or the BglII site (Bg, +4 bp). Coordinates are given with respect to the S.cerevisiae sequence, number 1 being the first nucleotide of the HpaI site. (B) Diagram of the genetic constructs involving RV and Bg.

Fig. 2. Sequence profile uncovering short-patch repair. (A) A partial sequence of ARG4 amplified from a single recombinant. The arrows point to sequence differences between the parental alleles, which are indicated underneath. Red arrows correspond to sites where the two parental bases are superimposed indicating an absence of MMR and uncovering a mixed clone. Green arrows indicate sites where only one of the parental bases is found. (B and C) The sequence of two subclones derived from the initial mixed clone.

Fig. 3. Length, position and structure of recombinant tracts. Donor DNA is in black and recipient DNA in gray. The maximal and minimal size (in base pairs) of the whole rearranged region is indicated on the left, and that of the internal rearrangements above the sequence. Gaps are regions of identity between the alternate S.cerevisiae and S.douglasii tracts. (A) Wild-type cells. Tracts nos 1–9 were obtained from mitotic events in ectopic haploids (FF18248). Tracts nos 10–15 were obtained from meiotic events in ectopic diploids (FF181387). (B) msh2 cells. The 12 first cases correspond to mitotic events in ectopic haploids (FF181378); the three others are meiotic events in ectopic cells (nos 13 and 14, FF181396) and in allelic cells (no. 15, Ec182). For recombinants 11, 12, 14 and 15, both strands of the original recombinant were recovered and sequenced. No. 15 was selected as an ARG⁺/arg^– sectored colony. Cases nos 12, 14 and 15 are detailed in Figure 4.

Fig. 4. Detailed structure of recombinant tracts. These three recombinants were recovered in msh2 cells. They correspond to one mitotic and two meiotic events (case nos 12, 14 and 15 in Figure 3B). The numbers are the coordinates, as defined in the legend of Figure 1, and indicate the position of the first and last polymorphic site that delimits rearranged segments. Numbers between the two strands of each duplex are the numbers of mismatches that were repaired in the conversion and restoration tracts.

Fig. 5. UV induction of ARG⁺ recombinants in wild-type and msh2 cells, heteroallelic for distant or close mutations in ARG4. Open symbols: heteroallelic cells with closely spaced mutations (wild type, Ec153; msh2, Ec144000; pms1, FF181617); closed symbols: heteroallelic cells with distant mutations (wild type, Ec160; msh2, FF181379; pms1, FF181614).