(A) Structure of pFP120 and predicted gene conversion events, with and without crossing over, following HO cleavage. P denotes PstI sites that distinguish crossover from noncrossover recombinants. A detailed description of the structure of pFP120 can be found in reference 53. (B) SSA between identical 205-bp URA3 gene segments that flank an HO-induced DSB. Following HO cleavage, 5′-3′ exonuclease digestion produces 3′ single-stranded tails that can anneal with complementary sequences. Removal of 3′ nonhomologous tails results in the formation of the deletion product. BglII sites that distinguish uncut (7.4 kb) and HO-cut (4.8 kb) chromosomal DNA from the deletion product (5.5 kb) are indicated. (C) Southern blot analysis showing the kinetics of HO-induced SSA in wild-type (tNS1817 containing pJH727 and pEAA54), msh2Δ (tNS1817 containing pJH727), and msh2-R730W (tNS1817 containing pJH727 and pEAA83) strains that contain repeats of the 205-bp URA3 gene segment illustrated in panel B. Chromosomal DNA from the indicated strains was isolated at the indicated time points after HO induction, digested with BglII, and probed with a DNA fragment centromere proximal to the URA3 gene (66). The ratios of plasmid-bearing colonies after and before induction were 0.09, 1.8, and 1.2 for msh2Δ, MSH2, and msh2-R730W strains, respectively. These values reflect the value of product formation, the extent of induction, and the growth of the culture during the 5-h induction (66).

Map of mutated Msh2p residues in S. cerevisiae and in human HNPCC kindreds. Residues corresponding to the carboxyl termini of the two homologs are shown. Identical and similar residues are shaded black and gray, respectively. The dominant negative yeast mutations are indicated by solid arrows above the yeast Msh2 (yMsh2) sequence; mutations identified in the human kindreds are indicated by open arrows below the human Msh2 (hMsh2) sequence. Of the 59 human MSH2 mutations that were examined, 31 mapped to the region shown (see Materials and Methods). Yeast mutations in residues identical to HNPCC mutated residues are indicated by the boxes. Mutations in yeast MSH2 that confer wild-type activity in the removal of nonhomologous DNA during DSB repair are indicated by circles. The ATP binding domain is located between residues 686 and 700 (region I), 739 and 745 (region II), 762 and 773 (region III), and 798 and 803 (region IV) with respect to the yeast Msh2p sequence (14, 48). The location of the msh2-D924V mutation in the double-mutant allele p[msh2-3] is not shown. ∗, nonsense mutation.

Model describing Msh2p-Msh3p interactions with duplex DNA containing nucleotide insertion-deletion mismatches or 3′ tails. In this model, binding of Msh2p-Msh3p (2–3) to a loop mismatch results in recruitment of the Mlh1p-Pms1p (L–M) complex and subsequent MMR steps. During SSA and gene conversion events, binding of Msh2p-Msh3p to duplex DNA containing 3′ tails results in stabilization of the recombination intermediate and allows the Rad1p-Rad10p (1–10) endonuclease to cleave the 3′ tails. We propose that msh2 dominant negative mutations displaying a separation-of-function phenotype do not affect the ability of corresponding Msh2p-Msh3p complexes to bind to DNA mispairs or to duplex DNA containing 3′ tails. However, we hypothesize that these mutant complexes are specifically defective in interactions with downstream MMR factors such as Mlh1p-Pms1p but are capable of stabilizing recombination intermediates for the Rad1p-Rad10p endonuclease.