In eukaryotes, mismatch repair plays a critical role in mutation avoidance and is carried out by the MutSLH family of proteins (for reviews, see references 13
, and 40
). During vegetative growth, these proteins recognize and bind DNA mispairs that result primarily from replication errors or DNA damage. In Escherichia coli
, MutS binding to DNA mispairs results in the recruitment of MutL, a matchmaker protein that functions in postreplicative mismatch repair by interacting with both the MutH endonuclease and UvrD helicase (22
). These interactions coordinate mispair recognition with DNA strand-specific signals so that mispairs are removed via excision and resynthesis steps that occur on the newly replicated strand.
Eukaryotes contain multiple MutS (Msh) and MutL (Mlh) homologs, with six Msh and four Mlh homologs present in Saccharomyces cerevisiae
). Genetic and biochemical studies have shown that the eukaryotic homologs display specialized functions with respect to the types of DNA substrates on which they act (10
). In S. cerevisiae
, the Mlh proteins form heterodimers (Mlh1p-Pms1p, Mlh1p-Mlh3p, and Mlh1p-Mlh2p) that display unique functions. Mlh1p is considered a central member of this group because heterodimers have not been identified among the other members (45
). The Mlh1p-Pms1p complex plays a major role in postreplicative mismatch repair, while the other two Mlh complexes appear to be redundant with Mlh1p-Pms1p and are required for the repair of only a limited set of DNA mispairs (18
Yeast mutants lacking Mlh1p or Pms1p display spontaneous mutation rates that are much higher than that of the wild type, and their mutations are epistatic to mutations deleting Msh2p, a central player in mispair recognition (36
). Like MutL, Mlh1p-Pms1p has been shown to bind and hydrolyze ATP in a reaction that drives conformational changes thought to be important for mismatch repair (26
). Also like MutL, Mlh1p-Pms1p has been shown to bind DNA (25
), though it is less clear how this activity functions in its matchmaking role.
While the role of E. coli
MutL has been relatively well characterized, our understanding of the mechanistic steps employed by the Mlh proteins is still in the early stages. As hypothesized for a matchmaking protein, Mlh1p has been shown to physically interact with Exo1p, a 5′-3′ double-stranded DNA exonuclease that is thought to act in excision steps of mismatch repair (65
). Consistent with a role in mismatch repair, high copy numbers of Exo1p suppress the mutator phenotype of specific mismatch repair mutants; however, exo1
mutants do not display a mismatch repair-like mutator phenotype (5
). Physical interactions have also been reported between Mlh1p and BLM/Sgs1p, a DNA helicase that has been hypothesized to repair stalled replication forks (37
). Mammalian cells defective in BLM/Sgs1p display a chromosome instability phenotype but do not display defects in mismatch repair (37
), suggesting that this helicase may not be required in mismatch repair but acts with Mlh proteins in other repair pathways.
In addition to mismatch repair, Msh and Mlh proteins have novel meiotic recombination functions. Genetic studies in yeast and mammalian cells have shown that the Msh4p-Msh5p and Mlh1p-Mlh3p complexes play important roles in meiotic crossing over (30
; reviewed in reference 10
). Yeast mutants lacking any one of these factors display approximately half the number of meiotic crossover events; in these mutants, spore viability is reduced as a result of nondisjunction events in meiosis I (reviewed in reference 10
). MLH1-deficient mice display a more severe crossover defect and are sterile (72
). In addition to its role in crossing over, Msh4p is required for establishing crossover interference (43
). Msh4p and Mlh1p interact physically and genetically (32
), suggesting that MutS and MutL homologs might function together to mediate crossing over in a mechanism that is still unclear.
Recent findings in yeast meiosis suggest that noncrossover (gene conversion) and crossover recombinants form through sequential and distinct pathways (4
). These studies proposed a model in which recombination is initiated through the formation of single-end invasion structures that later mature into double Holliday junction intermediates that can be resolved into crossovers. In this model, however, large portions of the single-ended invasions are processed to noncrossovers without ever forming stable Holliday junction intermediates. Msh4p has been hypothesized to bind Holliday junctions (30
); such a function could be important in stabilizing the Holliday junction intermediates proposed in such a model.
The role of MLH1
in mismatch repair has been studied primarily through the use of deletion and site-specific mutations; however, only the deletion mutation has been analyzed in meiotic crossing over. Furthermore, most site-specific mutations in MLH1
have been created in amino-terminal residues which have been suggested by crystallographic analysis to be important in ATP binding and/or hydrolysis (9
). This approach has been limited by the fact that only the first 349 residues of MutL have been crystallized and that the approximately 400-amino-acid carboxy-terminal regions of Mlh proteins show modest sequence homology. Previous studies have focused on the mismatch repair aspect of MLH1
function because of its implication in hereditary nonpolyposis colorectal cancer (17
) but a comprehensive characterization of the meiotic functions of MLH1
has yet to be pursued.
In this study, we employed systematic mutagenesis of MLH1 with the goal of identifying regions required for mismatch repair and meiotic functions. This analysis identified both previously studied and uncharacterized regions of MLH1 that are required for mismatch repair and crossing over. In addition, we isolated separation-of-function alleles that conferred defects in postreplicative mismatch repair but did not disrupt meiotic crossing over. We also identified mutants that were functional for meiotic but not vegetative mismatch repair, suggesting that these repair processes are distinct.