Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.     

MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

A role for the MutL homologue MLH2 in controlling heteroduplex formation and in regulating between two different crossover pathways in budding yeast

BACKGROUND AND AIMS: Mismatch repair proteins play important roles during meiotic recombination in the budding yeast Saccharomyces cerevisiae and most eukaryotic organisms studied to date. To study the functions of the mismatch repair protein Mlh2p in meiosis, we constructed mlh2Delta strains and measured rates of crossing over, gene conversion, post-meiotic segregation and spore viability. We also analysed mlh1Delta, mlh3Delta, msh4Delta, msh5Delta, exo1Delta and mus81Delta mutant strains singularly and in various combinations. RESULTS: Loss of MLH2 resulted in a small but significant decrease in spore viability and a significant increase in gene conversion frequencies but had no apparent effect on crossing over. Deletion of MLH2 in mlh3Delta, msh4Delta or msh5Delta strains resulted in significant proportion of the "lost" crossovers found in single deletion strains being regained in some genetic intervals. We and others propose that there are at least two pathways to generate crossovers in yeast (Ross-Macdonald and Roeder, 1994; Zalevsky et al., 1999; Khazanehdari and Borts, 2000; Novak et al., 2001; de los Santos et al., 2003). Most recombination intermediates are processed by the "major", Msh4-dependent pathway, which requires the activity of Mlh1p/Mlh3p/Msh4p/Msh5p as well as a number of other proteins. The minor pathway(s) utilizes Mms4p/Mus81p. We suggest that the absence of Mlh2p allows some crossovers from the MSH4 pathway to traverse the MUS81-dependent pathway.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

金属离子促进嗜热四膜虫错配修复蛋白Mlh3核酸内切酶活性

嗜热四膜虫有性生殖过程中生殖系小核延伸并活跃转录,减数分裂过程中染色体同源重组起始于程序化的DNA双链断裂的形成,DNA错配修复系统能够去除DNA复制过程中所引起的错配并促进同源重组。减数分裂特异表达的错配修复因子Mlh3对四膜虫的有性生殖是必需的,然而具体功能并不清楚。本研究人工合成MLH3（TTHERM_001044369）基因,构建重组表达质粒pGEX-MLH3, 转化大肠杆菌BL21（DE3）并获得重组表达的GST-Mlh3蛋白。纯化的GST-Mlh3蛋白在配位不同的金属离子Cu2+、Mn2+、Mg2+后,有效切割超螺旋质粒DNA。ATP和ADP可进一步促进Mlh3的核酸内切酶活性。突变Mlh3中离子结合模体DQHA(X)2E(E)4E中的D117和E123位点,Mlh3D117N/E123A的核酸内切酶活性降低。进一步删除离子结合和ATP结合位点的C端结构域,突变体的核酸内切酶活性进一步降低,表明Mlh3的核酸内切酶活性是离子依赖型。减数分裂期HA-Mlh3免疫共沉淀鉴定了Mlh3可能的相互作用因子链交换蛋白Dmc1、DSB修复蛋白Mnd1、MutS、染色体维持蛋白Smc2和Smc4。结果表明,四膜虫的Mlh3通过金属离子依赖的内切酶活性,以及与其他因子相互作用,在减数分裂错配修复和同源重组过程中发挥重要作用。     

Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.     

A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae

Interference-dependent crossing over in yeast and mammalian meioses involves the mismatch repair protein homologs MSH4-MSH5 and MLH1-MLH3. The MLH3 protein contains a highly conserved metal-binding motif DQHA(X)(2)E(X)(4)E that is found in a subset of MLH proteins predicted to have endonuclease activities (Kadyrov et al. 2006). Mutations within this motif in human PMS2 and Saccharomyces cerevisiae PMS1 disrupted the endonuclease and mismatch repair activities of MLH1-PMS2 and MLH1-PMS1, respectively (Kadyrov et al. 2006, 2007; Erdeniz et al. 2007). As a first step in determining whether such an activity is required during meiosis, we made mutations in the MLH3 putative endonuclease domain motif (-D523N, -E529K) and found that single and double mutations conferred mlh3-null-like defects with respect to meiotic spore viability and crossing over. Yeast two-hybrid and chromatography analyses showed that the interaction between MLH1 and mlh3-D523N was maintained, suggesting that the mlh3-D523N mutation did not disrupt the stability of MLH3. The mlh3-D523N mutant also displayed a mutator phenotype in vegetative growth that was similar to mlh3Delta. Overexpression of this allele conferred a dominant-negative phenotype with respect to mismatch repair. These studies suggest that the putative endonuclease domain of MLH3 plays an important role in facilitating mismatch repair and meiotic crossing over.     

Distinct functions of MLH3 at recombination hot spots in the mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1-MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3(-/-) exhibit a 85-94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3(-/-) meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3(-/-) spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that approximately 10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.     

Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.CROSSING over during meiosis not only generates variation but is also important for providing the necessary interactions between homologous chromosomes that ensure correct segregation at division I of meiosis. Recombination is initiated by the production of programmed double-strand breaks (DSBs), catalyzed by the covalently attached Spo11p (Bergerat et al. 1997; Keeney et al. 1997), aided by a number of proteins (reviewed in Keeney and Neale 2006). DSBs are made at a much higher frequency than crossovers, and designation of only a subset to yield crossovers is thought to occur during early stages of DSB repair (Borner et al. 2004). At least two distinct pathways contribute to the production of crossover events in Saccharomyces cerevisiae. The major pathway is dependent on Msh4p/Msh5p and the mismatch repair proteins Mlh1p and Mlh3p (Ross-MacDonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Wang et al. 1999; Abdullah et al. 2004) and the second pathway is dependent on Mus81p/Mms4p endonuclease (de los Santos et al. 2001, 2003).Mitotic mismatch repair (MMR) is the process by which mutations that arise during DNA replication and recombination are recognized and removed (reviewed in Kolodner 1996; Harfe and Jinks-Robertson 2000). Msh2p forms a heterodimer with Msh6p (MutSα) to repair base–base mismatches and small insertions and/or deletions and with Msh3p (MutSβ) to repair large insertions and/or deletions (reviewed in Jiricny 2006). Mlh1p forms heterodimers with Pms1p, Mlh2p, and Mlh3p to coordinate the removal of these mismatches (Prolla et al. 1994; Wang et al. 1999). Mlh1p/Pms1p (MutLα) are involved in the repair of all types of mismatches in combination with MutSα and MutSβ, and in the absence of either protein a mutator phenotype is observed (Habraken et al. 1997, 1998). Mlh1p/Mlh2p (MutLβ) and Mlh1p/Mlh3p (MutLγ) are involved in the MutSβ pathway only, which repairs frameshift mutations caused by insertions or deletions. Consequently mlh3Δ mutants only exhibit a weak mutator phenotype, due to a lesser involvement in mismatch repair and a partial overlap in function with Pms1p (Flores-Rozas and Kolodner 1998; Harfe et al. 2000).Although the MutL homologs interact primarily through their C-terminal domains (Pang et al. 1997; Ban and Yang 1998), it is thought that the N-terminal domains must also interact for the complex to be fully functional (Ban and Yang 1998). Binding of ATP causes the proteins to undergo conformational changes, which are essential for the interaction between the N termini (Ban et al. 1999; Tran and Liskay 2000; Sacho et al. 2008). ATP hydrolysis and subsequent release of ADP is required to allow the protein complex to return to its initial state, completing the cycle so that the subunits are ready to bind ATP again if required. Using mutants of MLH1 and PMS1 that are presumed to be defective for ATP binding and/or ATP hydrolysis, it has been shown that both of these functions are essential for fully effective mismatch repair (Tran and Liskay 2000). However, the ATP binding and ATP hydrolysis mutants of PMS1 exhibited lower mitotic mutation rates than the corresponding MLH1 ATPase mutants, suggesting that there is functional asymmetry within the Mlh1p/Pms1p heterodimer (Tran and Liskay 2000; Hall et al. 2002). Another example of the asymmetry in the contributions of these subunits to function can be seen in assays that measure recombination between diverged sequences (homeologous recombination). The Mlh1p ATPase activity has been shown to be more important for the suppression of homeologous recombination than Pms1p ATPase activity (Welz-Voegele et al. 2002). This functional asymmetry is supported by in vitro biochemical analysis that demonstrated Pms1p has a lower ATP binding affinity than Mlh1p (Hall et al. 2002).As mentioned above, Mlh1p/Mlh3p function in the Msh4p/Msh5p pathway for meiotic recombination (Hunter and Borts 1997; Santucci-Darmanin et al. 2000). The Msh4p/Msh5p complex is thought to act in the stabilization of Holliday junction intermediates to allow their resolution in a crossover configuration (Snowden et al. 2004). The Mlh1p/Mlh3p complex has been suggested to act in the resolution of these structures, either directly or indirectly. Human Pms2 and its yeast homolog, Pms1p, have been shown to possess a latent endonuclease activity, conferred by a motif that is conserved among some of the MutL homologs, including Mlh3p (Kadyrov et al. 2006, 2007). Mutations in the DHQA(X)₂E(X)₄E motif in yeast MLH3 cause defects in both mismatch repair and meiotic recombination equivalent to mlh3Δ, suggesting that Mlh3p may also possess an endonuclease activity that is important for the generation of crossovers (Nishant et al. 2008).ATP binding by Mlh1p has been shown to be important for both of its meiotic functions (crossing over and repair of heteroduplex DNA) (Pang et al. 1997; Tran and Liskay 2000; Hoffmann et al. 2003). In contrast, the ATP hydrolysis mutant mlh1-E31A/mlh1-E31A appears to have no effect on meiotic recombination (Tran and Liskay 2000; Hoffmann et al. 2003). This may partly be explained by in vitro studies demonstrating that this mutant exhibits a low level of ATPase activity (Hall et al. 2002).The meiotic functions of MLH1 can be functionally separated as shown by mutating the same residue, G98, to different amino acids (Hoffmann et al. 2003). The residue G98 is situated in the ATPase motif in the GFRGEAL box (GYRGDAL in Mlh3p), which forms the lid of the ATP binding pocket. Mutations in this motif are predicted to affect ATP binding and/or heterodimerization with Pms1p (Ban and Yang 1998; Ban et al. 1999). Mutating the residue G98 in the ATP binding lid to alanine resulted in defective repair of heteroduplex DNA while crossing over was unaffected, but when the same residue was mutated to valine both mismatch repair and crossover functions were defective (Hoffmann et al. 2003). The mlh1-G98V mutant disrupts the interaction of Mlh1p with Pms1p, while mlh1-G98A does not (Pang et al. 1997). This may contribute to the difference observed in the effect on crossing over as Mlh1p is thought to interact with Pms1p and Mlh3p through the same residues (Wang et al. 1999; Kondo et al. 2001). Consequently if the interaction with Pms1p is affected then it is likely that the interaction with Mlh3p is also disrupted.We constructed mlh3 mutants corresponding to the ATP binding and ATP hydrolysis mutants of mlh1 to explore the role of Mlh3p in meiotic recombination. We also constructed mlh3-G97A and mlh3-G97V mutants, equivalent to the mlh1-G98A/V pair that has been shown to differentially affect the mitotic and meiotic functions of Mlh1p. All mutants were assayed for mitotic mismatch repair, meiotic heteroduplex repair, crossing over, and chromosome segregation.     

Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes

Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively). Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps. To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles. Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain. To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates. The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs. recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions.     

The Unstructured Linker Arms of Mlh1-Pms1 Are Important for Interactions with DNA during Mismatch Repair

DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

Handcuffing intrinsically disordered regions in Mlh1–Pms1 disrupts mismatch repair

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker''s yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.     

Functional domains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatch repair proteins and their relevance to human hereditary nonpolyposis colorectal cancer-associated mutations.

The MutL protein is an essential component of the Escherichia coli methyl-directed mismatch repair system but has no known enzymatic function. In the yeast Saccharomyces cerevisiae, the MutL equivalent, an Mlh1p and Pms1p heterodimer, interacts with Msh2p bound to mismatch-containing DNA. Little is known of the functional domains of Mlh1p and Pms1p. In this report, we define the Mlh1p and Pms1p domains required for Mlh1p-Pms1p interaction. The Mlh1p-interactive domain of Pms1p is comprised of 260 amino acids near the carboxyl terminus while the Pms1p-interactive domain of Mlh1p resides in the final 212 residues. The two domains are sufficient for Mlh1p-Pms1p interaction, as determined by the two-hybrid assay and by in vitro protein affinity chromatography. Deletions within the domains completely eliminated Mlh1p-Pms1p interaction. Using site-directed mutagenesis, we altered a number of highly conserved residues in the Mlh1p and Pms1p proteins, including some alterations that mimic germline mutations observed for human hereditary nonpolyposis colorectal cancer. Alterations either in the consensus MutL box located in the amino-terminal portion of each protein or in the carboxyl-terminal homology motif of Mlh1p eliminated DNA mismatch repair function but had no effect on Mlh1p-Pms1p interaction. In addition, certain MLH1 and PMS1 mutant alleles caused a dominant negative mutator effect when overexpressed. We discuss the implications of these findings for the structural organization of the Mlh1p and Pms1p proteins and the importance of Mlh1p-Pms1p interaction.     

Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha

Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.     

Supercomplex formation between Mlh1-Mlh3 and Sgs1-Top3 heterocomplexes in meiotic yeast cells

The genome of Saccharomyces cerevisia encodes four mismatch repair MutL proteins and these proteins form three heterocomplexes: Mlh1-Mlh2, Mlh1-Mlh3, and Mlh1-Pms1. Only, the Mlh1-Mlh3 heterocomplex has been implicated specifically in promotion of meiotic crossing-over. In this report, we show that yeast Mlh3 co-immunoprecipitates with Sgs1 helicase in sporulating cells at late stage of meiotic prophase I. Sgs1, a member of the RecQ DNA helicase family, appears to form a stable complex with topoisomerase III (Top3) during meiosis. We suggest that Mlh1-Mlh3 heterocomplex may act as a molecular matchmaker to coordinate Sgs1-Top3 complex in the resolution of meiotic recombination intermediates.     

Conservation of functional asymmetry in the mammalian MutLα ATPase

The DNA mismatch repair (MMR) protein dimer MutLα is comprised of the MutL homologues MLH1 and PMS2, which each belong to the family of GHL ATPases. These ATPases undergo functionally important conformational changes, including dimerization of the NH₂-termini associated with ATP binding and hydrolysis. Previous studies in yeast and biochemical studies with the mammalian proteins established the importance of the MutLα ATPase for overall MMR function. Additionally, the studies in yeast demonstrated a functional asymmetry between the contributions of the Mlh1 and Pms1 ATPase domains to MMR that was not reflected in the biochemical studies. We investigated the effect of mutating the highly conserved ATP hydrolysis and Mg²⁺ binding residues of MLH1 and PMS2 in mammalian cell lines. Amino acid substitutions in MLH1 intended to impact either ATP binding or hydrolysis disabled MMR, as measured by instability at microsatellite sequences, to an extent similar to MLH1-null mutation. Furthermore, cells expressing these MLH1 mutations exhibited resistance to the MMR-dependent cytotoxic effect of 6-thioguanine (6-TG). In contrast, ATP hydrolysis and binding mutants of PMS2 displayed no measurable increase in microsatellite instability or resistance to 6-TG. Our findings suggest that, in vivo, the integrity of the MLH1 ATPase domain is more critical than the PMS2 ATPase domain for normal MMR functions. These in vivo results are in contrast to results obtained previously in vitro that showed no functional asymmetry within the MutLα ATPase, highlighting the differences between in vivo and in vitro systems.     

Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations

Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.