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Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease*

Open AccessPublished:January 08, 2014DOI:https://doi.org/10.1074/jbc.M113.534644
      Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

      Introduction

      DNA mismatch repair (MMR)
      The abbreviations used are:
      MMR
      mismatch repair
      MLH
      MutL homolog
      (d)HJs
      (double) Holliday junctions
      β-ME
      β-mercaptoethanol
      PCNA
      proliferating cell nuclear antigen
      RFC
      replication factor C.
      is a conserved mechanism that acts during DNA replication to remove DNA polymerase misincorporation errors such as base-base and insertion/deletion mismatches. In the well characterized Escherichia coli MMR system, MutS recognizes and binds mismatches, recruiting MutL. This interaction relays mismatch recognition by MutS to the MutH endonuclease, resulting in nicking at nearby GATC sites of the unmethylated, newly replicated strand. MutH nicking provides an entry point for UvrD helicase and single-stranded exonucleases that then excise the mismatch, resulting in a gap that is filled in by replicative DNA polymerases (
      • Kunkel T.A.
      • Erie D.A.
      DNA mismatch repair.
      ).
      Multiple MutS and MutL homologs (MSH and MLH, respectively) have been identified in eukaryotes. In Saccharomyces cerevisiae, the MSH complexes Msh2-Msh6 and Msh2-Msh3 bind to DNA mismatches and interact primarily with the MLH complex Mlh1-Pms1 to activate mismatch excision and DNA re-synthesis steps (
      • Kunkel T.A.
      • Erie D.A.
      DNA mismatch repair.
      ). Msh2-Msh6 primarily acts to repair base-base and small insertion/deletion mismatches, whereas Msh2-Msh3 primarily acts to repair larger (up to 17 nucleotides) insertion/deletion loop mismatches (
      • Kunkel T.A.
      • Erie D.A.
      DNA mismatch repair.
      ). A minor MMR pathway has been identified that involves recognition of insertion/deletion mismatches by Msh2-Msh3 followed by interaction with the Mlh1-Mlh3 complex (
      • Hunter N.
      Molecular Genetics of Recombination.
      ,
      • Flores-Rozas H.
      • Kolodner R.D.
      The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations.
      ). Interestingly, subsets of MSH and MLH proteins act in a distinct, interference-dependent, meiotic crossover pathway (see below).
      MutH-type endonucleases have not been identified in eukaryotes; however, a subset of MLH complexes displays DNA nicking activity (
      • Guarné A.
      The functions of MutL in mismatch repair. The power of multitasking.
      ). For example, human Mlh1-Pms2 (hMutLα) and bakers' yeast Mlh1-Pms1 display latent endonuclease activities that are essential for mismatch repair (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ). Studies on human MMR by Modrich and co-workers (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ) have suggested that hMutLα nicking activity, which is directed to an existing nick, provides access to exonucleases (e.g. Exo1) that act in mismatch excision and may also facilitate strand discrimination. Mutations in a DQHAX2EX4E metal binding motif present in both human Pms2 and yeast Pms1 disrupt both MLH endonuclease activity and MMR (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ,
      • Deschênes S.M.
      • Tomer G.
      • Nguyen M.
      • Erdeniz N.
      • Juba N.C.
      • Sepúlveda N.
      • Pisani J.E.
      • Liskay R.M.
      The E705K mutation in hPMS2 exerts recessive, not dominant, effects on mismatch repair.
      ). Recently, two groups reported the crystal structures of the endonuclease domain of MLH proteins (
      • Pillon M.C.
      • Lorenowicz J.J.
      • Uckelmann M.
      • Klocko A.D.
      • Mitchell R.R.
      • Chung Y.S.
      • Modrich P.
      • Walker G.C.
      • Simmons L.A.
      • Friedhoff P.
      • Guarné A.
      Structure of the endonuclease domain of MutL. Unlicensed to cut.
      ,
      • Gueneau E.
      • Dherin C.
      • Legrand P.
      • Tellier-Lebegue C.
      • Gilquin B.
      • Bonnesoeur P.
      • Londino F.
      • Quemener C.
      • Le Du M.H.
      • Márquez J.A.
      • Moutiez M.
      • Gondry M.
      • Boiteux S.
      • Charbonnier J.B.
      Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.
      ). For Bacillus subtilis MutL, the endonuclease domain consisted of dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Such an organization was proposed to serve as a regulatory module to prevent MutL from promiscuously nicking DNA. The authors also suggest that interactions involving MutL and other protein factors, such as MutS and processivity clamp, trigger conformational changes in this motif that modulate MutL endonuclease activity (
      • Pillon M.C.
      • Lorenowicz J.J.
      • Uckelmann M.
      • Klocko A.D.
      • Mitchell R.R.
      • Chung Y.S.
      • Modrich P.
      • Walker G.C.
      • Simmons L.A.
      • Friedhoff P.
      • Guarné A.
      Structure of the endonuclease domain of MutL. Unlicensed to cut.
      ).
      Crossing over in meiotic prophase in most sexually reproducing organisms creates physical connections between chromosome homologs that are critical for their proper segregation in Meiosis I (
      • Zickler D.
      From early homologue recognition to synaptonemal complex formation.
      ). In bakers' yeast, meiotic recombination is initiated by the formation of about 140–170 double-strand breaks located throughout the genome (
      • Cao L.
      • Alani E.
      • Kleckner N.
      A pathway for generation and processing of double strand breaks during meiotic recombination in.
      ,
      • Keeney S.
      • Giroux C.N.
      • Kleckner N.
      Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.
      ,
      • Chen S.Y.
      • Tsubouchi T.
      • Rockmill B.
      • Sandler J.S.
      • Richards D.R.
      • Vader G.
      • Hochwagen A.
      • Roeder G.S.
      • Fung J.C.
      Global analysis of the meiotic crossover landscape.
      ,
      • Robine N.
      • Uematsu N.
      • Amiot F.
      • Gidrol X.
      • Barillot E.
      • Nicolas A.
      • Borde V.
      Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae.
      ,
      • Pan J.
      • Sasaki M.
      • Kniewel R.
      • Murakami H.
      • Blitzblau H.G.
      • Tischfield S.E.
      • Zhu X.
      • Neale M.J.
      • Jasin M.
      • Socci N.D.
      • Hochwagen A.
      • Keeney S.
      A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation.
      ). Approximately 40% of these double-strand breaks are converted into crossovers between homologous chromosomes, and the remainder are repaired as noncrossovers or by using a sister chromatid as a template (
      • Schwacha A.
      • Kleckner N.
      Identification of double Holliday junctions as intermediates in meiotic recombination.
      ). These double-strand breaks are resected to form 3′ single-strand tails that primarily invade the unbroken homolog and undergo pairing with complementary sequences. Stabilized invasion intermediates (single-end invasion intermediates) are converted into double Holliday junctions (dHJs) and ultimately resolved into crossovers (
      • Hunter N.
      • Kleckner N.
      The single-end invasion. An asymmetric intermediate at the double-strand break to double-Holliday Junction transition of meiotic recombination.
      ,
      • Allers T.
      • Lichten M.
      Intermediates of yeast meiotic recombination contain heteroduplex DNA.
      ). Work in bakers' yeast has suggested that the majority of crossovers are formed in an interference-dependent pathway where dHJs, stabilized by Msh4-Msh5 (
      • Börner G.V.
      • Kleckner N.
      • Hunter N.
      Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis.
      ,
      • Snowden T.
      • Acharya S.
      • Butz C.
      • Berardini M.
      • Fishel R.
      hMSH4-hMSH5 recognizes Holliday junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes.
      ,
      • Nishant K.T.
      • Chen C.
      • Shinohara M.
      • Shinohara A.
      • Alani E.
      Genetic analysis of bakers' yeast Msh4-Msh5 reveals a threshold crossover level for meiotic viability.
      ), are resolved through the actions of the Sgs1 helicase, the Exo1 XPG nuclease (in steps not requiring its exonuclease activity), and a putative Mlh1-Mlh3 endonuclease activity (
      • Jessop L.
      • Rockmill B.
      • Roeder G.S.
      • Lichten M.
      Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of Sgs1.
      ,
      • Oh S.D.
      • Lao J.P.
      • Hwang P.Y.
      • Taylor A.F.
      • Smith G.R.
      • Hunter N.
      BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules.
      ,
      • De Muyt A.
      • Jessop L.
      • Kolar E.
      • Sourirajan A.
      • Chen J.
      • Dayani Y.
      • Lichten M.
      BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism.
      ,
      • Zakharyevich K.
      • Tang S.
      • Ma Y.
      • Hunter N.
      Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.
      ,
      • Nishant K.T.
      • Plys A.J.
      • Alani E.
      A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae.
      ,
      • Argueso J.L.
      • Wanat J.
      • Gemici Z.
      • Alani E.
      Competing crossover pathways act during meiosis in Saccharomyces cerevisiae.
      ).
      Little is known at the biochemical level about how Sgs1, Exo1, and Mlh1-Mlh3 act on crossover intermediates and how Msh4-Msh5 might coordinate Mlh1-Mlh3. One model gaining increasing attention is that Msh4-Msh5 restricts a putative Mlh1-Mlh3 endonuclease activity to recombination intermediates that are resolved to crossover products, and Sgs1 acts as a pro-crossover factor creating a specific dHJ structure that can be readily cleaved by an endonuclease activity intrinsic to Mlh1-Mlh3 (
      • Zakharyevich K.
      • Tang S.
      • Ma Y.
      • Hunter N.
      Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.
      ). These ideas are supported by the genetic and physical studies cited above as well as the following. 1) Msh4-Msh5 can bind to both single-end invasion and Holliday junction substrates in vitro (
      • Snowden T.
      • Acharya S.
      • Butz C.
      • Berardini M.
      • Fishel R.
      hMSH4-hMSH5 recognizes Holliday junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes.
      ). 2) Mlh3 contains a DQHAX2EX4E metal binding motif (Fig. 1A). 3) Cell biological assays show that Mlh1-Mlh3 acts downstream of Msh4-Msh5 in meiosis (
      • Lipkin S.M.
      • Moens P.B.
      • Wang V.
      • Lenzi M.
      • Shanmugarajah D.
      • Gilgeous A.
      • Thomas J.
      • Cheng J.
      • Touchman J.W.
      • Green E.D.
      • Schwartzberg P.
      • Collins F.S.
      • Cohen P.E.
      Meiotic arrest and aneuploidy in MLH3-deficient mice.
      ,
      • Moens P.B.
      • Kolas N.K.
      • Tarsounas M.
      • Marcon E.
      • Cohen P.E.
      • Spyropoulos B.
      The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination.
      ,
      • Kolas N.K.
      • Svetlanov A.
      • Lenzi M.L.
      • Macaluso F.P.
      • Lipkin S.M.
      • Liskay R.M.
      • Greally J.
      • Edelmann W.
      • Cohen P.E.
      Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.
      ). 4) Crossing over in bakers' yeast meiosis is reduced by ∼2-fold in mlh3Δ mutants and to a much greater extent (7–16-fold) in mms4Δ mlh3Δ mutants defective in both interference-dependent and -independent crossover pathways (
      • Nishant K.T.
      • Chen C.
      • Shinohara M.
      • Shinohara A.
      • Alani E.
      Genetic analysis of bakers' yeast Msh4-Msh5 reveals a threshold crossover level for meiotic viability.
      ,
      • Argueso J.L.
      • Wanat J.
      • Gemici Z.
      • Alani E.
      Competing crossover pathways act during meiosis in Saccharomyces cerevisiae.
      ,
      • Hunter N.
      • Borts R.H.
      Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis.
      ,
      • Wang T.F.
      • Kleckner N.
      • Hunter N.
      Functional specificity of MutL homologs in yeast. Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction.
      ,
      • Sonntag Brown M.
      • Lim E.
      • Chen C.
      • Nishant K.T.
      • Alani E.
      Genetic analysis of mlh3 mutations reveals interactions between crossover promoting factors during meiosis in bakers' yeast.
      ).
      Figure thumbnail gr1
      FIGURE 1Purification of the Mlh1-Mlh3 complex. A, organization of Mlh3 including the conserved ATP binding domain (dark blue boxes) in the highly conserved N-terminal domain (cyan), the 117-amino acid predicted linker domain (gray box), the Mlh1 interaction domain in the less well conserved C-terminal domain of Mlh3 (blue box), and residues within interaction domain that form part of the endonuclease active site (red box). The aspartic acid residue at amino acid 523 that was mutated to asparagine (D523N) is indicated with an arrow. B, left panel, schematic of the Mlh1-Mlh3 complex based on crystal structures (
      • Pillon M.C.
      • Lorenowicz J.J.
      • Uckelmann M.
      • Klocko A.D.
      • Mitchell R.R.
      • Chung Y.S.
      • Modrich P.
      • Walker G.C.
      • Simmons L.A.
      • Friedhoff P.
      • Guarné A.
      Structure of the endonuclease domain of MutL. Unlicensed to cut.
      ,
      • Gueneau E.
      • Dherin C.
      • Legrand P.
      • Tellier-Lebegue C.
      • Gilquin B.
      • Bonnesoeur P.
      • Londino F.
      • Quemener C.
      • Le Du M.H.
      • Márquez J.A.
      • Moutiez M.
      • Gondry M.
      • Boiteux S.
      • Charbonnier J.B.
      Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.
      ,
      • Ban C.
      • Yang W.
      Crystal structure and ATPase activity of MutL. Implications for DNA repair and mutagenesis.
      ,
      • Kosinski J.
      • Steindorf I.
      • Bujnicki J.M.
      • Giron-Monzon L.
      • Friedhoff P.
      Analysis of the quaternary structure of the MutL C-terminal domain.
      ,
      • Sacho E.J.
      • Kadyrov F.A.
      • Modrich P.
      • Kunkel T.A.
      • Erie D.A.
      Direct visualization of asymmetric adenine-nucleotide-induced conformational changes in MutLα.
      ), including predicted lengths of linker arms (144 amino acids for Mlh1, 117 amino acids for Mlh3) and location of His10, HA epitope, and FLAG epitope insertions. In this model Mlh1 and Mlh3 are each composed of two globular domains connected by a flexible linker, and Mlh1 and Mlh3 interact though their C-terminal domains. The N-terminal ATP binding domains of both proteins are highly conserved among the MutL homologs and undergo ATP-dependent conformational changes. Mlh1 is in green, and Mlh3 is in blue. Right panel, SDS-PAGE analysis of purified Mlh1-Mlh3 (WT) and Mlh1-mlh3-D523N (DN). Coomassie Blue R250-stained 8% Tris-glycine gel: 0.5 μg of Mlh1-mlh3-D523N; 0.5 μg of Mlh1-Mlh3; mw = molecular mass standards from top to bottom: 200, 116, 97, 66, 45, 31 kDa. C, mass spectrometry analysis of bands detected after Q-Sepharose chromatography.
      A DQHAX2EX4E metal binding motif found in MLH endonucleases was identified in the beginning of the C-terminal domain of Mlh3 (Fig. 1A). Nishant et al. (
      • Nishant K.T.
      • Plys A.J.
      • Alani E.
      A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae.
      ) showed that a mutation in this motif, mlh3-D523N, created a dominant negative allele and conferred a null phenotype for Mlh3 functions in both MMR and meiotic crossing over. They also suggested that Mlh1-Mlh3 possesses an endonuclease activity required for its meiotic crossing over and MMR functions. We present the purification of bakers' yeast Mlh1-Mlh3 and demonstrate that it is a metal-dependent endonuclease that nicks supercoiled DNA. Mlh1-Mlh3 also facilitates binding of Msh2-Msh3 to insertion/deletion mismatches in vitro, and its endonuclease activity on supercoiled DNA is stimulated by Msh2-Msh3. In conjunction with previous genetic data, our work supports a role for the Mlh1-Mlh3 endonuclease in MMR and in resolving recombination intermediates.

      DISCUSSION

      In this study we showed that the bakers' yeast Mlh1-Mlh3 complex displayed an endonuclease activity. We also provided the first purification protocol for Mlh1-Mlh3, which will be important in mechanistic studies in MMR and meiotic crossover control (outlined below). This activity was dependent on divalent metal but was not stimulated by ATP or RFC + PCNA. Interestingly Mlh1-Mlh3 bound to various oligonucleotide substrates but did not cleave them. Finally, we detected interactions between Mlh1-Mlh3 and Msh2-Msh3 in gel shift assays and showed that Msh2-Msh3 stimulated Mlh1-Mlh3 endonuclease activity.
      Genetic studies suggested that Mlh1-Mlh3 intrinsic endonuclease domain is important for resolving dHJs into crossovers, yet the biochemical mechanism remains unknown (
      • Zakharyevich K.
      • Tang S.
      • Ma Y.
      • Hunter N.
      Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.
      ,
      • Nishant K.T.
      • Plys A.J.
      • Alani E.
      A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae.
      ). To our knowledge MLH proteins have not been observed to cleave any type of model substrate constructed from oligonucleotides. However, a large number of MLH proteins that contain an endonuclease domain motif display endonuclease activity on supercoiled and nicked plasmids (
      • Guarné A.
      The functions of MutL in mismatch repair. The power of multitasking.
      ,
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ), suggesting a DNA binding specificity for MLH proteins that is not seen on small synthetic substrates. Our work shows that Mlh1-Mlh3 can nick both supercoiled and pre-nicked DNA; one way to explain our observations is that Mlh1-Mlh3-mediated endonuclease cleavage requires longer oligonucleotide substrates than those used in our study. MLH proteins have been shown to bind cooperatively to DNA (
      • Hall M.C.
      • Wang H.
      • Erie D.A.
      • Kunkel T.A.
      High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer.
      ); thus longer DNA substrates may be needed to stabilize MLH binding to promote DNA cleavage. Consistent with this idea, we hypothesize that Msh2-Msh3 stimulates Mlh1-Mlh3 endonuclease activity by binding non-specifically to supercoiled DNA or possibly to loops in supercoiled DNA created by the extrusion of inverted repeat sequences (
      • Giraud-Panis M.J.
      • Lilley D.M.
      Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII.
      ). In this model Mlh1-Mlh3 is specifically recruited to Msh2-Msh3 that is stably bound to DNA, thus promoting activation of the Mlh1-Mlh3 endonuclease.
      Unlike other MLH endonucleases, the Mlh1-Mlh3 endonuclease was not stimulated by ATP or RFC + PCNA. Based on these results we suggest that Mlh1-Mlh3 endonuclease activity on Holliday junctions requires novel and complex interactions with other pro-crossover factors. In support of this idea, Zakharyevich et al. (
      • Zakharyevich K.
      • Tang S.
      • Ma Y.
      • Hunter N.
      Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.
      ) suggested that Msh4-Msh5 interacts with an Exo1-Mlh1-Mlh3 nuclease ensemble to resolve dHJs and that Sgs1 acts as a pro-crossover factor to interact with Mlh1-Mlh3 and ZMM meiotic factors (ZIP, MSH, MER) to stabilize strand exchange intermediates and to coordinate the formation of dHJs (see also
      • De Muyt A.
      • Jessop L.
      • Kolar E.
      • Sourirajan A.
      • Chen J.
      • Dayani Y.
      • Lichten M.
      BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism.
      ). They also proposed that the Sgs1 helicase could act in this role by promoting a specific type of dHJ conformation that can be readily cleaved by an endonuclease. Such interactions between Mlh1-Mlh3 and the above factors may be necessary to facilitate large conformational changes within the Mlh1-Mlh3 complex to allow for cleavage of HJs. In support of this idea, Gueneau et al. (
      • Gueneau E.
      • Dherin C.
      • Legrand P.
      • Tellier-Lebegue C.
      • Gilquin B.
      • Bonnesoeur P.
      • Londino F.
      • Quemener C.
      • Le Du M.H.
      • Márquez J.A.
      • Moutiez M.
      • Gondry M.
      • Boiteux S.
      • Charbonnier J.B.
      Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.
      ) purified and crystallized the MutLα C-terminal domain. They did not observe an endonuclease activity associated with the C-terminal domain and suggested, as did others (e.g.
      • Pillon M.C.
      • Lorenowicz J.J.
      • Uckelmann M.
      • Klocko A.D.
      • Mitchell R.R.
      • Chung Y.S.
      • Modrich P.
      • Walker G.C.
      • Simmons L.A.
      • Friedhoff P.
      • Guarné A.
      Structure of the endonuclease domain of MutL. Unlicensed to cut.
      ), that conformational rearrangements involving DNA, the N-terminal domain of MutLα, and the linker regions connecting the N- and C-terminal domains and metal cations are required for this activity.
      We showed that Msh2-Msh3 stimulates the endonuclease activity of Mlh1-Mlh3. Interactions between MSH and MLH proteins has been characterized in several organisms and have been shown to involve the ATP binding domain of the MLH factor studied (
      • Mendillo M.L.
      • Hargreaves V.V.
      • Jamison J.W.
      • Mo A.O.
      • Li S.
      • Putnam C.D.
      • Woods Jr., V.L.
      • Kolodner R.D.
      A conserved MutS homolog connector domain interface interacts with MutL homologs.
      ,
      • Lenhart J.S.
      • Pillon M.C.
      • Guarné A.
      • Simmons L.A.
      Trapping and visualizing intermediate steps in the mismatch repair pathway in vivo.
      ), thereby reinforcing the idea that the crosstalk between the N- and C-terminal domains of Mlh1-Mlh3 is important to promote resolution of dHJs into crossovers. We have not explored whether post-translational modifications are required to activate Mlh1-Mlh3 during meiotic prophase. Such modifications were detected by Matos et al. (
      • Matos J.
      • Blanco M.G.
      • Maslen S.
      • Skehel J.M.
      • West S.C.
      Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis.
      ). They found that the Mus81-Mms4 endonuclease was phosphorylated and hyper-activated by Cdc5 during meiosis. This activation was critical to generate crossing over required for proper chromosome segregation in Meiosis I.

      Acknowledgments

      We are grateful to Aaron Plys for initiating this project, Petr Cejka and Joe Jiricny for sharing unpublished information and reagents, John Pagano and Manju Hingorani for technical advice, and members of the Alani laboratory for helpful comments.

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