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Regulation of the MLH1–MLH3 endonuclease in meiosis

A Publisher Correction to this article was published on 19 January 2021

This article has been updated

Abstract

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1–MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3,4,5,6,7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4–MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ–MutSγ–EXO1–RFC–PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC–PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.

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Fig. 1: MSH4–MSH5 and EXO1(DA) interact with and promote the MLH1–MLH3 endonuclease.
Fig. 2: RFC–PCNA promotes DNA cleavage by the MutLγ–MutSγ–EXO1(DA) ensemble.
Fig. 3: The stimulation of the MLH3 nuclease ensemble by PCNA requires a PIP box motif and is conserved in evolution.

Data availability

All relevant data generated or analysed during this study are included in this published article and its Supplementary Information file.

Change history

  • 19 January 2021

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by grants from the Swiss National Science Foundation (31003A_17544) and ERC (681-630) to P.C., Institut Curie and CNRS to V.B., Agence Nationale de la Recherche (ANR-15-CE11-0011) to V.B. and J.-B.C., the Novo Nordisk Foundation (NNF15OC0016662) and ERC (724718) to E.R.H., and the Swiss National Science Foundation (155823 and 176108) to J.M. We thank J. Jiricny (ETH Zurich) and members of the Cejka laboratory for helpful comments on the manuscript and N. Hunter for communicating results before publication. We thank ScopeM at ETH Zurich for instrument access.

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Authors

Contributions

E.C., A.S., R.A. and P.C. planned, performed and analysed the majority of the experiments and wrote the paper. L.R. and A.A. performed most of the experiments with yeast recombinant proteins and electrophoretic mobility shift assays. N.W. performed experiments to define simultaneous DNA binding by MLH1–MLH3 and MSH4–MSH5. J.H. performed experiments with yeast mlh1 and mlh3 variants mutated in PIP-box-like sequences, and analysed the data together with J.M. Chip experiments and Rfc1–Mlh1 and Rfc1–Mlh3 pulldown assays were carried out by C.A., and the data analysed together with V.B. J.-B.C. helped to prepare the MLH1–MLH3 expression construct and designed experiments with the PIP-box peptide. X.A.-G. and E.R.H. prepared the MSH4–MSH5 expression construct. All authors contributed to prepare the final version of the manuscript.

Corresponding author

Correspondence to Petr Cejka.

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Peer review information Nature thanks Andreas Hochwagen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 ATP hydrolysis promotes MLH1-MLH3 to nick scDNA.

a, A scheme of MLH1 and MLH3 constructs. The maltose-binding protein (MBP) on MLH3 was cleaved during protein purification. b, Recombinant MLH1-MLH3 used in this study. The 4–15% gradient polyacrylamide gel was stained with Coomassie blue. The experiments in this study used 3 preparations of MLH1-MLH3 that looked similar upon electrophoretic separation and exhibited similar activities. c, Nuclease assay with MLH1-MLH3 and 2.7 kbp-long supercoiled DNA (scDNA) as a substrate. The reaction with 5 mM manganese acetate was incubated without ATP at 37 °C. This experiment was carried out three times with similar results. d, Quantitation of assays such as in c. Averages shown; error bars, SEM; n = 3 independent experiments. e, Nuclease assay with MLH1-MLH3 (300 nM) and 2.7 kbp-long scDNA. Linear DNA was used as a marker. The assay was carried out at 37 °C and contained 5 mM manganese acetate and ATP (0.5 mM). The MLH1-MLH3 nuclease introduces nicks in dsDNA but does not linearize dsDNA. This experiment was carried out three times with similar results. f, Quantitation of nuclease assays with MLH1-MLH3 without or with ATP (0.5 mM), in the presence of manganese (5 mM). Averages shown; error bars, SEM; n = 4 independent experiments. g, Nuclease assay with MLH1-MLH3 and 5 mM magnesium acetate. The reaction buffer contained ATP (0.5 mM). The assay was carried out at 37 °C. The heterodimer exhibits barely detectable nuclease activity in magnesium. This experiment was carried out two times with similar results. h, Nuclease assay with MLH1-MLH3 and various nucleotide cofactors (ADP, ATP and non-hydrolysable ATP analogs ATP-γ-S and AMP-PNP, all 0.5 mM). The assay was carried out at 37 °C with 5 mM manganese acetate. The panel shows a representative experiment. This experiment was carried out four times with similar results. i, Quantitation of nuclease assays such as in panel h, supplemented with various nucleotide co-factors and their analogs (0.5 mM). Averages shown; error bars, SEM; n = 4 independent experiments. j, Purified MLH1-MLH3 variants used in this study. MLH1(EA), MLH1(E34A); MLH3(EA), MLH3(E28A); MLH3(3ND), MLH3(D1223N-Q1224K-E1229K). The 4–15% gradient polyacrylamide gel was stained with Coomassie blue. We used single preparations of the mutant MLH1-MLH3 variants. k, Alignment of MLH1 and MLH3 ATPase motifs. Conserved residues are highlighted in red. Alanine substitutions in MLH3 and MLH1 mutants used in this study are in italics. l, Nuclease assay with wild type MLH1-MLH3 and indicated variants deficient in ATP hydrolysis, without or with ATP (0.5 mM). The assay was carried out at 37 °C, with 5 mM manganese acetate. This experiment was carried out six times with similar results. m, Quantitation of nuclease assays as shown in panel l, without or with ATP (0.5 mM), with either wild type or MLH1-MLH3 variants mutated in conserved ATPase domain residues. Averages shown; error bars, SEM; n = 6 independent experiments. n, Electrophoretic mobility shift assay with indicated MLH1-MLH3 variants, oligonucleotide-based Holliday junction as the substrate, in the absence of ATP and no magnesium (with 3 mM EDTA). Asterisk (*) indicates the position of the 5' radioactive label. A representative experiment is shown at the bottom, a quantitation (averages shown, n = 3 independent experiments; error bars, SEM) at the top. o, Nuclease assays with wild type MLH1-MLH3 on oligonucleotide-based DNA substrates (Holliday junction, HJ and nicked Holliday junction, nicked HJ). The asterisk indicates the position of the 5' radioactive label. The assay was carried out at 37 °C, with 5 mM manganese or magnesium acetate, as indicated, with ATP (1 mM). The products were analysed by 10% native polyacrylamide gel electrophoresis. This experiment was carried out two times with similar results.

Extended Data Fig. 2 Human and yeast MutSγ complexes preferentially bind branched DNA intermediates.

a, Recombinant human MSH4-MSH5 used in this study. We used 3 preparations of MSH4-MSH5 that exhibited similar activities. b, Electrophoretic mobility shift assays with human MSH4-MSH5 and indicated DNA substrates. Asterisk (*) indicates the position of the 5' radioactive label. The assays were carried out in a buffer containing 2 mM magnesium acetate without ATP. The experiments were performed three times. c, Quantitation of DNA binding assays such as shown in panel b. Averages shown; error bars, SEM; n = 3 independent experiments. d, Electrophoretic mobility shift assays with yeast Msh4-Msh5 and indicated DNA substrates. Asterisk (*) indicates the position of the 5' radioactive label. The assays were carried out in a buffer containing 2 mM magnesium acetate without ATP. The experiments were performed two times. e, Quantitation of experiments such as shown in panel d. The lines represent average; n = 2 independent experiments. f, Quantification of electrophoretic mobility shift assays with yeast Msh4-Msh5 and indicated DNA substrates, without magnesium (with 3 mM EDTA). The lines represent average; n = 2 independent experiments.

Extended Data Fig. 3 MutSγ and MutLγ physically interact and moderately stabilize each other at DNA junctions.

a, To investigate the interplay of MutLγ and MutSγ at DNA junctions, we performed electrophoretic mobility shift assays with either or both complexes under more stringent conditions (75 mM NaCl, 2 mM magnesium acetate), separated on 0.6% agarose gels. Under these conditions, MSH4-MSH5 lost the capacity to stably bind Holliday junctions/D-Loops, but could help stabilize the MutSγ-MutLγ complex. The binding of MutLγ alone was not stable, as evidenced by a weak protein-DNA band and the presence of smear in the lanes indicative of complexes that dissociated during electrophoresis. The addition of MutSγ resulted in a moderate stabilization of the protein-DNA complex, and a minor super-shift in electrophoretic mobility of the stable protein-DNA band (indicated by the red and blue arrows). The experiment was performed three times with similar results. b, Electrophoretic mobility shift assays as in panel a, but without magnesium (with 3 mM EDTA). The experiment was performed five times with similar results. c, Quantitation of assays such as shown in panel b. The Y axis indicates relative protein-DNA complex stability, obtained upon dividing the protein-DNA band intensity (see blue or red arrows in panel b) by the intensity of the radioactive signal in the lane above the free substrate band, but below the protein-DNA band. Averages shown; error bars, SEM; n = 5 independent experiments. d, Assays as in a, with human MutLγ and either human or yeast MutSγ. The supershift was observed only when the cognate human complexes were combined. The experiment was performed two times with similar results. e, Electrophoretic mobility shift assays as in a, but with yeast MutLγ and MutSγ complexes. The experiment was performed two times with similar results. f, Protein interaction assays with immobilized MLH1-MLH3 (bait) and MSH4-MSH5 (prey). The 10% polyacrylamide gel was stained with silver. The experiment was performed two times with similar results. g, Protein interaction assays with immobilized human MSH4-MSH5 or yeast Msh4-Msh5 that were used as baits, and human MLH1-MLH3 (prey). The eluted proteins were analysed by silver staining. Although interaction between yeast Msh4-Msh5 and human MLH1-MLH3 was still detected, it was weaker than the interaction between the cognate MSH4-MSH5 and MLH1-MLH3 complexes. The experiment was performed two times with similar results. h, Protein interaction assay with immobilized yeast Msh4-Msh5 (bait) and yeast Mlh1-Mlh3 (prey). The eluted proteins were analysed by western blotting. The experiment was performed two times with similar results. i, Electrophoretic mobility shift assays with MLH1-MLH3 and MSH4-MSH5, as indicated, and oligonucleotide-based Holliday junction DNA substrate. 32P-labelled λDNA/HindIII digest was used as a marker. The DNA-bound MLH1-MLH3 and MSH4-MSH5 species migrate high up on the agarose gel where the resolution capacity is limited. The experiment was performed two times with similar results. j, Electrophoretic mobility shift assay with yeast Ku70-Ku80 heterodimer and Holliday junction DNA substrate. Ku bound the dsDNA ends of the four Holliday junction arms, resulting in up to 4 heterodimers bound to the DNA substrate (lanes 5-7). Comparison with λ DNA/HindIII and panel i revealed that the Ku-DNA complex migrates much faster than DNA-bound MLH1-MLH3 and MSH4-MSH5. This suggests that multiple units of MLH1-MLH3 and MSH4-MSH5 bind DNA. The experiment was performed two times with similar results.

Extended Data Fig. 4 MSH4-MSH5 promotes DNA cleavage by MLH1-MLH3, but the complex does not exhibit resolvase activity.

a, Quantitation of kinetic nuclease assays with MLH1-MLH3 (50 nM) without or with MSH4-MSH5 (50 nM) using 5.6 kbp-long scDNA. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 2 mM ATP. Averages shown; error bars, SEM; n = 3 independent experiments. b, Nuclease assays with MSH4-MSH5 and either wild type MLH1-MLH3 or nuclease-dead MLH1-MLH3 (D1223N-Q1224K-E1229K, 3ND). The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. The experiment was performed three times with similar results. c, Quantitation of nuclease assays with various MLH1-MLH3 and MSH4-MSH5 concentrations, as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. Averages shown; error bars, SEM, n = 3 independent experiments. The efficiency of nuclease cleavage was generally dependent on the concentrations used. When using 50 nM MLH1-MLH3, the maximal cleavage efficiency was achieved together with 50 nM MSH4-MSH5, no further increase when using 100 nM MSH4-MSH5 was observed. This suggests that both heterodimers may form a stoichiometric complex. Vice versa, when using 50 nM MSH4-MSH5, a further increase of DNA cleavage was observed when MLH1-MLH3 concentrations exceeded 50 nM, which is in agreement with the capacity of MLH1-MLH3 to cleave DNA on its own. d, Quantitation of nuclease assays with MLH1-MLH3 and MSH4-MSH5, as indicated, in the presence of various nucleotide co-factors or their analogs (2 mM). The assays were carried out at 30 °C in the presence of 5 mM manganese acetate. Averages shown; error bars, SEM; n = 6 independent experiments. e, Representative nuclease assays with MSH4-MSH5 and variants of MLH1-MLH3 deficient in ATP hydrolysis, as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. The experiment was performed three times with similar results. f, Representative nuclease assays with MLH1-MLH3 and variants of MSH4-MSH5 deficient in ATP hydrolysis, as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. The experiment was performed three times with similar results. g, Recombinant MSH4-MSH5 and its variants used in this study. MSH4(G685A), MSH4(GA); MSH5(G597A), MSH5(GA). The 4–15% gradient polyacrylamide gel was stained with Coomassie blue. We used a single preparation of the MSH4-MSH5 mutant variants. h, Quantitation of electrophoretic mobility shift assays with MSH4-MSH5 and its ATPase motif mutant variants. Oligonucleotide-based Holliday junction was used as the substrate. Asterisk (*) indicates the position of the 5' radioactive label. ATP was not included in the binding buffer. The mutations did not affect the capacity of MSH4-MSH5 to bind DNA. Averages shown; error bars, SEM; n = 3 independent experiments. i, Nuclease reactions were carried out with yeast or human MutSγ and MutLγ complexes, as indicated (50 nM), with 2.7 kbp-long scDNA substrate. While human MutSγ promoted DNA cleavage by human MutLγ (compare lanes 2 and 3), yeast MutSγ did not notably promote DNA cleavage by human MutLγ (compare lanes 2 and 5), and reciprocally, human MutSγ did not promote DNA cleavage by yeast MutLγ (compare lanes 7 and 8). The experiment was performed two times with similar results. j, Quantitation of nuclease assays with human and yeast MutSγ and MutLγ complexes as in panel i, but with 10.3 kbp-long scDNA substrate. Averages shown; error bars, SEM; n = 3 independent experiments. k, Cleavage of pIRbke8mut cruciform DNA (inverted repeats folding back to form a Holliday junction structure) by MutSγ and MutLγ complexes. The quantitation below the lanes represents an average from two independent experiments. Simultaneous cleavage of both strands at the junction point would lead to linear DNA. No linear DNA was observed with MutSγ and MutLγ, indicating a lack of canonical resolvase activity. The experiment was performed nine times with similar results. l, Representative nuclease assays with indicated proteins and oligonucleotide-based Holliday junction DNA. Asterisk (*) indicates the position of the 5' radioactive label. No DNA cleavage was observed, indicating a lack of structure-specific DNA cleavage activity on the oligonucleotide-based substrate. The products were analysed by 15% denaturing polyacrylamide gel electrophoresis. The experiment was performed two times with similar results.

Extended Data Fig. 5 MutSβ but not MutSα stimulates MutLγ to a similar extent as MutSγ.

a, Coomassie-stained polyacrylamide gel showing recombinant MutSβ (MSH2-MSH3). We used one MSH2-MSH3 preparation in this study. b, Coomassie-stained polyacrylamide gel showing recombinant MutSα (MSH2-MSH6) used in this study. We used one MSH2-MSH6 preparation in this study. c, Nuclease assays with MLH1-MLH3, MSH4-MSH5, and MSH2-MSH3 or MSH2-MSH6, as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 3 independent experiments; error bars, SEM) at the top.

Extended Data Fig. 6 Stimulation of the nuclease activity of MutSγ-MutLγ by EXO1(D173A).

a, Recombinant EXO1(D173A), used in this study. The 4–15% gradient polyacrylamide gel was stained with Coomassie blue. We used three EXO1(D173A) preparations in this work. b, Nuclease assays with MLH1-MLH3 and MSH4-MSH5, as indicated, without (left) or with EXO1(D173A) (right). The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 3 independent experiments; error bars, SEM) at the top. c, Nuclease assays with MLH1-MLH3 and/or EXO1(D173A), as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 4 independent experiments; error bars, SEM) at the top. EXO1(DA) does not promote the nuclease of MLH1-MLH3 alone. The limited DNA cleavage in lane 3 likely results from residual nuclease activity of EXO1(D173A) that becomes apparent at high protein concentrations (100 nM) in the presence of manganese. d, Quantitation of electrophoretic mobility shift assays with MLH1-MLH3, MSH4-MSH5 and EXO1(D173A), as indicated. The protein-DNA species were resolved in 1% agarose gels. Averages shown; error bars, SEM; n = 5 independent experiments. EXO1(D173A) did not notably affect DNA binding of MLH1-MLH3 and MSH4-MSH5. e, Nuclease assays with MLH1-MLH3, MSH4-MSH5 with either human EXO1(D173A) or yeast Exo1(D173A), as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 5 independent experiments; error bars, SEM) at the top. f, Nuclease assays with MLH1-MLH3, MSH2-MSH3 and EXO1(D173A), as indicated. The assays were carried out at 30 °C in the presence of 5 mM manganese acetate and 0.5 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 3 independent experiments; error bars, SEM) at the top.

Extended Data Fig. 7 RFC-PCNA promote the nuclease activity of the MutSγ-MutLγ-EXO1(DA) ensemble.

a, Recombinant human and yeast RFC and PCNA used in this study. The 4–15% gradient polyacrylamide gel was stained with Coomassie blue. We used two yeast RFC and PCNA preparations, and one human RFC preparation in this work. b, Nuclease assays with scDNA and indicated proteins (all 50 nM, except human PCNA, 100 nM) were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 5 independent experiments; error bars, SEM) at the top. c, Experiments as in panel b, comparing the efficacy of human and yeast RFC as a part of the MLH3 nuclease ensemble. Averages shown; n = 4 independent experiments; error bars, SEM. d, Nuclease reactions containing MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM, respectively) (column 1), without MSH4-MSH5 (column 2) or without EXO1(D173A) (column 3). Reactions were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM; n = 5 independent experiments. e, Kinetic nuclease assays with MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM, respectively), as indicated. Reactions were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM; n = 5 independent experiments. f, Nuclease assays with MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and hRFC-hPCNA (50-100 nM, respectively), as indicated, with supercoiled (left) or relaxed DNA (right). Reactions were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Shown is a representative experiment. RFC-PCNA do not stimulate the cleavage of relaxed DNA. The experiment was performed three times with similar results. g, Nuclease assays with MLH1-MLH3, MSH4-hMSH5, EXO1(D173A) without or with yRFC-hPCNA, as indicated. The assays were carried out at 37 °C in the presence of 5 mM manganese acetate and 2 mM ATP. A representative experiment is shown at the bottom, a quantitation (averages shown; n = 3 independent experiments; error bars, SEM) at the top. Without magnesium, no stimulation of DNA cleavage by RFC-PCNA was observed. h, Nuclease reactions with MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM, respectively), as indicated. Reactions were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM; n = 5 independent experiments. i, Nuclease assays with MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM, respectively) and 5 mM magnesium acetate, either with no nucleotide co-factor (lane 2), with ATP (2 mM, lane 3) or ADP (2 mM, lane 4). ATP is strictly required for DNA cleavage by the nuclease ensemble. The experiment was performed four times with similar results. j, Representative nuclease assays with MLH1-MLH3 (50 nM), MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM, respectively), lane 2. Lanes 3-7 contain instead MLH1-MLH3 or MSH4-MSH5 variants deficient in ATP hydrolysis, as indicated. See Fig. 1d, e for the specific mutations. Reactions were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. The experiment was performed four times with similar results. k, Nuclease assays with indicated oligonucleotide-based substrates carried out at 37 °C in the presence of 5 mM magnesium acetate and 2 mM ATP. All proteins 30 nM, as indicated. Asterisk (*) indicates the position of the 5' radioactive label. The reaction products were analysed on a 15% denaturing polyacrylamide gel. No DNA cleavage was observed. The experiment was performed two times with similar results.

Extended Data Fig. 8 PIP box-like motifs in EXO1, MLH3 and MLH1 facilitate the stimulatory effect of RFC-PCNA on the hMLH3 nuclease ensemble.

a, The MLH1P-MLH3P variant (see Fig. 3b) is not impaired in Holliday junction-binding. Electrophoretic mobility shift assay was carried out with 5 ng/reaction dsDNA competitor and 3 mM EDTA (no magnesium). Asterisk (*) indicates the position of the 5' radioactive label. The experiment was carried out three times with similar results. b, The MLH1P and MLH3P variant combinations are not impaired in nuclease activity without or with MSH4-MSH5 and EXO1(D173A) in the absence of RFC-PCNA. The nuclease assays were performed with 5 mM manganese acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM, n = 3 independent experiments. c, Nuclease assays with MSH4-MSH5 (50 nM), EXO1(D173A) (50 nM) and yRFC-hPCNA (50-100 nM), and a respective MLH1-MLH3 variant, as indicated (see Fig. 3b). Mutations in the PIP-box like motif reduce the stimulation of the nuclease ensemble by RFC-PCNA. The assays were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM, n = 5 independent experiments. d, The EXO1P(D173A) variant with mutated PIP-box motif (see Fig. 3b) is not affected in its ability to promote the nuclease of MLH1-MLH3 and MSH4-MSH5 (without RFC-PCNA). The assays were carried out with 5 mM manganese acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM, n = 4 independent experiments. e, The EXO1P(D173A) variant with mutated PIP-box motif (see Fig. 3b), in complex with MLH1-MLH3 and MSH4-MSH5 impairs the stimulatory function of yRFC-hPCNA (50-100 nM). The assays were carried out with 5 mM magnesium acetate and 2 mM ATP at 37 °C. Averages shown; error bars, SEM, n = 5 independent experiments.

Extended Data Fig. 9 RFC-PCNA promote meiotic recombination in yeast cells.

a, Spore viability upon tetrad microdissection, analysed in the wild type strain, mlh1Δ and mlh3Δ, and in strains complemented with a construct expressing untagged Mlh1P (Q572A-L575A-F578A) or Mlh3P (Q293A-V296A-F300A) at the endogenous chromosomal locus. At least 156 spores from 2 independent experiments were analysed for each genotype. b, western blot analysis of Mlh1P expression in yeast. TCA extracts were prepared from exponentially proliferating SK1 strains expressing MLH1, MLH1-FLAG or MLH1P-FLAG from the endogenous gene locus. The PIP-box-like mutation affects the stability of the FLAG-tagged Mlh1 protein. Blots were probed with anti-FLAG antibody (Sigma, F7425). Crm1 is a protein normalization control. Asterisk denotes a cross-reacting band. The western blot was carried out three times from two different TCA extractions with similar results. c, western blot analysis of Mlh3P expression in yeast. As in b, but with MLH3, MLH3-FLAG or MLH3P-FLAG constructs. Blots were probed with anti-FLAG antibodies: Sigma F7425 (left panel); A8592 (right panel). Crm1 is a protein normalization control. Mlh1-FLAG and Mlh3P-FLAG showed comparable expression levels. Asterisks denote cross-reacting bands. The western blot was carried out three (left panel) or two (right panel) times from two different TCA extractions with similar results. d, A pulldown of TAP-tagged yeast Rfc1-5 and associated proteins from meiotic cell extracts from pCUP1-IME1 cells 5 h 30 min after the induction of meiosis. The presence of Mlh1-HA and Mlh3-Myc in the TEV eluate was analysed by western blotting. This experiment was carried out once. e, Rfc1-TAP levels at the three indicated meiotic DSB hotspots relative to a negative control site (NFT1) were assessed by ChIP and qPCR in ndt80∆-arrested cells after 7 h in meiosis. Mlh3 is not required for the recruitment of RFC to the meiotic DSB hotspots. MLH3: VBD2136; mlh3∆: VBD2137. Averages shown; n = 2 independent experiments.

Extended Data Fig. 10 A possible model for biased resolution of recombination intermediates by the MLH3 nuclease ensemble.

Meiotic dsDNA breaks (a) are resected (1) and the resulting DNA overhang invades matching DNA on a homologous chromosome (2). The unstable D-Loop intermediates (b) are stabilized by MSH4-MSH5 (3), DNA synthesis by RFC-PCNA-Polδ (4) and branch migration (5), leading to more stable structures termed single-end invasions (c). This is followed by a second end capture (6), and more DNA synthesis (7) leading to precursors of double Holliday junctions (d) and later matured double Holliday junctions (e). As a result of the previous steps, MSH4-MSH5 and RFC-PCNA may be present asymmetrically at the (d) or (e) intermediates at the junctions points or their vicinity. The asymmetric presence of the co-factors then directs and stimulates the biased DNA cleavage (9) of (d) or (e) structures by MLH1-MLH3-EXO1. Upon final processing (10) and ligation (11), the ultimate result is a DNA crossover characterized by reciprocal exchange of the DNA arms of the recombining chromosomes.

Supplementary information

Supplementary Table

Supplementary Table 1. Oligonucleotides used in this study.

Reporting Summary

Supplementary Table

Supplementary Table 2. Saccharomyces cerevisiae strains used in this study. All strains are SK1 derivatives.

Supplementary Data

Gel source data (uncropped gel images).

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Cannavo, E., Sanchez, A., Anand, R. et al. Regulation of the MLH1–MLH3 endonuclease in meiosis. Nature 586, 618–622 (2020). https://doi.org/10.1038/s41586-020-2592-2

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