Regulation of the MLH1-MLH3 endonuclease in meiosis

Abstract

32During prophase of the first meiotic division, cells deliberately break their DNA. 33These DNA breaks are repaired by homologous recombination, which facilitates 34proper chromosome segregation and enables reciprocal exchange of DNA seg-35 ments between homologous chromosomes, thus promoting genetic diversity in 36 the progeny 1 . A successful completion of meiotic recombination requires nucleo-37 lytic processing of recombination intermediates. Genetic and cellular data impli-38 cated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in gen-39 erating crossovers, but mechanisms that lead to its activation were unclear 2-4 . 40Here, we have biochemically reconstituted key elements of this pro-crossover 41 pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to 42 support crossing over [5][6][7] , binds branched recombination intermediates and phys-43 ically associates with MutLγ. This helps stabilize the ensemble at joint molecule 44 structures and adjacent dsDNA. Second, we show that MutSγ directly stimulates 45 DNA cleavage by the MutLγ endonuclease, which demonstrates a novel and unex-46 pected function for MutSγ in triggering crossing-over. Third, we find that MutLγ 47 tion of yeast MutLγ is dependent on the integrity of the metal binding 75 DQHA(X)2E(X)4E motif within Mlh3, implicating the nuclease of Mlh3 in resolving 76 recombination intermediates 2,3,18,19,56 . Despite wealth of genetic and cellular data, 77 the mechanisms that control the MutLγ nuclease and lead to biased joint molecule 78 processing remained undefined. 79 80 81 82 83 84 4 Results 85 86 Human MutLγ is an ATP-stimulated endonuclease 87To study human MutLγ (hMLH1-hMLH3), we expressed and purified the hetero-88 dimer from insect cells ( Fig. 1a and Extended Data Fig. 1a,b). Similarly to the mis-89 match repair (MMR)-specific hMutLα (hMLH1-hPMS2) 20 , the hMLH1-hMLH3 90 complex non-specifically nicked double-stranded supercoiled DNA (scDNA) in the 91 presence of manganese without any other protein co-factor ( Fig. 1b,c, Extended 92 Data Fig. 1c), while almost no activity was observed with magnesium (Extended 93 Data Fig. 1d), which is believed to be the specific metal co-factor 20 . Mutations in 94 the conserved metal binding motif of hMLH3 abolished the endonuclease, indicat-95 ing that the DNA cleavage activity was intrinsic to the hMutLγ heterodimer ( Fig. 96 1d, see also Extended Data Fig. 1e). ATP promoted the nuclease activity >2-fold 97 ( Fig. 1d,e, Extended Data Fig. 1f-h). Experiments with various ATP analogs re-98 vealed that ATP hydrolysis by hMLH1-hMLH3 was required for the maximal stim-99 ulation of DNA cleavage (Fig. 1f, Extended Data Fig. 1h). The N-termini of both 100 hMLH1 and hMLH3 proteins contain conserved Walker motifs implicated in ATP 101 binding and hydrolysis 21 . To define whether the ATPase of hMLH1, hMLH3 or 102 both subunits of the heterodimer promotes its nucleolytic activity, we prepared 103 the respective hMutLγ variants with mutations in the conserved motifs of either 104 subu...

All three mammalian MutL complexes are required for repeat expansion in a mouse cell model of the Fragile X-related disorders

“…For example, we know that in the case of yeast MutLγ, polymer formation is required for MutLγ cleavage [ 50 ]. Polymer formation is a conserved property of MutL complexes in organisms ranging from bacteria to mammals, including human MutLα and yeast and human MutLγ [ 50 , 51 ]. Given the sequence similarities and many common properties of the MutL proteins, we speculate that MutLα and MutLβ have some ability to contribute to the polymers formed on a MutLγ-bound substrate.…”

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse

“…Two classes of mechanisms transform DNA-strand-invasion intermediates into crossovers. Most crossovers (90-95%) are generated by the class I pathway, which relies on the putative crossover-resolvase MutLγ complex (MLH1/MLH3) [3][4][5][6][7][8][9] . This pathway is subject to poorly understood regulatory mechanisms that differentiate at least one recombination intermediate into crossover on each chromosome (crossover assurance), and prevent the formation of crossovers in close proximity to one another (crossover interference; reviewed in ref.…”

“…Following their differentiation in early-mid pachytene, crossovercommitted recombination complexes mature by losing RNF212/ MutSγ and accumulating HEI10 15,18 , the cell-cycle kinase CDK2 20 and MutLγ 21 in mid/late pachytene. MutLγ is thought to resolve the recombination complex-associated strand-invasion intermediates as crossovers 3,4,[6][7][8][9]22 . In contrast, it is unclear how CDK2, HEI10 and CNTD1 contribute to crossover maturation beyond their upstream roles in homologous synapsis (CDK2) 23,24 and crossover differentiation (HEI10 and CNTD1) 15,16 .…”

Implications of Metastable Nicks and Nicked Holliday Junctions in Processing Joint Molecules in Mitosis and Meiosis

“…After the first draft of this manuscript was finished, two back-to-back papers in Nature led by Hunter and Cejka groups provided additional insights into the Mlh1-Mlh3 mechanism, as well as how the nuclease could pick the correct strands for CO-biased resolution [ 23 , 24 ]. Mlh1-Mlh3 does not work alone but as part of a multimeric complex that resembles the canonical MutLα MMR complex, and includes Msh4-Msh5 (MutSγ), Exo1, the proliferating cell nuclear antigen (PCNA), and the replication factor C (RFC).…”

“…Moreover, I also explore the effects of nicks in the vicinity of HJs. Recent literature underlines the importance of such nicks during the resolution of HJs in meiosis by the endonuclease MutLγ (MLH1-MLH3; Mlh1-Mlh3 in yeast) [ 23 , 24 , 25 ].…”