Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

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 mechanism3-7. 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.

Coupling DNA Damage and Repair: an Essential Safeguard during Programmed DNA Double-Strand Breaks?

DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the 'price to pay' to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.

Special issue on "recent advances in meiosis from DNA replication to chromosome segregation".

Meiosis is the special division that produces haploid gametes, such as sperm and eggs. It involves a complex series of events that integrate large structural changes at the chromosome scale with fine regulation of recombination events in localized regions. To evaluate the complexity of these processes, the meiosis field covers a variety of disciplines and model organisms, making it an exciting and rapidly changing area of research. The field as a whole highlights both the conserved aspects of meiosis, as well as the marked diversity of the means taken to ensure that, ultimately, gametes will contain a balanced number of chromosomes and genetic diversity will have been produced. Studying meiosis is also critically important for the improvement of our human condition as errors of meiosis are a leading cause of infertility, miscarriage, and developmental disabilities. Finally, the complex chromosome behavior of meiosis is a genetically tractable paradigm, the study of which improves our understanding of many fundamental cellular processes including DNA repair, genome stability, cancer etiology, chromatin structure, and chromosome dynamics.This special issue on meiosis contains twenty-two papers, of which five are in-depth reviews that complement and put in context the experimental data presented in the seventeen original research articles. The content of this issue illustrates the diversity of topics covered by researchers in the field, ranging from the effects of environment and external factors on the success of meiosis, the cell cycle actors that control the meiotic divisions, the mechanism of chromosome segregation, and the mechanisms that ensure proper homologous chromosome pairing, recombination, and synapsis. Multiple organisms are covered. Also evident is the fact that more and more studies use multicellular organisms as a model system, in large part due to the increased availability of tools that were previously restricted to studies in budding and fission yeasts.

Concerted action of the MutLß heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion.

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLß complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLß preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLß is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Interplay between modifications of chromatin and meiotic recombination hotspots.

Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called 'hotspots'. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Double functions for the Mre11 complex during DNA double-strand break repair and replication.

Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.