Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP.

In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

Absence of chromosome axis protein recruitment prevents meiotic recombination chromosome-wide in the budding yeast Lachancea kluyveri.

Meiotic recombination shows broad variations across species and along chromosomes and is often suppressed at and around genomic regions determining sexual compatibility such as mating type loci in fungi. Here, we show that the absence of Spo11-DSBs and meiotic recombination on Lakl0C-left, the chromosome arm containing the sex locus of the Lachancea kluyveri budding yeast, results from the absence of recruitment of the two chromosome axis proteins Red1 and Hop1, essential for proper Spo11-DSBs formation. Furthermore, cytological observation of spread pachytene meiotic chromosomes reveals that Lakl0C-left does not undergo synapsis. However, we show that the behavior of Lakl0C-left is independent of its particularly early replication timing and is not accompanied by any peculiar chromosome structure as detectable by Hi-C in this yet poorly studied yeast. Finally, we observed an accumulation of heterozygous mutations on Lakl0C-left and a sexual dimorphism of the haploid meiotic offspring, supporting a direct effect of this absence of meiotic recombination on L. kluyveri genome evolution and fitness. Because suppression of meiotic recombination on sex chromosomes is widely observed across eukaryotes, the mechanism for recombination suppression described here may apply to other species, with the potential to impact sex chromosome evolution.

Scaling Up Citizen Workshops in Public Libraries to Disseminate and Discuss Primary Care Research Results: Quasi-Experimental Study.

BACKGROUND: Little is known about engaging patients and stakeholders in the process of scaling up effective knowledge translation interventions targeting the public. OBJECTIVE: Using an integrated knowledge translation approach, we aimed to scale up and evaluate an effective pilot program to disseminate research results in public libraries. METHODS: We conducted a scaling-up study targeting the public. On the basis of our successful pilot project, we codeveloped and implemented a large-scale program of free citizen workshops in public libraries, in a close research partnership with stakeholders and patient representatives. Citizen workshops, each facilitated by 1 participating physician and 1 science communicator, consisted of a 45-minute computer-assisted presentation and a 45-minute open exchange. The intervention outcome was knowledge gained. The scale-up outcomes were satisfaction, appropriateness, coverage, and costs. An evaluation questionnaire was used to collect data of interest. Both quantitative and qualitative analyses were performed. RESULTS: The workshop theme chosen by the patient and stakeholder representatives was the high prevalence of medication overuse among people aged ≥65 years. From April to May 2019, 26 workshops were conducted in 25 public libraries reaching 362 people. The mean age of participants was 64.8 (SD 12.5) years. In total, 18 participating physicians and 6 science communicators facilitated the workshops. Participants reported significant knowledge gain (mean difference 2.1, 95% CI 2.0-2.2; P<.001). The median score for overall public satisfaction was 9 out of 10 (IQR 8-10). The public participants globally rated the workshops as having a high level of appropriateness. Coverage was 92% (25/27) of the total number of public libraries targeted. Costs were CAD $6051.84 (US $4519.69) for workshop design and CAD $22,935.41 (US $17,128.85) for scaling them up. CONCLUSIONS: This project successfully established a large-scale and successful implementation science or knowledge translation bridge among researchers, clinicians, and citizens via public libraries. This study provides a model for a dissemination practice that benefits the public by both engaging them in the dissemination process and targeting them directly.

The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly.

Meiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11, and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism.

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.

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLß heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLß recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLß ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLß may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLß ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

Methods to Map Meiotic Recombination Proteins in Saccharomyces cerevisiae.

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), catalyzed by the type II topoisomerase-like Spo11 protein. Meiotic DSBs are repaired by homologous recombination, which produces either crossovers or noncrossovers, this decision being linked to the binding of proteins specific of each pathway. Mapping the binding of these proteins along chromosomes in wild type or mutant yeast background is extremely useful to understand how and at which step the decision to repair a DSB with a crossover is taken. It is now possible to obtain highly synchronous yeast meiotic populations, which, combined with appropriate negative controls, enable to detect by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) the transient binding of diverse recombination proteins with high sensitivity and resolution.

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

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.

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination.

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

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.

Crossing and zipping: molecular duties of the ZMM proteins in meiosis.

Accurate segregation of homologous chromosomes during meiosis depends on the ability of meiotic cells to promote reciprocal exchanges between parental DNA strands, known as crossovers (COs). For most organisms, including budding yeast and other fungi, mammals, nematodes, and plants, the major CO pathway depends on ZMM proteins, a set of molecular actors specifically devoted to recognize and stabilize CO-specific DNA intermediates that are formed during homologous recombination. The progressive implementation of ZMM-dependent COs takes place within the context of the synaptonemal complex (SC), a proteinaceous structure that polymerizes between homologs and participates in close homolog juxtaposition during prophase I of meiosis. While SC polymerization starts from ZMM-bound sites and ZMM proteins are required for SC polymerization in budding yeast and the fungus Sordaria, other organisms differ in their requirement for ZMM in SC elongation. This review provides an overview of ZMM functions and discusses their collaborative tasks for CO formation and SC assembly, based on recent findings and on a comparison of different model organisms.

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1's N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3's localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1's roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.

Can patients be trained to expect shared decision making in clinical consultations? Feasibility study of a public library program to raise patient awareness.

INTRODUCTION: Shared decision making (SDM) is a process whereby decisions are made together by patients and/or families and clinicians. Nevertheless, few patients are aware of its proven benefits. This study investigated the feasibility, acceptability and impact of an intervention to raise public awareness of SDM in public libraries. MATERIALS AND METHODS: A 1.5 hour interactive workshop to be presented in public libraries was co-designed with Quebec City public library network officials, a science communication specialist and physicians. A clinical topic of maximum reach was chosen: antibiotic overuse in treatment of acute respiratory tract infections. The workshop content was designed and a format, whereby a physician presents the information and the science communication specialist invites questions and participation, was devised. The event was advertised to the general public. An evaluation form was used to collect data on participants' sociodemographics, feasibility and acceptability components and assess a potential impact of the intervention. Facilitators held a post-workshop focus group to qualitatively assess feasibility, acceptability and impact. RESULTS: All 10 planned workshops were held. Out of 106 eligible public participants, 89 were included in the analysis. Most participants were women (77.6%), retired (46.1%) and over 45 (59.5%). Over 90% of participants considered the workshop content to be relevant, accessible, and clear. They reported substantial average knowledge gain about antibiotics (2.4, 95% Confidence Interval (CI): 2.0-2.8; P < .001) and about SDM (4.0, 95% CI: 3.4-4.5; P < .001). Self-reported knowledge gain about SDM was significantly higher than about antibiotics (4.0 versus 2.4; P < .001). Knowledge gain did not vary by sociodemographic characteristics. The focus group confirmed feasibility and suggested improvements. CONCLUSIONS: A public library intervention is feasible and effective way to increase public awareness of SDM and could be a new approach to implementing SDM by preparing potential patients to ask for it in the consulting room.

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation.

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2-Zip4-Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF-ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals.

The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes.

Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we provide evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where Spp1 works in three ways to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by "reading" and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.

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.