Evaluation de l'évitabilité des infections en établissements d'hébergement pour personnes âgées dépendantes (comparaison de deux méthodes d'expertise de cas)

Les infections représentent un problème majeur dans les institutions gériatriques. L évaluation de l évitabilité de l événement infectieux est une étape clé pour orienter les efforts de prévention nécessaires à la maîtrise de ce problème. L objectif de cette étude a été de comparer à une méthode classique, une méthode originale d évaluation de l évitabilité des infections en Établissements d Hébergement pour Personnes Agées Dépendantes (EHPAD) basée sur la méthode Delphi, et de déterminer laquelle de ces deux méthodes utiliser dans le cadre d un programme de recherche sur l évitabilité du risque infectieux en EHPAD. L étude a consisté à recueillir, de façon prospective, 10 cas cliniques d infections en EHPAD, puis à en faire évaluer l évitabilité simultanément par deux groupes d experts. Un groupe a utilisé une méthode classique d expertise sur dossiers, l autre une méthode basée sur la méthode Delphi. Les résultats obtenus par la méthode Delphi ont été comparés qualitativement à ceux fournis par la méthode classique. Le taux de concordance entre les deux méthodes a été de 70%. Les deux méthodes ont retrouvé un taux d évitabilité de 20%. Cette étude nous montre que la méthode Delphi présente une concordance satisfaisante avec la méthode classique pour évaluer l évitabilité des infections en EHPAD par expertise de cas. Toutefois, en l absence de méthode de référence établie dans la littérature, nous ne pouvons pas conclure à sa validité, les deux méthodes présentant des limites. Nous proposons donc d utiliser une association des deux méthodes dans la poursuite du programme d étude de l évitabilité des infections en EHPAD.PARIS6-Bibl. St Antoine CHU (751122104) / SudocSudocFranceF

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 mechanism3,4,5,6,7. 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

Des mécanismes moléculaires aux applications cliniques. L’essentiel du Colloque Réplication-Réparation-Recombinaison 2019

International audienc

Genetic Evidence for the Involvement of Mismatch Repair Proteins, PMS2 and MLH3, in a Late Step of Homologous Recombination.

International audienceHomologous 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 non-meiotic 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 times decrease in the frequency of heteroallelic HR-dependent repair of a 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 g-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and three times decrease in the number of cisplatin-induced sister chromatid exchange (SCE). The ectopic expression of the Gen1 HJ resolvase 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

Concerted action of the MutL beta 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 MutL beta complex, Mlh1-Mlh2, specifically inter acts 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 cross over 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 MutL beta preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutL beta 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

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

International audienceIn 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