SafeHR

Homologous recombination (RH) is a major DNA double-strand break (DSB) repair mechanism conserved in all organisms. In eukaryotes, HR depends on the formation of nucleoprotein filaments of the Rad51 recombinase on single-stranded DNA. These filaments allow the genome to be scanned for homologous DNA which is used as a template for restorative synthesis. The goal of the SafeHR project is to define the mechanisms that regulate the formation, stability and dissociation of Rad51 filaments that prevent unscheduled toxic recombination events from occurring. These mechanisms are related to the action of negative and positive regulators that influence the dynamics of Rad51 filaments. Recently, Partner 1 showed that the major filament-forming factor, Rad52, remains associated with filaments and inhibits their dismantling by DNA helicase Srs2. The Rad51 paralog protein complexes, Rad55-Rad57 and SHU, are also involved in this inhibition. Through a multidisciplinary approach of genetics, molecular biology, biochemistry, protein structure and microscopy and using yeast as a model organism, our consortium plans to define the structure and dynamics of the association of these proteins with Rad51 filaments, to determine the regulatory mechanisms and the cause of their potential toxicity. Our consortium includes four teams working in the fields of DNA repair and HR using complementary approaches. Thanks to structural approaches (modelling, NMR, crystallography and cryo-EM) and genetics, partners 1 and 2 have started to characterize the assembly of Rad52, Rad55-Rad57 and SHU within the Rad51 filaments. These studies will be pursued in collaboration with partner 4 with highly integrative structural biology approaches. This strategy allows us to design mutations that affect specific interactions and to precisely assess the role of each of these interactions. This will be a significant advance over previous studies that only used whole gene deletion approaches. Associated with the construction of a Rad51-GFP fusion protein by Partners 2 and 3, which for the first time allows the study by microscopy of functional Rad51 filaments in vivo, these mutations will allow us to determine the role of Rad52 and different Rad51 paralogs in HR induced by DSBs or by DNA single-strand breaks (SSB) formed behind DNA replication forks blocked by DNA damage. This strategy will also make it possible to understand the mechanisms of inhibition of the Srs2 helicase by the positive effectors of the Rad51 filaments. Finally, Partner 1 has developed a cellular system to synchronously induce the formation of toxic Rad51 filaments, which will be used to study in depth the causes and consequences of their formation. The project is organized into four work packages (WP): WP1 aims to determine the structure of the Rad52-Rad51 interaction and its role in the formation and stabilization of the Rad51 filament. The newly developed microscopy tools by Partner 3 and the separation of function mutations identified by Partners 1 and 2 will be used to quantitatively assess the impact of Rad52 on Rad51 filament nucleation and stability. WP2 consists in pursuing the determination of the structure of the Rad55-Rad57 and SHU complexes in association with the Rad51 filament and in understanding the involvement of these complexes in the repair of DSBs or CSBs. WP3 consists of the determination of the mechanisms of inhibition of Srs2 by Rad52 and paralogs of Rad51. WP4 will establish the molecular basis of the Rad51 toxic filaments using the genetic system built by Partner 1.