RaFiStHol

Homologous recombination (HR) is a universal DNA break repair pathway involved in cell survival, cancer suppression, sexual reproduction, and of biotechnological interest for genome-editing. HR uniquely uses an intact duplex DNA molecule as a repair template, which entails a genome- and nucleus-wide homology search step carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF). Despite recent progress in deciphering the homology sampling process in vitro, how it operates in vivo remains elusive. Moreover, what role does the elongated filament structure and its dynamic formation/disassembly play? This notable gap in our basic understanding of homology search mainly stems from our technical inability to chart this step in cells. Our consortium developed novel assays to track transient HR intermediates (Piazza team) and to visualize functional Rad51-ssDNA filaments (Taddei team) that overcome these limitations in S. cerevisiae. Following formation of a single DSB we can, over time, (i) track NPF filaments’ structure, dynamics, and nuclear localization in individual cells, (ii) determine the genomic regions it contacts and (iii) quantify homology identification at the population level. Under the umbrella of an integrative polymer physics model (Jost team), these quantitative data of different nature put us in a unique position to undertake the structure-function analysis of Rad51-ssDNA filament in homology search in cells. We will specifically address the role of the catalytic activities of Rad51 and its conserved partners, of the NPF’s sequence composition, and of factors involved in the spatial organization of chromatin. This approach should allow to propose generic mechanisms and crack the central conundrum of the HR field: the mechanism of homology search and the contributions of conserved proteins in this process.