Pre-synaptic events in homologous recombination and maintenance of genomic stability - SUMMARY Lesions, breaks, and errors in DNA are drivers of genomic instability. Double-stranded DNA breaks (DSBs) are a particularly severe form of damage and are repaired by the homologous recombination (HR) pathway. On average, ~10 to 50 DSBs occur per cell per day and thus pose the highest risk to genomic integrity. Unrepaired DSBs lead to gross chromosomal rearrangements and are key drivers of cancers. HR is initiated when the DSB is resected to generate ssDNA. Rad51 is the recombinase that drives HR and forms a helical nucleoprotein filament on the resected ssDNA. This step, termed ‘pre-synaptic nucleoprotein filament formation’ commits to repairing the DSB through the HR pathway. Not surprisingly, this step is tightly regulated by mediator proteins. Pro-HR mediators such as Rad52 (yeast) and BRCA2 (humans) promote HR by aiding in the formation and stabilization of the Rad51 filament. In contrast, anti-HR mediators antagonize this step by displacing Rad51 from ssDNA. In addition, Rad51-paralogs further promote Rad51 filament formation by capping and stabilizing the filaments. The mechanisms of how mediators and Rad51-paralogs work to facilitate Rad51 filaments are poorly understood. Our recent discoveries on Rad52 revealed how two Rad51 binding modes are utilized to facilitate nucleoprotein filament formation. Mode-1 sorts Rad51 oligomers into monomeric units and Mode-2 stacks Rad51 at a defined position within the complex. We also uncovered that the Rad51-Rad52 complex recognizes the ss-dsDNA junction on an RPA-coated ssDNA, thus initiating filament formation from a single defined position on DNA. We propose a Sort, Stack, Extend, and Protect (SSEP) model for mediator and Rad51 paralogs in formation of the pre-synaptic Rad51 nucleoprotein filament. In this proposal, we seek to decipher a) how the junction is recognized through RPA-Rad52-Rad51 interactions; b) the mechanisms of how Rad51 paralogs (Rad55-Rad57 and Csm2-Psy3-Shu1-Shu2) promote Rad51 filament formation; and c) establish how the sorting and stacking properties are enacted in the human BRCA2 protein. Ensemble and single-molecule Förster resonance energy transfer (FRET) fluorescence microscopy, structural mass-spectrometry, and C-trap analysis are utilized to establish how double-strand DNA breaks are repaired through homologous recombination.
