Rad52-dependent recombination in response to replication stress

SUMMARY Genomic instability, one of the hallmarks of cancer, is driven by replication stress. Replication stress can result from oncogene activation, damage to the template strands, depletion of nucleotides, or from physical impediments to progression of replication forks, such as non-canonical DNA structures or tightly bound proteins. Homologous recombination (HR) at stalled or collapsed replication forks is important to restart replication, but at the same time can be an additional source of genomic instability by promoting chromosome rearrangements. Recent studies identified an unexpected role for Rad52 in facilitating DNA synthesis in response to replication stress in human cells. Although Rad52 is essential for all HR in budding yeast, its role in mammalian cells had seemed minor because mice lacking it are viable and show only mild defects in HR. The finding that Rad52 is required for viability of BRCA2-deficient cells has been interpreted as redundancy for mediating Rad51 filament assembly. However, the new studies showing that Rad52-promoted DNA synthesis in response to replication stress is independent of Rad51 suggest a novel function for Rad52. Because Rad52 has emerged as potential therapeutic target for BRCA-deficient tumors, it is important to understand its cellular functions. Here we apply the facile genetics of budding yeast and new technologies to create a site-specific replication fork stall or collapse to identify the mechanism for Rad52 dependent recombination in the context of replication stress. In the first aim of the proposal, we will use Tus/Ter or Flp/FRT systems to induce a replication fork stall or collapse, respectively, adjacent to a sensitive reporter to detect HR in dividing cells. We will determine the requirements for Rad51 and Rad52 for Tus/Ter and Flp/FRT-stimulated recombination, and identify recombination intermediates by two-dimensional gel electrophoresis. The role of converging forks in suppressing HR at the Tus/Ter block will also be tested. The second aim addresses the role of Rad51 stabilization of stalled forks, nascent strand degradation and Pol32-dependent DNA synthesis for Tus/Ter-induced recombination. !