Mechanisms of Recombinational DNA Repair

Project Summary/Abstract Homologous recombination maintains genomic stability through high-fidelity repair of double-strand DNA breaks and other complex DNA damage that is induced directly or indirectly by common anti-tumor agents including ionizing radiation, topoisomerase-targeted drugs, interstrand crosslinking agents, and those causing stalling of replication forks. Homologous recombination defects bear dual significance for cancer by first leading to genomic instability and increased cancer predisposition. Moreover, recombination defects cause specific cellular vulnerabilities that can be exploited therapeutically either by traditional DNA damage-based treatment or by targeted treatment (e.g., poly(ADP-ribose) polymerase inhibition). The same genome-preserving mechanism contributes to genomic instability and genome evolution through non-allelic homologous recombination between repeated DNA elements. The objectives of this R35 MIRA are to study the mechanisms of how homologous recombination maintains genomic stability, and how it induces genome rearrangements between repeated DNA sequences using biochemical, structural biology, genetic, cell biological, and genomic approaches. Our research uses the budding yeast Saccharomyces cerevisiae as the lead model to establish fundamental paradigms that we are transferring to human proteins, cells, and tumor genomics. We have developed new experimental tools to detect recombination intermediates and products that close important gaps in the toolbox to study recombination. We propose to expand these tools and adapt them to human cells. Building on substantial preliminary data, we will continue to address unresolved questions regarding the dynamic processing of displacement loops, a central recombination intermediate; and the roles of the double-stranded DNA motor proteins RAD54 and RAD54B in homologous recombination. We have discovered multi-invasion intermediates, where a single Rad51-single-strand DNA filament pairs with multiple donor loci, leading to a cascade of genomic rearrangements. We propose to define the mechanisms resolving such multi-invasion intermediates and define their mutagenic impact. More broadly, we will define the mechanisms and regulation of non-allelic homologous recombination. We will establish how mismatch repair functions in quality control to avoid recombination between near-identical repeats and establish the significance of this process for human tumor biology by analyzing genomes of mismatch repair-deficient tumors (Lynch Syndrome) for signatures of hyper-recombination between repeated DNA elements and multi-invasion-mediated rearrangements.