Abstract 1127 FANCD2 promotes HR outcomes during DSB repair by stabilizing template and DSB molecules

Eukaryotic cells repair DNA double strand breaks (DSBs) using two repair strategies: end joining (EJ), which ligates the ends of the lesion back together, and homologous recombination (HR), which uses a homologous template molecule to replace sequence spanning the DSB. Previous work by our lab and others established that the Fanconi Anemia (FA) pathway, and especially FANCD2, binds to DSBs and regulates DSB repair outcomes. However, the precise role played by FANCD2 in DSB repair remained undefined. Here we report that FANCD2 localizes to both the DSB molecule and the template molecule during DSB repair and that this localization depends on FANCD2 ubiquitination by the FA core complex. FANCD2 performs distinct activities on each substrate. The binding of FANCD2 to extrachromosomal template molecules improves template stability and nuclear abundance. These results support a model in which FANCD2 is loaded onto template molecules independently from DSB repair. At the DSB, FANCD2 localization does not contribute to resection activity, nor promote the formation of recombination intermediates, nor influence chromatin openness. Instead, DSB-bound FANCD2 promotes HR in two ways: first, by recruiting HR-permissive chromatin remodeling factors; and second, by delaying mitosis until DSB repair is complete. Altogether, our findings provide mechanistic detail for the role of the FA pathway in DSB repair. This work also suggests new strategies to bias DSB repair outcomes for gene editing applications. This work was supported by NIH R35 GM142975.