ABSTRACT DNA double-strand breaks (DSB)s occur upon exposure of cells to ionizing radiation and chemicals in the environment, and when DNA replication forks become impeded by lesions and obstacles then subsequently collapse. Two mechanistically distinct DSB repair pathways, namely, homology-directed repair (HDR) and non-homologous DNA end-joining (NHEJ), are responsible for the removal of the majority of DSBs. Whereas HDR is mostly accurate, NHEJ, while efficient, often entails loss of DNA sequence during repair, and can also generate chromosome translocations and replication fork fusions. Failure of NHEJ or HDR leads to heightened engagement of alternate end-joining (AltEJ) and single-strand annealing (SSA), highly mutagenic and otherwise minor pathways, as repair tools. As such, the choice of DSB repair pathway has a major impact on the maintenance of genome stability, preventing neoplastic transformation of cells, and oncogenesis. Our Program Project brings together seven leading NIH-funded laboratories to collaborate synergistically in delineating conserved mechanisms of HDR, replication fork repair, and replication fork maintenance, and how HDR is negatively regulated to favor the use of NHEJ as DSB repair tool. We have assembled three Shared Resource Cores to provide state-of-the-art services in the production of high quality protein preparations for mechanistic experiments, biophysical and structural analyses of protein-ligand interactions and precise measurement of binding constants, as well as cellular interrogations of mechanisms of DSB repair and replication fork maintenance. Altogether, we are exceptionally well poised to leverage our deep knowledge of DSB repair mechanisms and leadership to understand how the tumor suppressors BRCA1-BARD1 and BRCA2 function to promote HDR and to overcome the HDR restrictive action of the epigenetic mark reader 53BP1 and its associated Shieldin and CTC1-STN1-TEN1 complexes. As such, our Program Project will not only exert a major impact in elucidating mechanisms of DSB repair pathway choice and cancer drug resistance, but will also identify novel targets and pathway pivot points to guide the development of new therapeutic strategies to treat incalcitrant breast, ovarian and other cancers.