ABSTRACT Homology-directed DNA repair (HDR) is a universal, conserved mechanism for the repair of DNA double- strand breaks and the maintenance of DNA replication forks. While HDR is relatively well understood in bacteria and lower eukaryotes, glaring knowledge gaps remain in the appreciation of mechanisms in mammals, because of greatly added complexity including myriad factors that are unique. Notably, several breast and ovarian tumor suppressors, including BRCA1-BARD1, BRCA2, and PALB2, have been implicated in HDR in mammals, and emerging evidence provides glimpses of the roles that they fulfill. However, a holistic understanding the molecular mechanisms of human HDR has been hampered by the immense challenge of expressing and purifying these very large proteins and protein complexes for mechanistic workup. My laboratory has overcome this challenge, allowing us to demonstrate the pivotal role of BRCA2 in complex with its obligatory partner DSS1 in facilitating the assembly of catalytically active filaments of the RAD51 recombinase on single-stranded DNA and to reveal a late role of the BRCA1-BARD1 complex in HDR via the promotion of RAD51-mediated DNA strand invasion. In this proposal, we aim to fill several knowledge gaps regarding how BRCA1-BARD1, BRCA2-DSS1, and PALB2 synergize in two distinct stages of the HDR process. Specifically, we will apply an integrated approach that encompasses reconstitution biochemistry, single-molecule biophysics, structural biology, DNA fiber analysis, and advanced cell biology to interrogate how the aforementioned tumor suppressors and their partner HDR factors, such as RAD54, help promote the assembly of the RAD51-ssDNA nucleoprotein filament and the catalysis of DNA strand invasion within the context of chromatin. The results from our work will bring molecular and mechanistic clarity to RAD51- dependent HDR and the preservation of stressed DNA replication forks in human cells.