RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells.

RAD54L is a DNA motor protein with critical roles in homologous recombination DNA repair (HR). In vitro, RAD54L was also shown to catalyze the reversal and restoration of model replication forks. Little, however, is known about the role of RAD54L in regulating the dynamics of DNA replication in cells. Here, we show that RAD54L functions as a fork remodeler and restrains the progression of replication forks in human cells. Analogous to HLTF and FBH1, and consistent with a role in fork reversal, RAD54L catalyzes the slowing of fork progression in response to replication stress. In BRCA1/2-deficient cells, RAD54L activity leads to nascent strand DNA degradation, and loss of RAD54L reduces DNA double-strand break formation. Using a separation-of-function mutation, we show that RAD54L-mediated fork restraint depends on its ability to catalyze branch migration. Our results reveal a new role for RAD54L in regulating the dynamics of replication forks in cells and highlight the impact of RAD54L function on the treatment of patients with BRCA1/2-deficient tumors.

RAD51AP1 and RAD54L Can Underpin Two Distinct RAD51-Dependent Routes of DNA Damage Repair via Homologous Recombination.

Homologous recombination DNA repair (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory HR sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54L, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that concomitant deletion of RAD51AP1 and RAD54L further sensitizes human cancer cell lines to treatment with olaparib, a Poly (adenosine 5'-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which induces DNA replication stress. We also show that the RAD54L paralog RAD54B compensates for RAD54L deficiency, although, surprisingly, less extensively than RAD51AP1. These results, for the first time, delineate RAD51AP1- and RAD54L-dependent sub-pathways and will guide the development of inhibitors that target HR stimulators of strand invasion.