Alexander V. Mazin

The breast and ovarian tumor suppressor BRCA1 heterodimerizes with BARD1 to promote DNA double-strand break repair by homologous recombination (HR) and to protect stressed DNA replication forks against nuclease attack. The large, intrinsically disordered central region of BRCA1 harbors binding sites for DNA and multiple repair factors, but its lack of stable structure has hindered mechanistic dissection of these activities. Using biochemical mapping and NMR spectroscopy, we delineate the DNA binding and RAD51 interaction interfaces within this region and construct separation-of-function mutants that selectively ablate each activity. Both DNA binding and RAD51 interaction are required for BRCA1-BARD1 to promote RAD51-mediated DNA strand invasion, and DNA binding also contributes to BLM-DNA2 end resection. These findings provide mechanistic insights into how individual ligand binding activities within BRCA1 contribute to genome maintenance.

Replication fork reversal alleviates DNA replication stress and maintains genome stability. We previously showed that FBH1 and RAD54L cooperate to promote fork reversal in human cells, and that FBH1-dependent fork reversal requires the branch migration activity of RAD54L. However, the molecular basis of this cooperation remained unclear. Here, we identify both a physical and functional interaction between FBH1 and RAD54L. We demonstrate that purified FBH1 and RAD54L interact directly and form a complex at stalled replication forks in cells. Mapping studies revealed that RAD54L Lobe 1 is critical for interaction with the FBH1 2B subdomain. In cells, FBH1-RAD54L complex formation is enhanced in the absence of RAD51. Consistently, purified RAD54L displays a stronger affinity for RAD51 than for FBH1. Using biochemical reconstitution assays, we further show that FBH1 and RAD54L promote fork reversal more efficiently together than either protein alone, with maximal reversal observed when FBH1 acts before RAD54L. Collectively, our findings establish RAD54L as an essential functional partner of FBH1 in replication fork reversal and provide mechanistic insight into the sequential coordination of their activities.

Recent studies in eukaryotes have revealed an important role of RNA in DNA repair and identified the RAD52 protein as a central player in RNA-dependent repair of DNA. In vitro, RAD52 promotes inverse RNA strand exchange between dsDNA and homologous RNA. This reaction is strongly stimulated by the RAD52 partner, replication protein A (RPA). Here, using NMR and biochemical methods we investigated the mechanism of this stimulation. We identified two RPA-binding sites in the unstructured RAD52 C-terminal domain (CTD), which mediate interaction with RPA70 and RPA32 subunits. These interactions are critical for stimulation of inverse RNA strand exchange. Furthermore, we showed that stimulation of inverse RNA strand exchange requires formation of an RPA-RNA complex that strengthens the RPA-RAD52 interaction and serves to deliver RNA to the RAD52-dsDNA complex for strand exchange. These results elucidate the mechanism of novel inverse RNA strand exchange activity of RAD52 and the role of RAD52-RPA interaction in RNA-dependent DNA repair.

Antagonistic activities of the 53BP1 axis and the tumor suppressor BRCA1-BARD1 determine whether DNA double-strand breaks (DSBs) are repaired by end joining or homologous recombination. We show that the CTC1-STN1-TEN1 (CST) complex, a central 53BP1 axis component, suppresses DNA end resection by EXO1 and the BLM-DNA2 helicase-nuclease complex but acts by distinct mechanisms in restricting these entities. Whereas BRCA1-BARD1 alleviates the CST-imposed EXO1 blockade, it has little effect on BLM-DNA2 restriction. CST mutants impaired for DNA binding or BLM–EXO1 interaction exhibit a hyper-resection phenotype and render BRCA1-deficient cells resistant to poly(ADP–ribose) polymerase (PARP) inhibitors. Our findings mechanistically define the crucial role of CST in DNA DSB repair pathway choice and have implications for understanding cancer therapy resistance stemming from dysfunction of the 53BP1 axis.

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DNA binding domains in genome maintenance.

RAD54L is a DNA motor protein with multiple roles in homologous recombination DNA repair. In vitro, RAD54L was shown to also catalyze the reversal and restoration of model replication forks. In cells, however, little is known about how RAD54L may regulate the dynamics of DNA replication. Here, we show that RAD54L restrains the progression of replication forks and functions as a fork remodeler in human cancer cell lines and non-transformed cells. Analogous to HLTF, SMARCAL1 and FBH1, and consistent with a role in fork reversal, RAD54L decelerates fork progression in response to replication stress and suppresses the formation of replication-associated ssDNA gaps. Interestingly, loss of RAD54L prevents nascent strand DNA degradation in both BRCA1/2- and 53BP1-deficient cells, suggesting that RAD54L functions in both pathways of RAD51-mediated replication fork reversal. In the HLTF/SMARCAL1 pathway, RAD54L is critical, but its ability to catalyze branch migration is dispensable, indicative of its function downstream of HLTF/SMARCAL1. Conversely, in the FBH1 pathway, branch migration activity of RAD54L is essential, and FBH1 engagement is dependent on its concerted action with RAD54L. Collectively, our results reveal disparate requirements for RAD54L in two distinct RAD51-mediated fork reversal pathways, positing its potential as a future therapeutic target.

RAD54L is a DNA motor protein with multiple roles in homologous recombination DNA repair (HR). In vitro , RAD54L was shown to also catalyze the reversal and restoration of model replication forks. In cells, however, little is known about how RAD54L may regulate the dynamics of DNA replication. Here, we show that RAD54L restrains the progression of replication forks and functions as a fork remodeler in human cells. Analogous to HLTF, SMARCAL1, and FBH1, and consistent with a role in fork reversal, RAD54L decelerates fork progression in response to replication stress and suppresses the formation of replication-associated ssDNA gaps. Interestingly, loss of RAD54L prevents nascent strand DNA degradation in both BRCA1/2- and 53BP1-deficient cells, suggesting that RAD54L functions in both pathways of RAD51-mediated replication fork reversal. In the HLTF/SMARCAL1 pathway, RAD54L is critical, but its ability to catalyze branch migration is dispensable, indicative of its function downstream of HLTF/SMARCAL1. Conversely, in the FBH1 pathway, branch migration activity of RAD54L is essential, and FBH1 engagement is dependent on its concerted action with RAD54L. Collectively, our results reveal disparate requirements for RAD54L in two distinct RAD51-mediated fork reversal pathways, positing its potential as a future therapeutic target.

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.