Reframing BRCA1 Function: A Coordinator of Lagging Strand Synthesis

BRCA1-deficient cancers are acutely sensitive to a broad range of anti-cancer therapies, yet the underlying mechanisms driving this vulnerability remain incompletely defined. While 53BP1 has been traditionally implicated in drug sensitivity through its suppression of homologous recombination (HR), emerging evidence suggests an alternative pathway. Our preliminary findings reveal that 53BP1 disrupts lagging strand processing in BRCA1-deficient cells, leading to defective Okazaki fragment maturation and accumulation of post-replication single-stranded DNA (ssDNA) gaps—even in the absence of exogenous DNA damage. These gaps coincide with mislocalized FEN1, implicating a defect in flap processing, and are marked by aberrant enrichment of 53BP1 at replication forks. We further demonstrate that these gaps create a dependency on RAD51 for protection and repair, revealing a previously unrecognized therapeutic vulnerability. The central goal of this proposal is to define how BRCA1 regulates lagging strand DNA synthesis to suppress replication-associated gaps—and how this process is antagonized by 53BP1 and related factors to drive treatment sensitivity. We hypothesize that BRCA1 promotes lagging strand flap engagement by restricting 53BP1, thereby enabling proper Okazaki fragment processing and limiting gap formation that would otherwise require RAD51-mediated protection. To test this model, we propose three specific aims: (1) define the role of BRCA1 in lagging strand synthesis; (2) determine how lagging strand processing defects are bypassed in BRCA1-deficient cells; and (3) elucidate how RAD51 protects and resolves replication-associated gaps. These aims will be addressed using proximity ligation assays (PLA), iPOND, chromatin proteomics, and functional genetic models. Collectively, these studies will challenge prevailing models of BRCA1 and 53BP1 function and reframe the origins of therapeutic sensitivity in BRCA1-deficient tumors. By defining how lagging strand defects and RAD51 dependencies arise, this work will uncover new avenues for targeting BRCA1-deficient and PARP inhibitor–resistant cancers. Project Number: 1R01CA311692-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Sharon Cantor | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $469,628 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 BTC-V (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11343098