One-ended Break Repair in Mammalian Cells

/ABSTRACT DNA repair initiated at sites of replication fork breakage is critical for the prevention of genomic instability in cycling cells. Defects in the repair of stressed replication forks have been directly implicated in cancer, notably in hereditary breast and ovarian cancer (HBOC). We have developed innovative tools for quantifying error-free homologous recombination (HR) and error-prone HR at broken mammalian replication forks or at double strand breaks (DSBs) where completion of HR occurs in an error-prone fashion. Collectively, the types of HR that this proposal addresses could be characterized as ‘one-ended’. This phrase means either that a DSB arises as a one-ended break (e.g., at a broken replication fork), or that only one end of a 2-ended DSB has homology with the donor. In each case, the mechanisms that enable termination of HR are necessarily error- prone, but are poorly understood. Our previous studies suggest that such breaks may carry a high risk of misrepair with formation of translocations or other chromosome rearrangements, thereby fostering genomic instability and promoting tumorigenesis. The major goals of this proposal are to define whether mechanisms exist to limit genomic instability at sites of replication fork breakage and one-ended HR in mammalian cells. We have developed an array of cutting-edge tools to support this study, including unique, sophisticated HR reporters that can distinguish between error-free ‘short tract’ HR and error-prone ‘long tract’ HR—a replicative response likely analogous to break-induced replication. We have developed a new HR reporter in which HR is triggered by a DNA-protein crosslink (DPC)-induced DNA nick highly similar to the DPC nick induced by the Topoisomerase I inhibitor and cancer therapeutic, Camptothecin. This tool can be used to induce fork breakage in a strand-specific manner, enabling us to target breaks to the leading or lagging strand of the approaching replication fork. We have also developed new tools to analyze the fate of the HR reaction if HR termination occurs without the presence of a homologous second end. In support of these studies, we will use cutting-edge single molecule ‘long read’ nanopore sequencing to identify and analyze gene conversion tracts and complex breakpoints that arise as a result of HR at one-ended breaks. Success in this project will reveal the mechanisms that regulate HR repair of broken forks and the mechanisms governing repair pathway “choice” at sites of aberrant HR termination. This work may also identify new molecular targets for cancer therapy. Project Number: 1R01CA314254-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Ralph Scully | Institution: BETH ISRAEL DEACONESS MEDICAL CENTER, BOSTON, MA | Award Amount: $524,110 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 MGG-T (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11389768