BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks

Abstract

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases1–3. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes4–8. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks9. Although BRCA1 and BRCA2 affect replication fork processing10–12, direct evidence that BRCA genes regulate HR at stalled chromosomal replication forks is lacking due to a dearth of tools for studying this process. We report that the Escherichia coli Tus/Ter complex13–16 can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mammalian cells. Tus/Ter-induced HR entails processing of bidirectionally arrested forks. We find that the BRCA1 C-terminal tandem BRCT repeat and regions of BRCA1 encoded by exon 11—two BRCA1 elements implicated in tumor suppression—control Tus/Ter-induced HR. Inactivation of either BRCA1 or BRCA2 increases the absolute frequency of “long-tract” gene conversions at Tus/Ter-stalled forks—an outcome not observed in response to a restriction endonuclease-mediated chromosomal double strand break (DSB). Therefore, HR at stalled forks is regulated differently from HR at DSBs arising independently of a fork. We propose that aberrant long-tract HR at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.