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.

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…We found previously that Tus F140A induces higher levels of HR than wtTus in mammalian cells carrying a 6xTer array HR reporter. 15 This suggests that the C6 base-flipping lock mechanism is not required for Tus/Ter-induced HR in mammalian cells and that the affinity of Tus for duplex Ter is the critical determinant of HR induction in mammalian cells. In contrast, Larsen et al found that Tus/Ter mediates polar replication fork arrest in Saccharomyces cerevisiae.…”

“…16 The Tus/6xTer replication fork barrier was »70% effective in blocking the mammalian replisome in this setting. 15 Unexpectedly, encounter of the mammalian replisome at the permissive end of the Ter array also causes replisome arrest, indicating that fork arrest at a Tus/6xTer replication fork barrier in mammalian cells is at least in part non-polar. By placing the 6xTer array within a reporter of mammalian HR, targeted to a specific locus of mouse embryonic stem (ES) cells, we found that transient expression of wild type Tus triggers HR at the 6xTer array, while expression of a mutant form of Tus (H144A) that has a low affinity for Ter failed to induce HR in the same cells.…”

“…By placing the 6xTer array within a reporter of mammalian HR, targeted to a specific locus of mouse embryonic stem (ES) cells, we found that transient expression of wild type Tus triggers HR at the 6xTer array, while expression of a mutant form of Tus (H144A) that has a low affinity for Ter failed to induce HR in the same cells. 14,15 Several lines of evidence indicate that Tus/Ter-induced HR in mammalian cells entails the processing of 2 converging forks arrested at the Tus/Ter fork block. Tus/Ter-induced HR predominantly generates "short tract" gene conversions (STGC)-a conservative HR outcome.…”

“…Crucially, Southern blot analysis of Tus/Ter-induced STGC products revealed that STGC termination entailed interaction with a second homologous DNA end-presumably derived from the second arrested fork. 15 An alternative, aberrant HR outcome termed "long tract" gene conversion (LTGC) entails the copying of several kilobases from the donor sister chromatid and is considered an error-prone outcome. [17][18][19][20] One cause of LTGC can be a failure of termination of conventional HR, when the second (non-invading) DNA end is either missing-as might occur in a "one-ended" break-or imperfectly coordinated with the invading strand.…”

“…[8][9][10] We recently adapted the Escherichia coli Tus/Ter replication fork arrest complex [11][12][13][14] to provoke site-specific replication fork stalling and chromosomal HR in mammalian cells. 15 In E. coli, binding of the monomeric Tus protein to the asymmetrical »23 bp Ter site provokes polar replication fork arrest, depending on whether the replicative helicase DnaB arrives at the non-permissive or permissive end of Ter. We found that an array of 6 Ter sites (6xTer), when bound by wild type (wt)Tus, provokes site-specific replication fork arrest within a replicating episomal plasmid, driven by the Epstein-Barr Nuclear Antigen-1 (EBNA1).…”