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.

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.

Template switching (TS) mediates damage-bypass via a recombination-related mechanism involving PCNA polyubiquitylation and Polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and electron microscopy, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps in the range of 150-200 nucleotides, and not DNA ends, initiate TS by strand invasion. This causes re-annealing of the parental strands and exposure of the non-damaged newly synthesized chromatid as template for replication by the other blocked nascent strand. Structures resembling double Holliday Junctions, postulated to be central double-strand break repair intermediates, but so far only visualized in meiosis, mediate late stages of TS, before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA damage tolerance in mitotic cells and for replication under conditions of genotoxic stress.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.