title: Break-induced replication is highly inaccurate.
abstract: DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.
authors.affiliation: Department of Biology, School of Science, IUPUI, Indianapolis, Indiana, United States of America.
authors.forename: Angela
authors.initials: A
authors.lastname: Deem
authors.forename: Andrea
authors.initials: A
authors.lastname: Keszthelyi
authors.forename: Tiffany
authors.initials: T
authors.lastname: Blackgrove
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vayl
authors.forename: Barbara
authors.initials: B
authors.lastname: Coffey
authors.forename: Ruchi
authors.initials: R
authors.lastname: Mathur
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
authors.forename: Anna
authors.initials: A
authors.lastname: Malkova
mesh_terms: D001483:Base Sequence; D053960:DNA Breaks; D053843:DNA Mismatch Repair; D004260:DNA Repair; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000502:physiology; D003854:Deoxyribonucleotides / Q000378:metabolism; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D003854:Deoxyribonucleotides; D004259:DNA-Directed DNA Polymerase