Break-Induced Replication Is Highly Inaccurate

Abstract

DNA replication initiated by one-ended homologous recombination at a double-strand break is highly inaccurate, as it greatly stimulates frameshift mutations over the entire path of the replication fork.

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.

Author Summary

Accurate transmission of genetic information requires the precise replication of parental DNA. Mutations (which can be beneficial or deleterious) arise from errors that remain uncorrected. DNA replication occurs during S-phase of the cell cycle and is extremely accurate due to highly selective DNA polymerases coupled with effective error-correction mechanisms. In contrast, DNA synthesis associated with short-patch DNA repair is often error-prone. Break-induced replication (BIR) presents an interesting case of large-scale DNA duplication that occurs in the context of DNA repair. In this study we employed a yeast-based system to investigate the level of mutagenesis associated with BIR compared to mutagenesis during normal DNA replication. We report that frameshifts, which are the most deleterious kind of point mutation, are much more frequent during BIR than during normal DNA replication. Surprisingly, we observed that the majority of mutations associated with BIR were created by polymerases responsible for normal DNA replication, which are assumed to be highly precise. Overall, we propose that BIR is a novel source of mutagenesis that may contribute to disease genesis and evolution.