Break-Induced Replication Is Highly Inaccurate

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.基因组复制与DNA修复过程均需合成DNA。尽管前者是高度精准的过程，但与DNA损伤修复相关的短补丁合成（short-patch synthesis）往往容易出错。断裂诱导复制（Break-induced replication, BIR）是一种独特的细胞过程：其在合成持续性、速率以及复制数百千碱基对的能力上均与正常DNA复制相似，但它起始于双链断裂（double-strand breaks, DSBs）而非复制起点。本研究通过一系列移码报告基因（frameshift reporters），对酿酒酵母（Saccharomyces cerevisiae）中与BIR相关的诱变效应进行了定量检测。我们证实，BIR介导的DNA合成在复制叉（replication fork）的整个行进路径中均存在内在不精准性：BIR过程中的移码诱变率最高可达正常复制的2800倍。值得注意的是，这种高诱变率不仅出现在BIR稳定性较低的DSB近端区域，同样也存在于BIR复制叉运转快速且稳定的DSB远端区域。我们证实，聚合酶校对（polymerase proofreading）与错配修复（mismatch repair）机制可纠正BIR过程中产生的错误。此外，BIR过程中细胞内脱氧核苷三磷酸（deoxyribonucleoside triphosphates, dNTPs）水平升高，这同样加剧了BIR相关的诱变效应。我们提出，未被纠错机制完全代偿的高水平DNA聚合酶错误，是BIR过程中诱变效应的主要诱因，其中DNA聚合酶δ（Pol δ）介导了大量致突变性错误的产生。我们进一步推测，真核细胞中BIR的激活，可能会显著促进突变积累，进而推动癌症发生与生物演化进程。