Department of Physics, Harvard University, Cambridge, MA 02138, USA.
Department of Biology, Northeastern University, Boston, MA 02115, USA.
Nucleic Acids Res. 2019 Feb 28;47(4):1836-1846. doi: 10.1093/nar/gky1252.
Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.
细菌双链断裂的重组修复通常从在双链断裂 (DSB) 的每一侧创建起始的 3' 单链 DNA (ssDNA) 尾巴开始。重要的是，如果遵循 RecBCD 途径，则 RecBCD 在起始链 3' 端的序列之间产生缺口。该缺口位于 DSB 两侧，并沿每个链延伸至最近的 Chi 位点。一旦起始链形成 ssDNA-RecA 丝，每个 ssDNA-RecA 丝都会搜索同源双链 DNA (dsDNA)，将其用作 RecBCD 产生的缺口所需的 DNA 合成模板。我们的实验结果表明，DNA 合成需要形成异源双链 dsDNA，该 dsDNA 将起始链中>20 个连续的碱基与原始 dsDNA 中来自一条链的序列匹配碱基配对。为了触发合成，异源双链体必须靠近起始链的 3' 端。这些实验确定的合成要求与 RecBCD 功能的 Chi 位点依赖性以及细菌基因组中 Chi 位点的分布相结合，可能使 RecBCD 途径避免一些由直接诱导的 DSB 引起的基因组重排；然而，同样的三个因素也可能促进其他重排。
