Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
J Bacteriol. 2014 Apr;196(7):1359-68. doi: 10.1128/JB.01475-13. Epub 2014 Jan 17.
DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5'-3' helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.
DNA 解旋酶在基因组维护中起着重要作用。RecD 解旋酶作为异源三聚体 RecBCD 解旋酶-核酸酶的一个组成部分，在大肠杆菌双链断裂修复中起着重要作用，已得到深入研究。有趣的是，许多细菌缺乏 RecBC，而是含有 RecD2 解旋酶，而 RecD2 解旋酶是否作为更大复合物的一部分发挥作用尚不清楚。根据研究的生物体，RecD2 已被证明能提供对广泛的 DNA 损伤剂的抗性，同时也有助于错配修复 (MMR)。在这里，我们研究了枯草芽孢杆菌 RecD2 解旋酶对基因组完整性的重要性。我们发现，recD2 的缺失赋予了自发突变率的适度增加，并且 ΔrecD2 细胞中的突变特征与 MMR 缺陷不一致，表明 RecD2 在枯草芽孢杆菌中有新的功能。为了进一步表征 RecD2 的作用，我们测试了缺失菌株对 DNA 损伤剂的敏感性。我们发现，枯草芽孢杆菌中 RecD2 的缺失使细胞对几种能阻止或损害复制叉运动的 DNA 损伤剂敏感。体内复制叉进展的测量表明，ΔrecD2 细胞中的复制叉更频繁地崩溃，这支持了 RecD2 对正常复制叉进展很重要的假设。枯草芽孢杆菌 RecD2 的生化特性表明，它是一种 5'-3'解旋酶，并且它直接结合单链 DNA 结合蛋白。总之，我们的结果突出了 RecD2 在 DNA 复制中的新作用，有助于在正常生长和复制叉遇到 DNA 损伤时维持复制叉的完整性。
