Department of Microbial Biotechnology, Centro Nacional de Biotecnología, (CNB-CSIC), Cantoblanco 28049, Madrid, Spain.
Nucleic Acids Res. 2018 Aug 21;46(14):7206-7220. doi: 10.1093/nar/gky541.
The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ssDNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA-dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation. The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.
无处不在的 RarA/Mgs1/WRNIP 蛋白在维持基因组稳定方面发挥着关键作用，但它的作用机制尚未完全阐明。我们发现枯草芽孢杆菌中的 RarA 在无辅基状态下优先结合单链 DNA（ssDNA）而非双链 DNA（dsDNA）。与 ssDNA 结合的 SsbA 蛋白能够负载 RarA，且 SsbA 的疏水性 C 端结构域对这种负载作用是必需的。RarA 是一种 DNA 依赖的 ATP 酶，ssDNA-dsDNA 连接物、SsbA 或 dsDNA 末端能够强烈地激活 RarA 的 ATP 酶活性。RarA 可能与 PriA 相互作用，但不刺激 PriA 解链。在一个重建的 PriA 依赖的 DNA 复制系统中，RarA 抑制起始，但不抑制链延伸。该抑制作用依赖于 SsbA 的存在，若缺乏 SsbA，或当宿主编码的预引发体和 DNA 解旋酶被具有相似功能的 SPP1 噬菌体编码的蛋白取代时，RarA 则无法发挥作用。我们提出，RarA 装配在受阻的复制叉上以维持基因组的完整性。通过与 SsbA 以及预引发体组件的相互作用，RarA 可能阻碍复制性解旋酶的组装，以防止重组中间体导致病理性的 DNA 复制起始。
