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Interaction with single-stranded DNA-binding protein localizes ribonuclease HI to DNA replication forks and facilitates R-loop removal.
Molecular Microbiology ( IF 3.6 ) Pub Date : 2020-05-19 , DOI: 10.1111/mmi.14529
Christine Wolak 1 , Hui Jun Ma 2 , Nicolas Soubry 2 , Steven J Sandler 3 , Rodrigo Reyes-Lamothe 2 , James L Keck 1
Affiliation  

DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R‐loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R‐loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R‐loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between Escherichia coli RNase HI and the single‐stranded DNA‐binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation of rnhA to encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media‐dependent slow‐growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R‐loop accumulation enhance or suppress, respectively, the growth phenotype of rnhAK60E rep::kan strains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R‐loops that block DNA replication.

中文翻译:

与单链 DNA 结合蛋白的相互作用将核糖核酸酶 HI 定位于 DNA 复制叉并促进 R 环去除。

DNA 复制复合体(复制体)经常会遇到可能阻碍其进展的蛋白质和不寻常的核酸结构。屏障可能包括转录复合物和 R 环,这些复合物和 R 环是在 RNA 与 RNA 聚合酶后面的互补 DNA 模板杂交时形成的。细胞编码多种 RNA 聚合酶和 R 环清除机制,以限制复制体暴露于这些潜在障碍。其中一种机制是核糖核酸酶 HI (RNase HI) 水解 R 环。在这里,我们研究了大肠杆菌RNase HI 和单链 DNA 结合蛋白 (SSB)之间相互作用在此过程中的细胞作用。与 SSB 的相互作用将 RNase HI 焦点定位于 DNA 复制位点。rnhA突变编码不能与 SSB 相互作用但保持酶活性 ( rnhAK60E ) 的 RNase HI 变体,消除了 RNase HI 病灶。该突变还会在缺乏 Rep 解旋酶的细胞中产生介质依赖性缓慢生长表型和激活的 DNA 损伤反应,Rep 解旋酶是一种破坏停滞转录复合物的酶。RNA聚合酶变体被认为会增加或减少R环积累,从而分别增强或抑制rnhAK60Erep::kan菌株的生长表型。这些结果确定了 RNase HI/SSB 相互作用在帮助清除阻碍 DNA 复制的 R 环方面的细胞作用。
更新日期:2020-05-19
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