Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.
Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.
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DOI：
10.1371/journal.pone.0128092
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kreuzer KN
中科院分区：
文献类型：
--
作者：
Henderson ML;Kreuzer KN
突变体EcoRII甲基转移酶蛋白（m.e EcoRII- c186a）在大肠杆菌中的表达导致紧密结合的dna -蛋白复合物（tbc）零星地位于染色体上，而不是串联阵列。这种散发性tbc致死性背后的机制尚未得到很好的研究，也不清楚非常紧密结合但非共价复合物的加工方式是否与共价dna -蛋白交联（DPCs）相同。通过二维凝胶电泳，我们发现m.e ecorii - c186a诱导的tbc在体内阻断复制分叉。只有在诱导m.e EcoRII- c186a时，在2D凝胶上才能检测到特定的气泡分子，并且消除特定EcoRII甲基化位点的突变导致相应的斑点消失。我们还对M.EcoRII-C186A诱导的tbc敏感突变体进行了候选基因筛选。我们发现了几种保护这些tbc所必需的基因产物，这些基因产物已知也可以保护野生型M.EcoRII诱导的DPCs（在5-氮胞苷结合后）：RecA, RecBC, RecG, RuvABC, UvrD, FtsK， XerCD和SsrA （tmRNA）。相比之下，RecFOR途径和Rep解旋酶对tbc的保护作用是必需的，而不需要M.EcoRII诱导的DPCs。我们提出，RecFOR和RecA暂停叉加工促进紧密结合（但非共价）阻断蛋白的释放，可能是通过授权Rep解旋酶驱动阻断M.EcoRII-C186A的解离。我们的研究也反对参与一些可能被认为可以防止tbc的蛋白质。我们借此机会直接比较了所有测试突变体对两种喹诺酮类抗生素的敏感性，这两种抗生素针对细菌II型拓扑异构酶并诱导一种独特形式的DPC。我们发现rep， ftsK和xerCD是新的喹诺酮类过敏突变体，并且也获得了一些可能对喹诺酮类药物有保护作用的功能参与的证据。
Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.
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