Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
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DOI：
10.1093/nar/gks264
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发表时间：
2012-07-01
影响因子：
14.9
通讯作者：
Maki, Hisaji
中科院分区：
文献类型：
--
作者：
Furukohri, Asako;Nishikawa, Yoshito;Maki, Hisaji
DNA聚合酶IV （Pol IV）是大肠杆菌中的三种翻译聚合酶之一。质谱的一项研究显示,单链dna结合蛋白(单边带)溶菌产物准备从指数增长细胞有很强的亲和力column-immobilized波尔IV。我们发现纯化单边带直接绑定到第四波尔拉试验,而单边带δC8, c端尾突变蛋白缺乏,未能与波尔IV。这些结果表明,波尔IV和单边带之间的相互作用是由单边带的c端尾。当在SSB δ c8包被模板上测试聚合酶活性时，我们观察到对Pol IV活性的强烈抑制。利用含有SSB尾部氨基酸序列的合成肽进行竞争实验发现，当Pol IV与SSB尾部的相互作用被抑制时，Pol IV的链长能力大大受损。这些结果表明，当模板ssDNA被SSB覆盖时，Pol IV需要与SSB的c端尾部相互作用才能有效地复制DNA。我们推测，在引物/模板连接处，Pol IV与模板上最近的SSB四聚体的尾部相互作用，并且这种相互作用允许聚合酶在拆卸SSB时沿着模板移动。
DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSB delta C8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSB delta C8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.