C-terminal deletions of the Escherichia coli RecA protein -: Characterization of in vivo and in vitro effects
C-terminal deletions of the Escherichia coli RecA protein -: Characterization of in vivo and in vitro effects
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DOI：
10.1074/jbc.m212917200
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发表时间：
2003-05-02
影响因子：
4.8
通讯作者：
Cox, MM
中科院分区：
文献类型：
--
作者：
Lusetti, SL;Wood, EA;Cox, MM
获得、表达和纯化了一组大肠杆菌RecA蛋白的C端缺失突变体，该突变体可逐步去除6、13、17和25个氨基酸残基。在体内，13到17个C末端残基的缺失导致对丝裂霉素C的敏感性增加。在体外，这些缺失增强了与双链DNA的结合，正如之前观察到的那样。我们证明，这种增强在很大程度上涉及删除第339位和346位之间的残基。此外，C末端的缺失导致DNA链交换反应的pH反应谱显著上移。超过13个氨基酸残基的C末端缺失导致在pH 7以下对DNA链交换的强烈抑制，此时野生型蛋白促进了熟练的反应。然而，同时，13-17个C-末端残基的缺失消除了在pH值在7.5到9之间时与野生型蛋白DNA链交换的减少。结果表明，C末端与蛋白质的其他部分之间存在广泛的相互作用，可能涉及多个盐桥。这些相互作用影响参与DNA链交换的关键基团的PK(A)，以及RecA蛋白与双链DNA的直接结合。
A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.