Regulation of Single-stranded DNA Binding by the C Termini of Escherichia coli Single-stranded DNA-binding (SSB) Protein
Regulation of Single-stranded DNA Binding by the C Termini of Escherichia coli Single-stranded DNA-binding (SSB) Protein
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DOI：
10.1074/jbc.m110.118273
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发表时间：
2010-05-28
影响因子：
4.8
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Kozlov, Alexander G.;Cox, Michael M.;Lohman, Timothy M.
同源四聚体大肠杆菌单链 DNA 结合 (SSB) 蛋白在 DNA 复制、修复和重组中发挥着核心作用。除了与瞬时形成的单链 (ss) DNA 结合的基本活性外，SSB 还结合一系列伙伴蛋白，并利用其四个本质上无序的 C 末端尾部将它们招募到其作用位点。在这里，我们表明，ssDNA 与 SSB 的结合受到 SSB C 末端尾部的抑制，特别是最后 8 个高酸性氨基酸的抑制，这些氨基酸构成了其多个伙伴蛋白的结合位点。我们检查了单链DNA与短寡脱氧核苷酸结合的能量学，发现在中等盐浓度下，酸性C末端的去除增加了单链DNA的内在亲和力，并增强了单链DNA结合位点之间的负协同性，表明C末端对单链DNA结合具有抑制作用。这种抑制作用随着盐浓度的增加而减弱。 ssDNA 与大约一半的 SSB 亚基的结合减轻了所有亚基的抑制作用。 C 末端的抑制主要是由于 ssDNA 结合时不太有利的熵变化。这些观察结果解释了为什么单链DNA与SSB的结合增强了SSB与其伙伴蛋白的亲和力，并表明SSB的C末端可能与其单链DNA结合位点至少短暂地相互作用。这种抑制及其通过 ssDNA 结合的缓解表明了一种机制，可以增强 SSB 选择性地将其伙伴蛋白招募到 DNA 位点的能力。
The homotetrameric Escherichia coli single-stranded DNA-binding (SSB) protein plays a central role in DNA replication, repair, and recombination. In addition to its essential activity of binding to transiently formed single-stranded (ss) DNA, SSB also binds an array of partner proteins and recruits them to their sites of action using its four intrinsically disordered C-terminal tails. Here we show that the binding of ssDNA to SSB is inhibited by the SSB C-terminal tails, specifically by the last 8 highly acidic amino acids that comprise the binding site for its multiple partner proteins. We examined the energetics of ssDNA binding to short oligodeoxynucleotides and find that at moderate salt concentration, removal of the acidic C-terminal ends increases the intrinsic affinity for ssDNA and enhances the negative cooperativity between ssDNA binding sites, indicating that the C termini exert an inhibitory effect on ssDNA binding. This inhibitory effect decreases as the salt concentration increases. Binding of ssDNA to approximately half of the SSB subunits relieves the inhibitory effect for all of the subunits. The inhibition by the C termini is due primarily to a less favorable entropy change upon ssDNA binding. These observations explain why ssDNA binding to SSB enhances the affinity of SSB for its partner proteins and suggest that the C termini of SSB may interact, at least transiently, with its ssDNA binding sites. This inhibition and its relief by ssDNA binding suggest a mechanism that enhances the ability of SSB to selectively recruit its partner proteins to sites on DNA.