Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex
Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex
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DOI：
10.1074/jbc.m110.107656
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发表时间：
2010-06-04
影响因子：
4.8
通讯作者：
Richardson, Charles C.
中科院分区：
文献类型：
--
作者：
Ghosh, Sharmistha;Hamdan, Samir M.;Richardson, Charles C.
由噬菌体T7编码的DNA聚合酶具有低的持续合成能力。大肠杆菌硫氧还蛋白与聚合酶拇指亚结构域中的76个残基的区段结合并增加持续合成能力。硫氧还蛋白的结合导致两个基本环的形成，环A和B，位于硫氧还蛋白结合结构域（TBD）内。两个环都与T7解旋酶的酸性C末端相互作用。相对弱的静电模式涉及解旋酶和TBD的C-末端尾部，而当聚合酶处于聚合模式时，发生不涉及C-末端尾部的高亲和力相互作用。T7基因2.5单链DNA结合蛋白（gp2.5）也有一个酸性的C-末端尾。gp2.5也有两种与聚合酶相互作用的模式，但都涉及gp2.5的C-末端尾。静电相互作用需要环A和B中的碱性残基，并且如通过表面等离子体共振测量的，gp2.5以相似的亲和力结合到两个环。当聚合酶处于聚合模式时，基因2.5蛋白的C末端在TBD外部的区域中与聚合酶相互作用。GP2.5在前导链合成期间增加聚合酶-解旋酶复合物的持续合成能力。当TBD的环B改变时，在前导链合成期间观察到失败的DNA产物。环B似乎在与解旋酶和gp2.5的通信中起重要作用，而环A在这些相互作用中起稳定作用。
The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.