Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA
Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA
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DOI：
10.1073/pnas.0912664107
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发表时间：
2010-02-02
影响因子：
11.1
通讯作者：
van Oijen, Antoine M.
中科院分区：
文献类型：
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作者：
Etson, Candice M.;Hamdan, Samir M.;van Oijen, Antoine M.
参与DNA复制的DNA聚合酶通过与合成因子形成复合物来实现核苷酸掺入的高合成能力。噬菌体T7 DNA聚合酶（gp 5）是复制型DNA聚合酶的模型系统，由基因5编码，与大肠杆菌硫氧还蛋白形成紧密的1：1复合物。硫氧还蛋白作为一种持续合成因子，通过一种尚未完全理解的机制，将gp 5从一种分布性聚合酶转化为一种高度持续性聚合酶。我们使用单分子成像方法来可视化荧光标记的T7 DNA聚合酶与双链DNA的相互作用。我们已经观察到T7 gp 5，无论有无硫氧还蛋白，非特异性结合双链DNA和扩散沿着双链体。gp 5/硫氧还蛋白复合物在扩散时保持与DNA紧密结合，而没有硫氧还蛋白的gp 5经历频繁的从DNA解离和与DNA重新结合。这些观察结果表明，硫氧还蛋白通过抑制DNA上和下的微观跳跃并保持复合物与双链体紧密结合来增加T7 DNA聚合酶的持续合成能力。
The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1: 1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.