Mechanisms of DNA replication elongation

有效的DNA复制涉及DNA聚合酶、多种因子和DNA之间的协调相互作用。 从T4噬菌体到真核生物，这些因素包括在复制叉之前解开DNA的解旋酶、结合滞后链上ssDNA的单链结合蛋白（SSB）以及与复制叉、解旋酶和SSB相关的解旋酶装载剂。 先前报道的T4系统中的解旋酶装载器的结构，基因产物（gp）59，揭示了N-末端结构域，其与来自真核生物的高迁移率族（HMG）蛋白具有结构同源性。用叉状DNA对这种结构进行建模表明，HMG样结构域可以在叉状之前与双链体DNA结合，而gp 59的C末端部分将为解旋酶（T4 gp 41）、SSB（T4 gp 32）和ssDNA叉臂提供对接位点。 为了测试这个模型，我们使用随机和靶向诱变来产生整个gp 59的突变。 我们已经测定了突变蛋白结合叉，引物叉，和ssDNAs，与SSB相互作用，刺激解旋酶活性，并在前导和滞后链DNA合成的功能的能力。 我们的研究结果提供了强有力的生化支持的N-末端gp 59 HMG基序在叉结合和gp 59的C-末端部分与解旋酶和SSB的相互作用的作用。 我们的研究结果还表明，进行性复制可能涉及gp 59之间的开关与解旋酶和SSB的相互作用。

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, the helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, while the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.