Mechanisms of DNA replication elongation

有效的DNA复制涉及DNA聚合酶、多因子和DNA之间的协调相互作用。从T4噬菌体到真核生物，这些因子包括在复制叉前解开DNA的解旋酶，与后链上的ssDNA结合的单链结合蛋白（SSB），以及与叉、解旋酶和SSB相关的解旋酶装载物。先前报道的T4系统解旋酶装载物基因产物（gp）59的结构揭示了一个n端结构域，该结构域与真核生物的高迁移基团（HMG）蛋白具有结构同源性。用叉DNA对这种结构进行建模表明，hmg样结构域可以与叉前面的双链DNA结合，而gp59的c端部分将为解旋酶（T4 gp41）、SSB （T4 gp32）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在gp59中产生突变。我们分析了突变蛋白与叉、引物叉和ssdna结合的能力，与SSB相互作用的能力，刺激解旋酶活性的能力，以及在前导链和滞后链DNA合成中的功能。我们的研究结果为n端gp59 HMG基序在叉结合中的作用以及gp59 c端与解旋酶和SSB的相互作用提供了强有力的生化支持。我们的研究结果还表明，过程复制可能涉及gp59与解旋酶和SSB相互作用之间的切换。

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.