Ermakova O V, Frappier L, Schildkraut C L
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.
J Biol Chem. 1996 Dec 20;271(51):33009-17. doi: 10.1074/jbc.271.51.33009.
We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.
我们之前已经表明，在一个小质粒以及完整的爱泼斯坦-巴尔病毒基因组中，复制叉会在爱泼斯坦-巴尔病毒潜伏复制起点oriP内的一族重复序列（FR）处停滞。FR内的20个重复序列中的每一个都包含爱泼斯坦-巴尔核抗原1（EBNA-1）的一个结合位点，EBNA-1是潜伏复制所需的唯一病毒蛋白。我们表明EBNA-1蛋白会增强FR处暂停的复制叉的积累。在本研究中，我们研究了一系列截短的EBNA-1蛋白，以确定EBNA-1蛋白中负责FR处复制叉暂停的部分。在存在全长EBNA-1或各种缺失的蛋白的情况下，对体外复制反应的产物进行二维琼脂糖凝胶电泳，以评估FR处复制叉暂停的程度。我们得出结论，DNA结合结构域的一部分对于复制叉暂停很重要。我们还提供了证据表明EBNA-1蛋白或截短的EBNA-1衍生物的磷酸化对于暂停并非必不可少。为了研究EBNA-1介导的复制叉暂停机制，我们询问EBNA-1是否能抑制复制性解旋酶的DNA解旋活性。我们发现，当与FR结合时，EBNA-1以方向独立的方式在体外抑制SV40 T抗原和大肠杆菌dnaB解旋酶的DNA解旋。
