ROLE OF EBNA-1 IN ARRESTING REPLICATION FORKS AT THE EPSTEIN-BARR-VIRUS ORIP FAMILY OF TANDEM REPEATS
ROLE OF EBNA-1 IN ARRESTING REPLICATION FORKS AT THE EPSTEIN-BARR-VIRUS ORIP FAMILY OF TANDEM REPEATS
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DOI：
10.1128/mcb.11.12.6268
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发表时间：
1991-12-01
影响因子：
5.3
通讯作者：
SCHILDKRAUT, CL
中科院分区：
文献类型：
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作者：
DHAR, V;SCHILDKRAUT, CL
位于EB病毒（EBV）oriP区域内的20个成员的30-bp串联重复序列家族可以在EBV核抗原1（EBNA-1）存在下充当转录增强子。 在人B细胞中的复制叉屏障和质粒复制的终止位点也发现在EBV串联重复内或附近。 在每个串联重复序列内是EBNA-1蛋白的共有结合序列，其是含有oriP的质粒的染色体外维持所需的。 要调查的因素，有助于逮捕的复制叉和终止在该地区的家庭的重复序列，我们已经使用了体外复制系统，其中EBV重组质粒的复制是从猿猴病毒40（SV 40）的DNA复制起点在SV 40 T抗原和可溶性提取物的存在下制备的人细胞。 该系统可以支持双向复制，从SV 40 DNA起点开始，终止发生在起点对面的区域。 使用二维琼脂糖凝胶电泳，我们观察到在EBV重复区域中存在EBNA-1时复制叉的屏障。 终止发生在串联重复序列处或附近，其方式与体内观察到的方式相似（T. A. Gahn和C. L. Schildkraut，Cell 58：527-535，1989）。 将重复次数从20减少到6对复制叉屏障的强度几乎没有影响。 在没有EBNA-1的情况下，复制叉也在EBV重复处被阻止，但效率低得多。 添加含有EBV重复序列家族的竞争DNA几乎可以完全消除在EBNA-1存在下产生的复制屏障。
The 20-member family of 30-bp tandem repeats located within the oriP region of Epstein-Barr virus (EBV) can act as a transcriptional enhancer in the presence of EBV nuclear antigen 1 (EBNA-1). A replication fork barrier and a termination site of plasmid replication in human B cells is also found within or near the EBV tandem repeats. Within each tandem repeat is a consensus binding sequence for the EBNA-1 protein that is required for extrachromosomal maintenance of oriP-containing plasmids. To investigate the factors that contribute to the arrest of replication forks and termination in the region of the family of repeats, we have used an in vitro replication system in which replication of EBV recombinant plasmids is initiated from the simian virus 40 (SV40) DNA replication origin in the presence of SV40 T antigen and soluble extracts prepared from human cells. The system can support bidirectional replication, initiating from the SV40 DNA origin with termination occurring in a region opposite the origin. Using two-dimensional agarose gel electrophoresis, we observed a barrier to replication forks in the presence of EBNA-1 in the region of the EBV repeats. Termination occurs at or near the tandem repeats in a manner similar to that observed in vivo (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989). Reducing the number of repeats from 20 to 6 had little effect on the strength of the replication fork barrier. In the absence of EBNA-1, replication forks also arrested at the EBV repeats, but at a much lower efficiency. The addition of competitor DNA containing the EBV family of repeats can almost completely abolish the replication barrier produced in the presence of EBNA-1.