CONSTITUTIVE BINDING OF EBNA1 PROTEIN TO THE EPSTEIN-BARR-VIRUS REPLICATION ORIGIN, ORIP, WITH DISTORTION OF DNA-STRUCTURE DURING LATENT INFECTION
CONSTITUTIVE BINDING OF EBNA1 PROTEIN TO THE EPSTEIN-BARR-VIRUS REPLICATION ORIGIN, ORIP, WITH DISTORTION OF DNA-STRUCTURE DURING LATENT INFECTION
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DOI：
10.1002/j.1460-2075.1993.tb06187.x
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发表时间：
1993-12-15
期刊：
影响因子：
11.4
通讯作者：
YATES, JL
中科院分区：
文献类型：
--
作者：
HSIEH, DJ;CAMIOLO, SM;YATES, JL
EB病毒（EBV）的环状170 kb基因组在潜伏感染期间的复制由细胞周期控制下的细胞复制机制进行。单个病毒蛋白EBNA 1指导细胞复制装置在基因定义的复制起点oriP内在四个EBNA 1结合位点的簇（在此称为双向复制的物理起点或OBR）处启动复制。oriP内的EBNA 1结合位点的第二簇，即30 bp重复序列，作为复制增强子发挥重要作用，并且还提供与复制无关的独特附加体维持功能。我们研究了oriP的功能元件结合EBNA 1和可能的其他蛋白质在增殖的Raji细胞产生体内足迹，使用两种试剂，硫酸二甲酯（DMS）和KMnO 4。 我们还采用了脱氧核糖核酸酶I（DNase I）与透化细胞。EBNA 1结合位点处的体内和透化细胞足迹，特别是使用DMS获得的那些，给出了所有这些位点在异步分裂细胞中被EBNA 1结合的强有力证据。没有一致的证据表明其他蛋白质在oriP的功能区域内的任何其他位点的结合。当细胞用高锰酸盐处理时，oriP内OBR的对称位置处的胸腺嘧啶被氧化，这表明在这些位置处的DNA结构的弯曲或其他扭曲; EBNA 1在体外与Raji细胞的总DNA的结合在相同的位置处诱导对高锰酸盐的反应性。使用异步分裂细胞获得的结果的最简单的解释是EBNA 1结合到其在oriP的位点，并在整个细胞周期的大部分时间内保持OBR处于扭曲的构象，这意味着复制是由细胞机制启动的，并且不受EBNA 1与oriP结合的可用性的限制。
Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.