SEPARATION OF THE COMPLEX DNA-BINDING DOMAIN OF EBNA-1 INTO DNA RECOGNITION AND DIMERIZATION SUBDOMAINS OF NOVEL STRUCTURE
SEPARATION OF THE COMPLEX DNA-BINDING DOMAIN OF EBNA-1 INTO DNA RECOGNITION AND DIMERIZATION SUBDOMAINS OF NOVEL STRUCTURE
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DOI：
10.1128/jvi.67.8.4875-4885.1993
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发表时间：
1993-08-01
影响因子：
5.4
通讯作者：
HAYWARD, SD
中科院分区：
文献类型：
--
作者：
CHEN, MR;MIDDELDORP, JM;HAYWARD, SD
EBNA-1对于EB病毒基因组潜伏的上体形式的复制是必不可少的，并参与病毒潜伏启动子的调节。EBNA-1的活性是通过直接DNA结合介导的。EBNA-1的DNA结合和二聚功能已经定位在羧基末端，氨基酸(AA)459到607。为了识别和定义这两个功能的亚域，我们在EBNA-1(AA 408到641)背景下创建了一系列广泛的缺失和点突变。用能识别EBNA-1(AA 408至641)而不能识别EBNA-1(AA 459至641)的单抗EBNA-OT1x进行免疫沉淀试验，检测EBNA-1突变体与野生型EBNA-1(AA 459至641)异源二聚的能力。这些实验表明，影响二聚化的突变发生在两个不同的区域，AA 501到532和AA 554到598。DNA结合在针对一组寡核苷酸结合位点的迁移率变化分析中进行测试。二聚化是DNA结合的先决条件。DNA识别结构域定位于二聚化结构域上游的一个单独的区域，AA 459至487。EBNA-1变异体带有AA 467和468位以及AA 477位的替换，给出了一种与突变的寡核苷酸探针结合的模式，这意味着这些特定的氨基酸在DNA识别中。EBNA-1似乎利用了新的机制来识别DNA和二聚化，因为这两个结构域都不符合之前描述的结构基序。
EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) polypeptide was tested in immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.