Thorner L K, Lim D A, Botchan M R
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Virol. 1993 Oct;67(10):6000-14. doi: 10.1128/JVI.67.10.6000-6014.1993.
The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.
牛乳头瘤病毒1型的E1蛋白是乳头瘤病毒DNA复制所需的多功能酶。它作为位点特异性DNA结合蛋白和DNA解旋酶，协助复制的起始。先前的研究表明，在E1浓度有限时，E2蛋白是E1有效结合复制起点所必需的。在本研究中，我们确定了E1蛋白位点特异性DNA结合所需的结构域。对一系列截短蛋白的实验表明，最初的299个氨基末端氨基酸包含DNA结合结构域；然而，与E1前129个氨基酸同源的共末端M蛋白并不结合起点DNA。E1的DNA结合结构域中的一系列小的内部缺失和取代突变表明，该蛋白区域中特定的碱性残基（在乳头瘤病毒家族的所有E1蛋白中保守）可能在结合DNA中起直接作用，并且侧翼保守的疏水亚结构域对DNA结合也很重要。E1与E2相互作用以进行协同DNA结合的区域在羧基末端截短蛋白中也保留着，并且我们表明全长E1与E2复合的能力对冷敏感。E1取代突变蛋白由哺乳动物表达载体表达，以确定E1的位点特异性DNA结合是否是细胞中瞬时DNA复制所必需的。这些E1蛋白表现出一系列突变表型，这与E1的位点特异性结合很重要的观点一致。有趣的是，一种E1突变体对起点结合有缺陷，但可以被E2挽救这种活性，它在细胞中支持显著的复制。
