Wang Y S, Hall J D
Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721.
J Virol. 1990 May;64(5):2082-9. doi: 10.1128/JVI.64.5.2082-2089.1990.
We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.
我们研究了单纯疱疹病毒1型的主要DNA结合蛋白（ICP8），以确定其DNA结合位点。由于我们从携带多个染色体定位的ICP8基因拷贝的细胞系中获得该蛋白，我们首先对该蛋白进行分析，以评估其与相应病毒蛋白的相似性。我们的蛋白在分子量、对抗体的反应、对单链DNA结合的偏好以及降低聚（dA-dT）解链温度的能力方面与病毒蛋白相似。为了确定DNA结合结构域，我们对该蛋白进行有限的胰蛋白酶消化，并在十二烷基硫酸钠-聚丙烯酰胺凝胶上分离肽产物。然后将这些片段转移到硝酸纤维素膜上，原位复性，并测试它们结合DNA的能力。通过该测定，我们鉴定出四个既结合DNA又表现出对单链DNA预期结合偏好的片段。确定了这些片段中最小的片段的序列，其对应于完整蛋白中跨越300至849位残基的多肽。基于来自其他疱疹病毒的单链DNA结合蛋白中的序列相似性，并且在一种情况下基于在更远缘的原核和真核蛋白中发现的保守序列，该肽包含几个可能对DNA结合很重要的区域。
