Leinbach S S, Heath L S
Department of Pathology, University of North Carolina, Chapel Hill.
Biochim Biophys Acta. 1989 Aug 14;1008(3):281-6. doi: 10.1016/0167-4781(89)90017-1.
The DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is multifunctional in vivo and binds preferentially to single-stranded DNA (ssDNA) in vitro. To define the ssDNA-binding domain of ICP8, peptides were produced and analyzed. Portions of the ICP8 gene were cloned into the transcription vector pSP64, and RNA was synthesized in vitro. Translation of this RNA in rabbit reticulocyte lysates produced peptides of 29, 35 and 30 kDa, representing amino-acid residues 332-564, 571-899 and 900-1196, respectively, of intact ICP8 (128 kDa, 1196 amino acids). These peptides were analyzed by ssDNA-cellulose column chromatography. About 55% of the 29 kDa peptide bound to ssDNA-cellulose columns, and the majority which bound eluted with 1.0 M NaCl. About 5% of the 35 kDa peptide and 12% of the 30 kDa peptide bound and eluted with 0.3 M NaCl. Thus, three regions of ICP8 were associated with ssDNA-binding activity. The ssDNA-binding domain of ICP8 was not completely defined, however, because a 95 kDa peptide which included these regions did not bind to or elute from ssDNA-cellulose in the same way as intact ICP8. Amino-acid residues 332-564 and 571-899 not only were associated with ssDNA-binding activity but also contain the altered amino acids of four ICP8 molecules which are deficient in DNA binding.
单纯疱疹病毒1型（HSV-1）的DNA结合蛋白ICP8在体内具有多种功能，在体外优先结合单链DNA（ssDNA）。为了确定ICP8的ssDNA结合结构域，制备并分析了肽段。将ICP8基因的部分片段克隆到转录载体pSP64中，并在体外合成RNA。在兔网织红细胞裂解物中翻译该RNA产生了29 kDa、35 kDa和30 kDa的肽段，分别代表完整ICP8（128 kDa，1196个氨基酸）的332 - 564、571 - 899和900 - 1196氨基酸残基。通过ssDNA纤维素柱色谱分析这些肽段。约55%的29 kDa肽段与ssDNA纤维素柱结合，结合的大部分肽段用1.0 M NaCl洗脱。约5%的35 kDa肽段和12%的30 kDa肽段结合并用0.3 M NaCl洗脱。因此，ICP8的三个区域与ssDNA结合活性相关。然而，ICP8的ssDNA结合结构域并未完全确定，因为包含这些区域的95 kDa肽段与完整ICP8在与ssDNA纤维素结合或洗脱的方式上不同。氨基酸残基332 - 564和571 - 899不仅与ssDNA结合活性相关，还包含四个DNA结合缺陷的ICP8分子的改变氨基酸。
