Barnett J W, Reinke C M, Turk S R, Drach J C
Biochim Biophys Acta. 1984 Feb 24;781(1-2):130-42. doi: 10.1016/0167-4781(84)90131-3.
Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.
对从感染单纯疱疹病毒2型（HSV）的KB细胞中分离出的细胞核进行了检测，以评估其作为疱疹DNA聚合酶原位来源的能力。与添加了模板的纯化酶不同，在分离出的细胞核中合成的DNA约80%是病毒DNA。体外标记的DNA片段的平均大小为3.2×10⁶道尔顿。基于当细胞核在存在BrdUTP而非dTTP的情况下孵育时DNA密度的增加，16%的核苷酸是在体外反应期间添加的。对分离出的细胞核提取物中的DNA聚合酶活性进行蔗糖梯度分析表明，几乎只存在疱疹DNA聚合酶。四种脱氧核苷三磷酸（dNTP）的米氏常数（Km）浓度为0.14至0.55微摩尔。阿糖腺苷（ara - A）和阿糖胞苷（ara - C）的5'-三磷酸竞争性抑制DNA合成（抑制常数Ki分别为0.03和0.22微摩尔），但双脱氧胸苷的5'-三磷酸不抑制。α-三磷酸腺苷（αATP）也作为该反应的底物（Km = 0.014微摩尔）。我们得出结论，来自HSV感染细胞的细胞核对于详细研究疱疹病毒复制抑制剂具有显著优势。
