Cheng N, Merrill B M, Painter G R, Frick L W, Furman P A
Division of Virology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Biochemistry. 1993 Aug 3;32(30):7630-4. doi: 10.1021/bi00081a005.
We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.
我们利用紫外线诱导的[甲基 - ³H]胸苷三磷酸（dTTP）交联来鉴定异二聚体HIV - 1逆转录酶（RT）上的核苷酸结合位点。通过照射含有[甲基 - ³H]dTTP和纯化的重组RT的溶液10分钟，使RT衍生化。dTTP与RT之间的紫外线诱导交联反应在长达10分钟的紫外线照射时间内呈线性，并且先前已经确定在90微摩尔时dTTP交联达到最大值的一半[程，N.，佩因特，G. R.，& 弗曼，P. A.（1991年）生物化学与生物物理研究通讯174，785 - 789]。在这些反应条件下，只有66 kDa/51 kDa RT异二聚体的66 kDa亚基被dTTP标记。用胰蛋白酶和天冬氨酸蛋白酶Asp - N对[甲基 - ³H]dTTP标记的RT进行酶解，肽段通过反相高效液相色谱法纯化。对与[甲基 - ³H]dTTP共价连接的肽段进行氨基酸序列分析。测序数据将RT的核苷酸结合位点定位到与抗病毒药物抗性相关的几个突变位点附近的赖氨酸 - 73处。由于大多数有效的抗艾滋病化合物是RT的抑制剂，关于其脱氧核苷三磷酸（dNTP）结合位点的信息可能有助于理解核苷类似物如齐多夫定（AZT）、双脱氧肌苷（ddI）和双脱氧胞苷（ddC）抗病毒活性的基础。这些信息对于更合理地设计抗HIV药物也可能是有用的。
