Snyder R O, Im D S, Muzyczka N
Department of Microbiology, State University of New York, Stony Brook Medical School 11794-8621.
J Virol. 1990 Dec;64(12):6204-13. doi: 10.1128/JVI.64.12.6204-6213.1990.
We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.
我们已经证明，当线性腺相关病毒（AAV）DNA的共价连接末端在体外被解析时，病毒编码的Rep蛋白会共价连接到DNA的5'末端。共价键存在于AAV Rep蛋白的一个酪氨酸残基与AAV基因组中一个胸苷残基的5'磷酸之间。只有由AAV p5启动子编码的Rep蛋白Rep68能够共价连接到AAV基因组的末端；由p19启动子编码的Rep蛋白则不能。我们还研究了完全体外解析反应的一些要求。抑制剂研究表明，末端解析需要DNA聚合酶δ、ATP和脱氧核糖核苷三磷酸，但不需要其余的核糖核苷三磷酸、DNA聚合酶α、RNA聚合酶II或拓扑异构酶I和II。最后，将纯化的AAV Rep68添加到未感染的HeLa细胞的粗提胞质溶胶中，足以进行解析。这表明末端解析依赖于宿主酶和病毒编码的p5 Rep蛋白。
