Snyder R O, Samulski R J, Muzyczka N
Department of Microbiology, SUNY Stony Brook Medical School 11794.
Cell. 1990 Jan 12;60(1):105-13. doi: 10.1016/0092-8674(90)90720-y.
We have developed an assay for a key step in the replication of adeno-associated virus (AAV) DNA. We demonstrate the covalently joined ends of linear AAV DNA can be resolved in vitro to the open duplex configuration. Only extracts prepared from human cells that have been infected with both adenovirus and AAV are capable of carrying out the reaction. The reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the AAV terminal palindrome. During resolution the orientation of the terminal palindrome is inverted, and the 3' viral strand is extended by DNA synthesis. The size of the newly synthesized 3' strand is nearly identical to that found in viral particles. These observations provide direct biochemical evidence for an essential step in the model for AAV DNA replication.
我们开发了一种针对腺相关病毒（AAV）DNA复制关键步骤的检测方法。我们证明线性AAV DNA的共价连接末端在体外可解析为开放双链体构型。只有从同时感染了腺病毒和AAV的人类细胞中制备的提取物才能进行该反应。该反应由在AAV末端回文序列末端附近的末端分辨率位点进行的位点特异性和链特异性内切核酸酶切割引发。在分辨率过程中，末端回文序列的方向会反转，并且3'病毒链通过DNA合成进行延伸。新合成的3'链的大小与病毒颗粒中的大小几乎相同。这些观察结果为AAV DNA复制模型中的一个关键步骤提供了直接的生化证据。
