Hong G, Ward P, Berns K I
Department of Microbiology, Cornell University Medical College, New York, NY 10021.
Proc Natl Acad Sci U S A. 1992 May 15;89(10):4673-7. doi: 10.1073/pnas.89.10.4673.
An in vitro assay for adeno-associated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the plasmid by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I digestion. The following results were obtained. (i) Only extracts of cells coinfected with AAV and adenovirus replicated the excised insert. (ii) Density label experiments showed semiconservative replication. (iii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.
已开发出一种用于腺相关病毒（AAV）DNA复制的体外检测方法。底物是一种在pBR322中含有AAV DNA双链形式的质粒。通过未感染细胞的提取物将AAV插入片段从质粒中切除或拯救出来。通过产生对Dpn I消化有抗性的全长切除的AAV DNA来检测复制情况。得到了以下结果。（i）只有与AAV和腺病毒共感染的细胞提取物能复制切除的插入片段。（ii）密度标记实验显示为半保留复制。（iii）只有切除的AAV插入片段被复制；pBR322序列未被复制。（iv）复制依赖于AAV末端重复序列的存在。（v）如果从AAV插入片段的两端删除末端55个碱基，则不会发生拯救：发生了复制，并且AAV和pBR322序列都被复制。我们得出结论，AAV末端重复序列对于DNA复制至关重要，但在某些条件下可能会使用一种不涉及发夹引物的起始机制。
