Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.
Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.
复制标题
腺相关病毒 DNA 体外复制：麦芽糖结合蛋白/Rep 68 融合蛋白激活。
DOI：
10.1128/jvi.68.9.6029-6037.1994
复制
发表时间：
1994
影响因子：
5.4
通讯作者：
Berns,KI
中科院分区：
文献类型：
--
作者：
Ward,P;Urcelay,E;Kotin,R;Safer,B;Berns,KI
腺相关病毒（AAV）非结构蛋白Rep 68是病毒DNA复制所必需的。在体外实验中，将Rep 68添加到未感染的HeLa细胞的提取物中，支持AAV DNA复制。在本文中，我们报告了在大肠杆菌中表达的麦芽糖结合蛋白与Rep 68融合时的复制过程的表征。当模板是线性双链AAV DNA或含有完整AAV DNA的质粒构建时，可以观察到复制。以重组质粒构建物为模板时，pBR322 DNA和AAV DNA均有复制；然而，线性pBR322 DNA没有被复制。当质粒作为模板时，复制似乎在完整的质粒上启动，并导致AAV序列与载体的序列分离，这一过程被称为拯救。没有证据表明可以在救援产物上开始复制。Rep 68可以从AAV DNA的3'端产生124个核苷酸的位点特异性缺口；尼克的站点被称为终端解决站点。我们的数据与起始发生在末端分解位点并向3′端移动最为一致。当模板是质粒构建时，要么延伸继续通过连接处进入pBR322序列，要么将新合成的序列夹住，交换模板链，复制AAV DNA。在4小时内呈线性复制，在此期间70%的最大合成发生。另一个发现是Rep融合可以在不合成DNA的情况下将AAV二聚体长度的双链中间体分解为单体双链。
The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.