Ni T H, Zhou X, McCarty D M, Zolotukhin I, Muzyczka N
Department of Microbiology, State University of New York, Stony Brook Medical School 11794.
J Virol. 1994 Feb;68(2):1128-38. doi: 10.1128/JVI.68.2.1128-1138.1994.
The study of eukaryotic viral DNA replication in vitro has led to the identification of cellular enzymes involved in DNA replication. Adeno-associated virus (AAV) is distinct from previously reported systems in that it is believed to replicate entirely by leading-strand DNA synthesis and requires coinfection with adenovirus to establish completely permissive replication. In previous work, we demonstrated that two of the AAV nonstructural proteins, Rep78 and -68, are site-specific endonucleases and DNA helicases that are capable of resolving covalently closed AAV termini, a key step in AAV DNA replication. We have now cloned the AAV nonstructural proteins Rep78, Rep68, and Rep52 in the baculovirus expression system. Using the baculovirus-expressed proteins, we have developed an efficient in vitro AAV DNA replication system which mimics the in vivo behavior of AAV in every respect. With no-end AAV DNA as the starting substrate, the reaction required an adenovirus-infected cell extract and the presence of either Rep78 or Rep68. Rep52, as expected, did not support DNA replication. A mutant in the AAV terminal resolution site (trs) was defective for DNA replication in the in vitro assay. Little, if any, product was formed in the absence of the adenovirus-infected HeLa cell extract. In general, uninfected HeLa extracts were less efficient in supporting AAV DNA replication than adenovirus-infected extracts. Thus, the requirement for adenovirus infection in vivo was partially duplicated in vitro. The reduced ability of uninfected HeLa extracts to support complete DNA replication was not due to a defect in terminal resolution but rather to a defect in the reinitiation reaction or in elongation. Rep78 produced a characteristic monomer-dimer pattern of replicative intermediates, but surprisingly, Rep68 produced little, if any, dimer replicative form. The reaction had a significant lag (30 min) before incorporation of 32P-deoxynucleoside triphosphate could be detected in DpnI-resistant monomer replicative form and was linear for at least 4 h after the lag. The rate of incorporation in the reaction was comparable to that in the simian virus 40 in vitro system. Replication of the complete AAV DNA molecule was demonstrated by the following criteria. (i) Most of the monomer and dimer product DNAs were completely resistant to digestion with DpnI. (ii) Virtually all of the starting substrate was converted to heavy-light or heavy-heavy product DNA in the presence of bromo-dUTP when examined on CsCl density gradients.(ABSTRACT TRUNCATED AT 400 WORDS)
对真核病毒DNA体外复制的研究已促使人们鉴定出参与DNA复制的细胞酶。腺相关病毒（AAV）与先前报道的系统不同，据信它完全通过前导链DNA合成进行复制，并且需要与腺病毒共感染才能建立完全允许的复制。在先前的工作中，我们证明了AAV的两种非结构蛋白Rep78和Rep68是位点特异性内切核酸酶和DNA解旋酶，它们能够解析共价闭合的AAV末端，这是AAV DNA复制中的关键步骤。我们现在已在杆状病毒表达系统中克隆了AAV非结构蛋白Rep78、Rep68和Rep52。利用杆状病毒表达的蛋白，我们开发了一种高效的体外AAV DNA复制系统，该系统在各个方面都模拟了AAV的体内行为。以无端AAV DNA作为起始底物，该反应需要腺病毒感染的细胞提取物以及Rep78或Rep68的存在。正如预期的那样，Rep52不支持DNA复制。AAV末端分辨率位点（trs）的突变体在体外测定中DNA复制存在缺陷。在没有腺病毒感染的HeLa细胞提取物的情况下，几乎不形成产物。一般来说，未感染的HeLa提取物在支持AAV DNA复制方面比腺病毒感染的提取物效率低。因此，体内对腺病毒感染的需求在体外部分得到了重现。未感染的HeLa提取物支持完全DNA复制的能力降低不是由于末端分辨率缺陷，而是由于重新起始反应或延伸方面的缺陷。Rep78产生了特征性的复制中间体单体 - 二聚体模式，但令人惊讶的是，Rep68几乎不产生二聚体复制形式。在DpnI抗性单体复制形式中检测到32P - 脱氧核苷三磷酸掺入之前，该反应有明显的延迟（30分钟），并且在延迟后至少4小时呈线性。该反应中的掺入速率与猿猴病毒40体外系统中的相当。完整AAV DNA分子的复制通过以下标准得以证明。（i）大多数单体和二聚体产物DNA对DpnI消化完全抗性。（ii）当在CsCl密度梯度上检查时，在存在溴代 - dUTP的情况下，几乎所有起始底物都转化为重 - 轻或重 - 重产物DNA。
