Dobbs L, Zhao L J, Sripad G, Padmanabhan R
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Virology. 1990 Sep;178(1):43-51. doi: 10.1016/0042-6822(90)90377-4.
Three distinct domains, A, the minimal origin, as well as B and C, the binding sites for the host nuclear factors, are required for efficient initiation of adenovirus (Ad) DNA replication at the termini. The initiation reaction was examined using partially purified nuclear extracts and various single-stranded oligomers as DNA templates. We observed that single-stranded oligomers containing Ad2 minimal origin (Ori) sequences (bp 1-18) from the I-strand of the Ad2 genome supported preterminal protein-dCMP complex formation in vitro. Using oligomers containing point mutations in the Ad2 minimal Ori sequence, six positions were identified as important to the function of the Ad2 minimal Ori sequence. Point mutations at position 7, 8, or 11 virtually abolished the ability of the oligomer to support the initiation reaction. Point mutations at position 4, 9, or 17 were found to decrease the ability of the oligomers to support the initiation reaction to 33, 67, and 58% of control, respectively. An oligomer complementary to the I-strand of the Ad2 minimal Ori was found to block initiation on minimal Ori template. A number of randomly selected nonspecific oligomers did not, in general, serve as templates for initiation with the exception of two oligomers, one of which was found to be about threefold more active than the control minimal Ori template. The biological significance of the in vitro initiation of Ad2 DNA replication on single-stranded DNA templates is discussed.
腺病毒（Ad）DNA在末端高效起始复制需要三个不同的结构域：A，即最小起始点，以及B和C，即宿主核因子的结合位点。使用部分纯化的核提取物和各种单链寡聚物作为DNA模板来检测起始反应。我们观察到，含有来自Ad2基因组I链的Ad2最小起始点（Ori）序列（碱基对1 - 18）的单链寡聚物在体外支持前末端蛋白 - dCMP复合物的形成。使用在Ad2最小Ori序列中含有点突变的寡聚物，确定了六个位置对Ad2最小Ori序列的功能很重要。第7、8或11位的点突变几乎消除了寡聚物支持起始反应的能力。发现第4、9或17位的点突变分别将寡聚物支持起始反应的能力降低至对照的33%、67%和58%。发现与Ad2最小Ori的I链互补的寡聚物可阻断在最小Ori模板上的起始反应。除了两种寡聚物外，一般来说，许多随机选择的非特异性寡聚物不能作为起始反应的模板，其中一种寡聚物的活性比对照最小Ori模板高约三倍。本文讨论了在单链DNA模板上体外起始Ad2 DNA复制的生物学意义。
