Leegwater P A, Rombouts R F, van der Vliet P C
Laboratory for Physiological Chemistry, State University of Utrecht, The Netherlands.
Biochim Biophys Acta. 1988 Dec 20;951(2-3):403-10. doi: 10.1016/0167-4781(88)90113-3.
Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.
腺病毒DNA通过取代一条亲代链，随后复制被取代的亲代单链（互补链合成）来进行复制。取代合成已在由病毒和细胞蛋白组成的重组系统中进行，使用病毒DNA-末端蛋白复合物作为模板或含有起始位点的线性化质粒。此前，有证据表明，在体内互补链合成需要形成源自反向末端重复序列杂交的柄状结构。为了研究体外互补链合成的条件，我们构建了一种人工柄状分子，它包含一个双链反向末端重复（ITR）区域和一个源自Ad2左右末端XmaI片段的单链环。这样的分子似乎是一种有效的模板，并且可以通过与双链DNA相同的蛋白质引发机制起始，利用前体末端蛋白。两种类型模板的效率相当。与双链分子的复制一样，柄状复制的起始受到核因子I和III的刺激，这两种蛋白与ITR的特定双链区域结合。腺病毒DNA结合蛋白是必需的，并且该蛋白具有DNA结合特性的39 kDa C末端结构域足以发挥其功能。这些结果支持了柄状结构形成是被取代链复制所必需的这一假说。
