de Vries E, van Driel W, Tromp M, van Boom J, van der Vliet P C
Nucleic Acids Res. 1985 Jul 11;13(13):4935-52. doi: 10.1093/nar/13.13.4935.
The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I.
在一个由含有腺病毒2型（Ad2）起源区域的克隆DNA片段作为模板的体外重组系统中，研究了高效腺病毒DNA复制的模板要求。复制可通过核因子I增强，核因子I是一种能特异性结合Ad2起源区域的细胞蛋白。结果表明，这种刺激强烈依赖于腺病毒DNA结合蛋白的浓度。我们使用合成寡核苷酸构建了在核因子I结合区域存在碱基替换的质粒。足迹分析和竞争滤膜结合研究表明，该区域三个保守核苷酸小片段中的两个参与了核因子I的结合。这两个片段外侧简并区域的碱基组成可影响结合亲和力。对这些突变体进行的体外起始和DNA链延伸实验表明，核因子I与Ad2起源区域的结合是刺激所必需的。然而，仅仅结合并不总是足够的，因为一个仅轻微干扰结合的突变在核因子I刺激DNA复制时受到严重损害。
