Rosenfeld P J, O'Neill E A, Wides R J, Kelly T J
Mol Cell Biol. 1987 Feb;7(2):875-86. doi: 10.1128/mcb.7.2.875-886.1987.
The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.
腺病毒DNA复制起点包含三个功能不同的序列结构域（A、B和C），它们对于DNA合成的起始至关重要。先前的研究表明，结构域B包含核因子I（NF-I）的识别位点，NF-I是一种细胞蛋白，是最佳起始所必需的。在本文报道的研究中，我们使用通过DNA识别位点亲和层析制备的高度纯化的NF-I（P. J. 罗森菲尔德和小T. J. 凯利，《生物化学杂志》261:1398 - 1408，1986）来研究病毒DNA复制起始所需的细胞蛋白。我们的数据表明，虽然NF-I对于体外高效起始至关重要，但也需要其他细胞因子。来自HeLa细胞核提取物的一个组分（BR-FT组分）被证明包含起始反应完全重建所需的所有其他细胞蛋白。通过凝胶电泳DNA结合测定对该互补组分进行分析，发现存在两种位点特异性DNA结合蛋白，ORP-A和ORP-C，它们分别识别病毒起点结构域A和C中的序列。这两种蛋白都通过DNA识别位点亲和层析进行了纯化，并且它们结合位点的边界通过DNase I足迹分析得以确定。通过测定这些蛋白对含有缺失和碱基取代突变的病毒起点的亲和力，完成了对ORP-A、NF-I和ORP-C识别序列的进一步表征。ORP-C识别腺病毒基因组第41至51个核苷酸之间的序列，对突变起点的分析表明，高效的复制起始依赖于高亲和力的ORP-C结合位点的存在。ORP-A识别位点定位于最小复制起点内病毒基因组的前12个碱基对。这些数据提供了证据，表明腺病毒DNA复制的起始涉及起点处的多种蛋白质 - DNA相互作用。
