Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro.
Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro.
复制标题
腺病毒 H5ts125 和 H5ts107 DNA 结合蛋白对体外 DNA 复制的影响。
DOI：
10.1016/0042-6822(83)90354-9
复制
发表时间：
1983
期刊：
影响因子：
3.7
通讯作者：
Horwitz,MS
中科院分区：
文献类型：
--
作者：
Friefeld,BR;Krevolin,MD;Horwitz,MS
体外腺病毒 (Ad) DNA 合成的遗传和生化研究表明，Ad DNA 结合蛋白 (Ad DBP) 对于 Ad DNA 合成的起始不是必需的，但对于链延长是必需的。 DBP 可以将第 26 个脱氧核苷酸的早期延伸增强大约两到四倍，随着链延伸的进一步进行，DBP 是绝对必需的。 Ad DNA 合成在需要 Ad DNA 在每个 5' 末端共价连接至蛋白质 (Ad DNA-pro)、Ad 感染的细胞质的各个部分以及未感染的 Hela 细胞核提取物的系统中进行测定。 Ad DNA 复制的起始通过 80,000 道尔顿前末端蛋白 (pTP) 和 5' dCMP 之间形成共价复合物来测量。来自 Twots 突变体 H5ts125 和 H5ts107 的 DNA 结合蛋白已被纯化，并显示在依赖于纯化蛋白的体外延伸系统中，在 30° 下有功能，但在 38° 下无活性。 72K 野生型 Ad2 DBP 的糜蛋白酶裂解产生 34K 羧基末端片段，该片段在 Ad DNA 体外延伸中保留了全部活性。
Genetic and biochemical studies of adenovirus (Ad) DNA synthesisin vitrodemonstrate that the Ad DNA binding protein (Ad DBP) is not necessary for the initiation of Ad DNA synthesis but is required for chain elongation. The DBP, which enhances early elongation to the 26th deoxynucleotide by approximately two- to fourfold, is absolutely required as chain elongation proceeds further. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5′ terminus to a protein (Ad DNA-pro), various fractions of Ad-infected cytoplasm, and an extract of uninfected Hela nuclei. Initiation of Ad DNA replication was measured by the formation of a covalent complex between the 80,000 dalton preterminal protein (pTP) and 5′ dCMP. DNA binding proteins from twotsmutants, H5ts125 and H5ts107, have been purified and shown to be functional at 30° but inactive at 38° in anin vitroelongation system dependent on purified proteins. Chymotryptic cleavage of the 72K wild-type Ad2 DBP produces a 34K carboxyl terminal fragment which retains full activity in thein vitroelongation of Ad DNA.