Bernstein J A, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1989 Aug 5;264(22):13066-73.
Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.
噬菌体T7 DNA复制叉处前导链和后随链的DNA合成需要基因4蛋白的解旋酶和引发酶活性。基因4蛋白由两个分子量分别为56 kDa和63 kDa的共线性多肽组成。我们之前已经证明，56 kDa的蛋白具有解旋酶活性，但缺乏引发酶活性（伯恩斯坦，J. A.，和理查森，C. C.（1988年）《美国国家科学院院刊》85，396 - 400）。现在已从T7感染细胞的提取物中纯化出63 kDa的基因4蛋白。如使用针对纯化的56 kDa蛋白的多克隆抗体进行的蛋白质免疫印迹分析所示，该制剂含有5 - 10%的56 kDa污染蛋白。63 kDa的蛋白催化依赖于DNA的dTTP水解并具有解旋酶活性；这两种比活性与针对56 kDa蛋白测定的相似。63 kDa的蛋白能高效合成序列特异性的二核苷酸、三核苷酸和四核苷酸，并刺激T7 DNA聚合酶对四核苷酸的延伸。虽然单独的56 kDa蛋白缺乏引发酶活性，但它能将63 kDa蛋白的引发酶活性提高4倍。这种刺激作用可以通过在56 kDa蛋白存在的情况下，63 kDa蛋白合成的引物量有类似增加来解释。
