Matson S W, Richardson C C
J Biol Chem. 1983 Nov 25;258(22):14009-16.
The gene 4 protein of bacteriophage T7 is both a primase and a helicase. In this paper, we present a detailed description of a third activity, single-stranded DNA-dependent nucleoside 5'-triphosphate hydrolysis, and show that this activity is coupled to the unidirectional translocation of the gene 4 protein on single-stranded DNA (Tabor, S., and Richardson, C.C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 205-209). The competitive inhibitor of NTP hydrolysis, beta, gamma-methylene dTTP, is also a potent inhibitor of gene 4 protein-dependent, RNA-primed DNA synthesis; inhibition is not due to a direct inhibition of T7 DNA polymerase or RNA primer synthesis. We conclude that the energy derived from the hydrolysis of NTPs by the gene 4 protein is required for translocation of the protein to primase recognition sites. Measurement of the rates of hydrolysis of NTPs using a variety of DNAs of known structure and length support the unidirectional translocation of the gene 4 protein on single-stranded DNA. Duplex DNA, RNA, and single-stranded DNA coated with single-stranded DNA-binding protein do not serve as effectors for the nucleoside triphosphatase of the gene 4 protein. Kinetic data suggest that the gene 4 protein does not remain bound to newly synthesized oligoribonucleotide primers but continues to search for other primase recognition sites. Although all the predominant naturally occurring NTPs except rCTP are hydrolyzed by the gene 4 protein, the enzyme shows specificity for dTTP with a Km of 0.4 mM. In the accompanying paper (Matson, S.W., Tabor, S., and Richardson, C.C. (1983) J. Biol. Chem. 258, 14017-14024), we show that the hydrolysis of NTPs is also required for the protein to function as a helicase in duplex regions of DNA.
噬菌体T7的基因4蛋白既是一种引发酶，也是一种解旋酶。在本文中，我们详细描述了其第三种活性，即单链DNA依赖性核苷5'-三磷酸水解，并表明这种活性与基因4蛋白在单链DNA上的单向移位相关联（泰伯，S.，和理查森，C.C.（1981年）《美国国家科学院院刊》78，205 - 209）。NTP水解的竞争性抑制剂β，γ-亚甲基dTTP，也是基因4蛋白依赖性、RNA引发的DNA合成的有效抑制剂；抑制作用并非由于对T7 DNA聚合酶或RNA引物合成的直接抑制。我们得出结论，基因4蛋白水解NTP所产生的能量是该蛋白移位至引发酶识别位点所必需的。使用各种已知结构和长度的DNA测量NTP水解速率，支持了基因4蛋白在单链DNA上的单向移位。双链DNA、RNA以及包被有单链DNA结合蛋白的单链DNA，都不能作为基因4蛋白核苷三磷酸酶的效应物。动力学数据表明，基因4蛋白不会一直结合在新合成的寡核糖核苷酸引物上，而是继续寻找其他引发酶识别位点。尽管除rCTP外，所有主要的天然存在的NTP都能被基因4蛋白水解，但该酶对dTTP表现出特异性，Km值为0.4 mM。在随附的论文中（马特森，S.W.，泰伯，S.，和理查森，C.C.（1983年）《生物化学杂志》258，14017 - 14024），我们表明，NTP水解对于该蛋白在DNA双链区域作为解旋酶发挥功能也是必需的。
