Bernstein J A, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1988 Oct 15;263(29):14891-9.
Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of a 56- and a 63-kDa species that provides primase and helicase activities for T7 DNA replication. The 56-kDa species has been purified 1800-fold from Escherichia coli cells containing a plasmid that encodes this gene 4 protein. The purified 56-kDa protein is homogeneous, as determined by denaturing gel electrophoresis, and is monomeric in its native form, as indicated by gel filtration. The binding of the 56-kDa protein to single-stranded DNA is stimulated by nucleoside 5'-triphosphates, as is the case for a mixture of the two molecular weight species. In the presence of DNA, the 56-kDa protein preferentially hydrolyzes dTTP (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 396-400). Since nucleoside 5'-triphosphatase activity is necessary for both helicase activity and for translocation of gene 4 protein to primase recognition sites, we have characterized this activity using the 56-kDa protein alone. In the DNA-dependent hydrolysis reaction, the enzyme displays a Km of 10 mM for dTTP, and a Vmax of 2.9 x 10(-5) M/min/mg of protein (at 2.5 micrograms/ml). There is little cooperativity with respect to dTTP binding (Hill coefficient = 1.1) except in the presence of ribonucleoside 5'-triphosphate, an inhibitor of dTTP hydrolysis (Hill coefficient greater than 1.5). The apparent KD for single-stranded circular DNA is 0.2 microM. The active species in dTTP hydrolysis is an oligomer of at least two subunits, as indicated by the effect of enzyme concentration upon the rate of DNA-dependent hydrolysis. The 56-kDa protein also catalyzes DNA-independent hydrolysis of dTTP with a Km of 0.11 mM and a Vmax of 1.3 x 10(-7) M/min/mg of protein (at 8 micrograms/ml). The active species in DNA-independent dTTP hydrolysis is also an oligomer.
从噬菌体感染的细胞中纯化得到的噬菌体T7基因4蛋白，由56 kDa和63 kDa两种蛋白组成的混合物，为T7 DNA复制提供引发酶和解旋酶活性。已从含有编码该基因4蛋白的质粒的大肠杆菌细胞中，将56 kDa的蛋白纯化了1800倍。经变性凝胶电泳测定，纯化后的56 kDa蛋白是均一的，凝胶过滤表明其天然形式为单体。56 kDa蛋白与单链DNA的结合受到核苷5'-三磷酸的刺激，两种分子量蛋白的混合物也是如此。在有DNA存在的情况下，56 kDa蛋白优先水解dTTP（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）。由于核苷5'-三磷酸酶活性对于解旋酶活性以及基因4蛋白向引发酶识别位点的转位都是必需的，我们仅使用56 kDa蛋白对该活性进行了表征。在依赖DNA的水解反应中，该酶对dTTP的Km为10 mM，Vmax为2.9×10⁻⁵ M/分钟/毫克蛋白（在2.5微克/毫升时）。除了在核糖核苷5'-三磷酸（dTTP水解的抑制剂）存在的情况下，dTTP结合几乎没有协同性（希尔系数 = 1.1）（希尔系数大于1.5）。单链环状DNA的表观解离常数KD为0.2 microM。如酶浓度对依赖DNA的水解速率的影响所示，dTTP水解中的活性物种是至少两个亚基的寡聚体。56 kDa蛋白还催化不依赖DNA的dTTP水解，Km为0.11 mM，Vmax为1.3×10⁻⁷ M/分钟/毫克蛋白（在8微克/毫升时）。不依赖DNA的dTTP水解中的活性物种也是寡聚体。
