Livingston D M, Hinkle D C, Richardson C C
J Biol Chem. 1975 Jan 25;250(2):461-9.
DNA polymerase III has been purified 4,500-fold from the Escherichis coli mutant, HMS83, which lacks DNA polymerases I and II. When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered. Polyacrylamide gel electrophoresis under denaturing conditions produces two protein bands with molecular weights of 140,000 and 40,000. The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A. Taken together these tow parameters indicate a native molecular weight of 180,000. Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand. The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand. Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single-stranded regions. The most purified enzyme preparation is devoid of endonuclease activities. In addition to the two exonuclease activities described in the accompanying paper, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates. DNA polymerase III has also been isolated from wild type E. coli containing the other two known DNA polymerases. Futhermore, the enzyme purified from three different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III (Gefter, M., Hirota, Y., Kornberb, T., Wechsler, J., and Barnoux, C. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 3150-3153).
已从缺乏DNA聚合酶I和II的大肠杆菌突变体HMS83中纯化出4500倍的DNA聚合酶III。当进行圆盘凝胶电泳时，纯化程度最高的级分显示出一条主要蛋白质带，可从中回收酶活性。在变性条件下进行聚丙烯酰胺凝胶电泳会产生两条分子量分别为140,000和40,000的蛋白质带。该酶的沉降系数为7.0 S，斯托克斯半径为62 Å。这两个参数综合起来表明其天然分子量为180,000。当提供DNA模板和互补引物链时，纯化的DNA聚合酶III催化核苷酸聚合成DNA。新合成的DNA共价连接到引物链的3'末端。由于聚合程度仅为10至100个核苷酸，因此最佳底物是具有小单链区域的天然DNA分子。纯化程度最高的酶制剂没有内切酶活性。除了随附论文中描述的两种外切酶活性外，纯化的聚合酶III还催化焦磷酸解以及焦磷酸与脱氧核苷三磷酸的交换。DNA聚合酶III也已从含有其他两种已知DNA聚合酶的野生型大肠杆菌中分离出来。此外，从三个不同的polC突变体中纯化的酶表现出改变的聚合酶III活性，证实polC是DNA聚合酶III的结构基因（盖弗特，M.，广田，Y.，科恩伯格，T.，韦克斯勒，J.，和巴努克斯，C.（1971年）美国国家科学院院刊68，3150 - 3153）。
