Yang X M, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Mar 7;272(10):6599-606. doi: 10.1074/jbc.272.10.6599.
T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA.
T7基因5 DNA聚合酶与大肠杆菌硫氧还蛋白（其持续性因子）形成复合物，基因5蛋白特有的一段76个氨基酸的序列（第258 - 334位氨基酸残基）参与了这种相互作用。我们研究了该区域氨基酸取代对T7噬菌体生长以及聚合酶与硫氧还蛋白相互作用的影响。在基因5的突变中，我们发现用谷氨酸或丙氨酸取代赖氨酸-302产生的蛋白质不能互补缺乏基因5的T7噬菌体（T7Delta5），使其在硫氧还蛋白水平降低的大肠杆菌上生长。一个三重突变体（K300E、K302E、K304E）即使在野生型细胞中也不能支持T7Delta5的生长。这种改变后的聚合酶受硫氧还蛋白的刺激程度比野生型酶低4倍，并且聚合酶 - 硫氧还蛋白复合物的持续性降低。改变后的聚合酶的核酸外切酶活性受硫氧还蛋白刺激的程度与野生型酶不同。观察到的基因5蛋白K(300,302,304)E - 硫氧还蛋白复合物的解离常数比野生型复合物高7倍。改变后的聚合酶对双链DNA的结合亲和力也较低。
