Yang X, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Sep 27;271(39):24207-12. doi: 10.1074/jbc.271.39.24207.
The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.
噬菌体T7编码的80 kDa基因5蛋白与大肠杆菌DNA聚合酶I的大片段（克列诺片段）具有显著的氨基酸同源性。与克列诺片段一样，T7基因5蛋白同时具有DNA聚合酶活性和3'至5'核酸外切酶活性。然而，与克列诺片段不同的是，T7基因5蛋白与大肠杆菌硫氧还蛋白紧密结合形成一种复合物，该复合物具有很高的核苷酸聚合持续合成能力。为了确定基因5蛋白中负责聚合、水解以及与硫氧还蛋白结合的结构域，我们分析了基因5蛋白的蛋白水解片段。在一个蛋白酶敏感区域（第250 - 300位氨基酸残基）内切割该蛋白，产生了两种分子量的肽段，分别为32 - 37 kDa和43 - 51 kDa，分别来源于N端和C端区域。DNA聚合酶活性存在于C端片段中，核酸外切酶活性存在于N端片段中。硫氧还蛋白可刺激C端片段的DNA聚合酶活性。所有片段均能与DNA结合。除了确定聚合酶和核酸外切酶结构域，蛋白酶敏感区域似乎还与大肠杆菌硫氧还蛋白相互作用。硫氧还蛋白可保护该区域不被蛋白酶水解，该区域的改变会降低硫氧还蛋白刺激聚合酶活性的能力。
