Kumar A, Abbotts J, Karawya E M, Wilson S H
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1990 Aug 7;29(31):7156-9. doi: 10.1021/bi00483a002.
Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.
大鼠DNA聚合酶β（β-pol）是一种39 kDa的蛋白质，由两个紧密折叠的结构域组成，即8 kDa的N端结构域和31 kDa的C端结构域，通过一个短的蛋白酶敏感区域相连。8 kDa结构域有助于完整蛋白质与模板结合，我们现在报道31 kDa的C端结构域具有催化活性。我们的结果表明，该结构域作为纯化的蛋白水解片段，在适当条件下进行DNA合成，但其催化常数较低，引物延伸特性与完整酶不同。对31 kDa催化结构域片段进行蛋白水解截短，从NH2末端去除一个60个残基的片段，导致活性几乎完全丧失，表明该片段的重要性。总体而言，这些结果表明β-pol的结构域分别具有不同的功能作用，即模板结合和核苷酸转移酶活性；然而，完整蛋白质在每种功能上比分离的单个结构域片段更具活性。
