MAMMALIAN DNA POLYMERASE-BETA - CHARACTERIZATION OF A 16-KDA TRANSDOMAIN FRAGMENT CONTAINING THE NUCLEIC ACID-BINDING ACTIVITIES OF THE NATIVE ENZYME
MAMMALIAN DNA POLYMERASE-BETA - CHARACTERIZATION OF A 16-KDA TRANSDOMAIN FRAGMENT CONTAINING THE NUCLEIC ACID-BINDING ACTIVITIES OF THE NATIVE ENZYME
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DOI：
10.1021/bi00157a014
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发表时间：
1992-10-27
期刊：
影响因子：
2.9
通讯作者：
WILSON, SH
中科院分区：
文献类型：
--
作者：
CASASFINET, JR;KUMAR, A;WILSON, SH
39-kDa DNA 聚合酶 β (beta-Pol) 分子可以通过温和的蛋白水解轻松转化为两个组成结构域；这些结构域以 8-kDa N 端片段和 31-kDa C 端片段表示 [Kumar 等人，2015]。 (1990a) J.Biol。化学。 265、2124-2131]。完整的 beta-Pol 是一种序列非特异性核酸相互作用蛋白，可结合双链 (ds) 和单链 (ss) 多核苷酸。这两种活性似乎是由酶的不同部分贡献的，因为 31-kDa 结构域结合 ds DNA，但不结合 ss DNA，相反，8-kDa 结构域结合 ss DNA，但不结合 ds DNA [Casas-Finet 等人，2017]。 (1991) J.Biol。化学。 266、19618-19625]。用胰凝乳蛋白酶截断 N 末端的 31 kDa 结构域，产生 27 kDa 片段（残基 140-334），消除了所有 DNA 结合活性。这表明31-kDa结构域的双链DNA结合能力可能在31-kDa结构域的N-末端片段中携带。我们使用 CNBr 制备了一个 16 kDa 片段（残基 18-154），该片段跨越 8 kDa 结构域的 ss DNA 结合区以及 31 kDa 结构域的 N 端部分。发现纯化的 16-kDa 片段具有单链和双链多核苷酸结合能力。这些活性的热力学结合特性与完整酶的热力学结合特性相似。结果表明，β-Pol 的所有核酸结合能力均位于前 154 个残基中，并允许将单链和双链结合能力分别定位到该蛋白质区域内的连续片段。
The 39-kDa DNA polymerase beta (beta-Pol) molecule can be readily converted into two constituent domains by mild proteolysis; these domains are represented in an 8-kDa N-terminal fragment and a 31-kDa C-terminal fragment [Kumar et al. (1990a) J. Biol. Chem. 265, 2124-2131]. Intact beta-Pol is a sequence-nonspecific nucleic acid-interactive protein that binds both double-stranded (ds) and single-stranded (ss) polynucleotides. These two activities appear to be contributed by separate portions of the enzyme, since the 31-kDa domain binds ds DNA but not ss DNA, and conversely, the 8-kDa domain binds ss DNA but not ds DNA [Casas-Finet et al. (1991) J. Biol. Chem. 266, 19618-19625]. Truncation of the 31-kDa domain at the N-terminus with chymotrypsin, to produce a 27-kDa fragment (residues 140-334), eliminated all DNA-binding activity. This suggested that the ds DNA-binding capacity of the 31-kDa domain may be carried in the N-terminal segment of the 31-kDa domain. We used CNBr to prepare a 16-kDa fragment (residues 18-154) that spans the ss DNA-binding region of the 8-kDa domain along with the N-terminal portion of the 31-kDa domain. The purified 16-kDa fragment was found to have both ss and ds polynucleotide-binding capacity. Thermodynamic binding properties for these activities are similar to those of the intact enzyme. The results indicate that all of the nucleic acid-binding capacity of beta-Pol is carried in the first 154 residues and permit localization of the ss- and ds-binding capacities, respectively, to contiguous segments within this region of the protein.