Prasad R, Kumar A, Widen S G, Casas-Finet J R, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-0851.
J Biol Chem. 1993 Oct 25;268(30):22746-55.
Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe.
大鼠DNA聚合酶β（β-pol）是一种39 kDa的单体蛋白，由两个结构和功能不同的结构域组成。8 kDa的NH2末端结构域结合单链（ss）DNA，而31 kDa的COOH末端结构域则不结合。为了便于研究β-pol的ssDNA结合结构-功能关系，我们在大肠杆菌中过表达了8 kDa结构域，并将重组蛋白纯化至同质。发现重组8 kDa结构域的单链核酸结合与先前报道的通过完整β-pol蛋白水解制备的8 kDa片段相似（库马尔，A.，威登，S.G.，威廉姆斯，K.R.，凯达尔，P.，卡佩尔，R.L.，和威尔逊，S.H.（1990b）《生物化学杂志》265，2124-2131；卡萨-菲内特，J.R.，库马尔，A.，莫里斯，G.，威尔逊，S.H.，和卡佩尔，R.L.（1991）《生物化学杂志》266，19618-19625）。通过光化学交联到[32P] p(dT)16来检测重组8 kDa结构域的DNA结合口袋内或附近的残基。交联定位在一个跨越28至35位残基的胰蛋白酶片段和一个跨越27至58位残基的V8蛋白酶片段上。对各种[32P]p(dT)16标记蛋白的序列分析表明，Ser30和His34通过与p(dT)16交联而被修饰。因此，β-pol的ssDNA结合结构域的这些残基似乎与这种核酸探针紧密接触。
