Srivastava D K, Evans R K, Kumar A, Beard W A, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068, USA.
Biochemistry. 1996 Mar 26;35(12):3728-34. doi: 10.1021/bi952632h.
Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N-terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido-dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta-pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263-280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol.
大鼠DNA聚合酶β（β-pol）经轻度蛋白水解产生一个N端8 kDa结构域和一个C端31 kDa结构域；通过进一步的蛋白水解，31 kDa结构域被降解为6 kDa和27 kDa的片段[库马尔，A.，威登，S.G.，威廉姆斯，K.R.，凯达尔，P.，卡佩尔，R.L.，&威尔逊，S.H.（1990年）《生物化学杂志》265，2124 - 2131]。在本研究中，我们发现用胰蛋白酶对β-pol的27 kDa片段进行更剧烈的消化会产生10 kDa和12 kDa的亚结构域。因此，大鼠β-pol有四个不同的蛋白水解片段，分别为8 kDa、6 kDa、10 kDa和12 kDa，从N端延伸至C端。为了确定dNTP结合位点的位置，在有或没有竞争性dNTP（dATP）存在的情况下，用8 - 叠氮基 - ATP或5 - 叠氮基 - dUTP对完整的β-pol进行光亲和标记。对标记的酶进行可控的蛋白水解，然后分离并测序得到的标记肽段。与dATP的竞争表明，溶液中β-pol的三个区域结合形成dNTP结合口袋，如下所示：8 kDa结构域的4 - 40位残基；10 kDa亚结构域的142 - 206位残基；以及12 kDa亚结构域（α-螺旋M和N）的263 - 280位残基。根据最近与大鼠β-pol结合的dATP的晶体结构对这些结果进行了讨论。
