Influence of DNA structure on DNA polymerase β active site function -: Extension of mutagenic DNA intermediates
Influence of DNA structure on DNA polymerase β active site function -: Extension of mutagenic DNA intermediates
复制标题
DOI：
10.1074/jbc.m404016200
复制
发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Beard, WA;Shock, DD;Wilson, SH
在DNA聚合酶（pol）β的三元底物复合物中，新生碱基对（模板和引入的核苷酸）夹在双链体DNA末端和聚合酶之间。为了探测dNTP结合口袋中的分子相互作用，我们分析了野生型pol β在修饰的DNA底物上的动力学行为，所述修饰的DNA底物改变DNA末端的结构并代表诱变中间体。对DNA底物进行修饰，以1）改变双链体末端的序列（匹配和错配），2）在新生碱基对附近引入脱碱基位点，3）在引物或模板链中插入额外的碱基以模拟移码中间体。结果表明，核苷酸插入效率（k（cat）/K-m，dGTP-dC）高度依赖于匹配（即Watson-Crick碱基对）DNA末端的序列同一性（模板/引物，G/C类似于A/T > T/A类似于C/G）。引物末端的错配强烈降低正确的核苷酸插入效率，但不影响DNA结合亲和力。过渡中间体通常比颠换更容易延伸。大多数错配的引物末端降低了插入率和引入核苷酸的结合亲和力。相比之下，在双链体DNA末端处的高嘌呤错配的催化效率的损失完全是由于不能插入引入的核苷酸，因为K-d（dGTP）不受影响。双链体末端中和周围的非碱性位点和额外的核苷酸降低催化效率，并且当位于引物链中时比模板链中的等同位置对新生碱基对结合口袋更有害。
In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus ( matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K-m, dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C similar to A/T > T/A similar to C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K-d(dGTP) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.