文章基本信息

标题：Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation
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作者：Dana T. Minnick ; Lixing Liu ; Nigel D. F. Grindley 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：3
页码：1194-1199
DOI：10.1073/pnas.032457899
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.