A new paradigm for DNA polymerase specificity
A new paradigm for DNA polymerase specificity
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DOI：
10.1021/bi060993z
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发表时间：
2006-08-15
期刊：
影响因子：
2.9
通讯作者：
Johnson, Kenneth A.
中科院分区：
文献类型：
--
作者：
Tsai, Yu-Chih;Johnson, Kenneth A.
我们证明 T7 DNA 聚合酶以三种不同的结构状态存在，如附着在识别（指）结构域上的构象敏感荧光团所报告的那样。由正确的核苷酸诱导的构象变化使底物进行正向反应，并且构象变化的缓慢逆转消除了化学步骤的速率对酶特异性的任何贡献。通过从三元 E、DNA、脱氧核苷三磷酸复合物中快速释放错配核苷酸，并通过使用底物结合能主动错配催化残基以降低错掺率，增强了对错配的辨别力。我们改进的酶选择性模型扩展了传统的热力学形式，包括底物诱导的结构排列或催化残基的错位作为自由能曲线的第三个维度，以及底物解离速率作为关键的动力学参数。
We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the rate of the chemistry step from any contribution toward enzyme specificity. Discrimination against mismatches is enhanced by the rapid release of mismatched nucleotides from the ternary E, DNA, deoxynucleoside triphosphate complex and by the use of substrate-binding energy to actively misalign catalytic residues to reduce the rate of misincorporation. Our refined model for enzyme selectivity extends traditional thermodynamic formalism by including substrate-induced structural alignment or misalignment of catalytic residues as a third dimension on the free-energy profile and by including the rate of substrate dissociation as a key kinetic parameter.