RB69 DNA polymerase structure, kinetics, and fidelity.
RB69 DNA polymerase structure, kinetics, and fidelity.
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RB69 DNA 聚合酶结构、动力学和保真度。
DOI：
10.1021/bi4014215
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发表时间：
2014-05-06
期刊：
影响因子：
2.9
通讯作者：
Konigsberg, William H.
中科院分区：
文献类型：
--
作者：
Xia, Shuangluo;Konigsberg, William H.
本文将总结我们的RB 69 DNA聚合酶（RB 69 pol）的结构和动力学的研究，以及选择的野生型酶的变种，进行了更深入的了解精致的高保真度的B家庭复制型DNA聚合酶。我们讨论了如何可以使用的各种RB 69 pol三元复合物的结构，以合理化从预稳态动力学分析得到的结果。我们的主要研究结果可归纳如下。(i)碱基间氢键相互作用可以将催化效率提高5000倍;同时，碱基选择性不仅仅取决于引入的dNTP和模板碱基之间的氢键数量。(ii)在RB 69 pol三元复合物中引物链的位置n - 1和n - 2以及模板链的位置n - 1处的小沟氢键相互作用对于有效的引物延伸和碱基选择性是必不可少的。(iii)传入的dNTP、倒数第二个碱基对和传入的dNTP周围的水合壳之间的部分电荷相互作用调节核苷酸插入效率和碱基选择性。(iv)在新生碱基对结合口袋（NBP）中具有氨基酸侧链的错配的传入dNTP和模板碱基之间的空间冲突以及传入dNTP和模板碱基之间的弱相互作用和大间隙是不正确的dNTP被野生型RB 69 pol如此低效地掺入的一些原因。此外，我们开发了tC°-tC硝基Förster共振能量转移测定法，以监测聚合酶和核酸外切酶亚结构域之间引物末端的分配。
This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n – 1 and n – 2 of the primer strand and position n – 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°–tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.
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影响因子：
15
作者：
Betz K;Malyshev DA;Lavergne T;Welte W;Diederichs K;Romesberg FE;Marx A
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影响因子：
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DOI：
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发表时间：
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影响因子：
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作者：
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