Functional evidence for a small and rigid active site in a high fidelity DNA polymerase - Probing T7 DNA polymerase with variably sized base pairs
Functional evidence for a small and rigid active site in a high fidelity DNA polymerase - Probing T7 DNA polymerase with variably sized base pairs
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DOI：
10.1074/jbc.m510744200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Kool, ET
中科院分区：
文献类型：
--
作者：
Kim, TW;Brieba, LG;Kool, ET
关于复制型DNA聚合酶高保真起源的假说最近集中在几何或空间效应在这种选择性中的重要性上。在这里，我们报告了一个系统的研究，在T7 DNA聚合酶（pol），复制酶的噬菌体T7的碱基对大小的影响。我们通过使用一系列大小逐渐增加的非极性胸苷形状模拟物，以非常小的增量（0.25埃）改变碱基对大小。在与硫氧还蛋白的1：1复合物中评价5A7A核酸外切酶缺陷突变体的稳态动力学。对于T7 pol，我们研究了与模板中的大尺寸T类似物相对的天然核苷酸的插入，并且相反地，对于大尺寸dTTP类似物与天然模板碱基相对。该酶对特定的碱基对大小显示出极高的选择性，当超过接近天然碱基对大小的最佳大小时，增加0.4埃，效率下降多达280倍。该酶也强烈拒绝比最佳值小0.3埃的对。T7 DNA pol的大小偏好一般较小，空间排斥大于DNA pol I Klenow片段，与前者的高保真度相关。不同的活性部位的大小和刚度的假设影响进行了讨论。数据提供直接支持的概念，即活性位点的紧密性是复制型聚合酶的高保真度的主要决定因素，并且刚性较低（较松）和较大的活性位点可以导致较低的保真度。
Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 angstrom) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1: 1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 angstrom beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 angstrom. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid ( looser) and larger active site can lead to lower fidelity.