Variation in Mutation Rates Caused by RB69pol Fidelity Mutants Can Be Rationalized on the Basis of Their Kinetic Behavior and Crystal Structures
Variation in Mutation Rates Caused by RB69pol Fidelity Mutants Can Be Rationalized on the Basis of Their Kinetic Behavior and Crystal Structures
复制标题
DOI：
10.1016/j.jmb.2010.12.033
复制
发表时间：
2011-03-04
影响因子：
5.6
通讯作者：
Konigsberg, William
中科院分区：
文献类型：
--
作者：
Xia, Shuangluo;Wang, Mina;Konigsberg, William
我们之前观察到，在RB69 DNA聚合酶（RB69pol）的新生碱基对结合袋中，Ala或Gly逐步取代氨基酸残基，扩大了该袋中的空间，导致错误结合的逐渐增加。然而，类似RB69pol新生碱基对结合口袋突变体的体内结果显示，通过T4噬菌体rI正向实验和rII逆转实验确定的RB69pol S565G/Y567A双突变体的突变率明显低于Y567A单突变体，这与我们的预测相反。为了研究这一意想不到的结果的原因，我们确定了相关三元配合物的预稳态动力学参数和晶体结构。我们发现，S565G/Y567A突变体通常比Y567A突变体具有更高的碱基选择性，并且dNMP插入、3'端核苷酸残基切除和引物延伸到错配之外的动力学参数不仅在这两个突变体之间存在差异，而且在T4 rI互补链中两个高度可变的序列之间也存在差异。通过比较正确和不正确输入dNTPs的两种突变体的晶体结构，可以深入了解S565G/Y567A双突变体保真度的意外提高。综上所述，动力学和结构结果为整合和解释体内和体外观察提供了基础。(C) 2011 Elsevier Ltd.版权所有。
We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3'-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations. (C) 2011 Elsevier Ltd. All rights reserved.