Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase β.
Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase β.
复制标题
DOI：
10.1021/acs.biochem.0c00847
复制
发表时间：
2021-02-09
期刊：
影响因子：
2.9
通讯作者：
Freudenthal BD
中科院分区：
文献类型：
--
作者：
Varela FA;Freudenthal BD
DNA聚合酶通过以核苷三磷酸为底物合成新的核苷酸聚合物，在基因组DNA的维持和复制中起着至关重要的作用。脱氧核苷三磷酸盐(DNTPs)是DNA聚合酶的典型底物，然而，一些细菌聚合酶已被证明插入脱氧核苷二磷酸盐(DNDP)，它缺乏第三个磷酸基团，即γ-磷酸。真核聚合酶是否能有效地结合dNDP还没有被研究，关于dNTP的γ-磷酸所起的化学或结构作用还不清楚。使用哺乳动物模型聚合酶(POL)β，我们利用动力学和结晶学方法研究了POLβ如何结合缺乏γ-磷酸[脱氧鸟苷二磷酸]的底物。使用单周转动力学，我们确定了通过POLβ跨模板DC的dGDP插入与dGTP插入相比显著受损。我们发现最显著的损害是表观插入率(KPOL)，与dGTP插入组相比，表观插入率(KPOL)降低了32000倍。X射线晶体结构显示dGDP和dGTP的酶-底物接触相似。这些发现表明，由于聚合酶化学的损伤，dGDP的插入效率大大降低。这项工作是哺乳动物聚合酶插入二磷酸核苷酸的第一个实例，并通过突出这些酶是如何进化到使用三磷酸核苷酸底物来深入了解聚合酶机制的性质。
DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (kpol), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme–substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.
登录
查看更多内容
发现聚合酶诱导的氧化核苷酸的细胞毒性。
影响因子：
64.8
作者：
Freudenthal, Bret D.;Beard, William A.;Perera, Lalith;Shock, David D.;Kim, Taejin;Schlick, Tamar;Wilson, Samuel H.
通讯作者：
Wilson, Samuel H.
DNA聚合酶β的结构和机制。
影响因子：
2.9
作者：
Beard WA;Wilson SH
通讯作者：
Wilson SH
影响因子：
5.7
作者：
Batra, VK;Beard, WA;Wilson, SH
通讯作者：
Wilson, SH
影响因子：
2.9
作者：
Pelletier, H;Sawaya, MR;Kraut, J
通讯作者：
Kraut, J
影响因子：
5.7
作者：
Krahn, JM;Beard, WA;Wilson, SH
通讯作者：
Wilson, SH