Mahmoud Mariam M, Schechter Allison, Alnajjar Khadijeh S, Huang Ji, Towle-Weicksel Jamie, Eckenroth Brian E, Doublié Sylvie, Sweasy Joann B
Department of Therapeutic Radiology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Department of Microbiology and Molecular Genetics, University of Vermont , Burlington, Vermont 05405, United States.
Biochemistry. 2017 Oct 17;56(41):5550-5559. doi: 10.1021/acs.biochem.7b00869. Epub 2017 Oct 2.
DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase β (Pol β) functions in base excision repair to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. To probe the mechanism that may underlie E288K's loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT because of an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure.
DNA聚合酶在DNA复制和修复过程中合成新的DNA，其忠实地执行此功能的能力对于维持基因组完整性至关重要。DNA聚合酶β（Polβ）在碱基切除修复中发挥作用，以填补单核苷酸间隙，并且Polβ的变体与癌症相关。具体而言，已在结肠肿瘤中发现E288K Polβ变体，并已证明其具有序列特异性诱变活性。为了探究E288K保真度丧失背后的机制，采用了一种荧光共振能量转移系统，该系统利用Polβ指状结构域上的荧光团和DNA底物上的猝灭剂。我们的结果表明，在掺入正确的脱氧核苷酸三磷酸（dNTP）时，E288K采用的总体机制与野生型（WT）Polβ相似。然而，在插入正确的dNTP时，与WT Polβ相比，E288K表现出指状结构域更快的闭合速率以及核苷酸释放速率较慢。我们还检测到E288K与错误的dNTP混合时酶的闭合，但WT Polβ没有。综上所述，我们的结果表明，由于其活性位点内dNTP快速异构化导致的固有缺陷，E288K Polβ比WT更容易掺入所有dNTP。结构建模表明，这种固有缺陷是由于E288K与DNA的相互作用，导致稳定的闭合酶结构。
