Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS,66160, USA.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS,66160, USA.
DNA Repair (Amst). 2020 Jul-Aug;91-92:102867. doi: 10.1016/j.dnarep.2020.102867. Epub 2020 May 16.
Under conditions of oxidative stress, reactive oxygen species (ROS) continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. When the nucleobase structure is altered, its base-pairing properties may also be altered, promoting mutations. Consequently, oxidative DNA damage is a major source of the mutation load that gives rise to numerous human maladies, including cancer. Base excision repair (BER) is the primary pathway tasked with removing and replacing mutagenic DNA base damage. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central enzyme with AP-endonuclease and 3' to 5' exonuclease functions during BER, and therefore is key to maintenance of genome stability. Polymorphisms, or SNPs, in the gene encoding APE1 (APEX1) have been identified among specific human populations and result in variants of APE1 with modified function. These defects in APE1 potentially result in impaired DNA repair capabilities and consequently an increased risk of disease for individuals within these populations. In the present study, we determined the X-ray crystal structures of three prevalent disease-associated APE1 SNPs (D148E, L104R, and R237C). Each APE1 SNP results in unique localized changes in protein structure, including protein dynamics and DNA binding contacts. Combined with comprehensive biochemical characterization, including pre-steady-state kinetic and DNA binding analyses, variant APE1:DNA complex structures with both AP-endonuclease and exonuclease substrates were analyzed to elucidate how these SNPs might perturb the two major repair functions employed by APE1 during BER.
在氧化应激条件下，活性氧（ROS）不断攻击 DNA 结构，导致核碱基的氧化和断裂。当核碱基结构发生改变时，其碱基配对性质也可能发生改变，从而促进突变。因此，氧化 DNA 损伤是导致许多人类疾病（包括癌症）的突变负荷的主要来源。碱基切除修复（BER）是负责清除和替换具有诱变作用的 DNA 碱基损伤的主要途径。脱嘌呤/脱嘧啶内切核酸酶 1（APE1）是 BER 过程中具有 AP 内切核酸酶和 3'至 5'外切核酸酶功能的核心酶，因此是维持基因组稳定性的关键。在编码 APE1（APEX1）的基因中，已经在特定人群中鉴定出多态性（或 SNP），导致 APE1 功能发生改变的变体。APE1 的这些缺陷可能导致 DNA 修复能力受损，从而增加这些人群中个体患病的风险。在本研究中，我们确定了三种常见疾病相关 APE1 SNP（D148E、L104R 和 R237C）的 X 射线晶体结构。每个 APE1 SNP 都会导致蛋白质结构的独特局部变化，包括蛋白质动力学和 DNA 结合接触。结合全面的生化特性分析，包括预稳态动力学和 DNA 结合分析，分析了具有 AP 内切核酸酶和外切核酸酶底物的变体 APE1:DNA 复合物结构，以阐明这些 SNP 如何干扰 APE1 在 BER 过程中使用的两种主要修复功能。
