Department of Advanced Technology Fusion, Konkuk University, Seoul, Republic of Korea.
DNA Repair (Amst). 2009 Oct 2;8(10):1190-200. doi: 10.1016/j.dnarep.2009.06.004. Epub 2009 Jul 16.
Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.
Rad9-Rad1-Hus1（9-1-1）是一种检查点蛋白复合物，在 DNA 损伤感应、细胞周期阻滞、DNA 修复或细胞凋亡中发挥作用。人类 8-氧鸟嘌呤 DNA 糖基化酶（hOGG1）是负责修复特定异常氧化核苷酸 7,8-二氢-8-氧鸟嘌呤（8-oxoG）的主要 DNA 糖基化酶。在这项研究中，我们鉴定了 hOGG1 与人类 9-1-1 之间的一种新相互作用，并研究了这种相互作用的功能后果。使用瞬时转染的 HEK293 细胞进行的共免疫沉淀分析表明 hOGG1 与 9-1-1 蛋白之间存在相互作用。随后，使用细菌表达和纯化的 hOGG1-His 和 GST 融合的 9-1-1 亚基（GST-hRad9、GST-hRad1 和 GST-hHus1）进行 GST 下拉测定表明 hOGG1 直接与人类 9-1-1 复合物的各个亚基相互作用。使用含有 8-oxoG/C 错配的 DNA 双链体进行的体外切除测定表明 hRad9、hRad1 和 hHus1 增强了 hOGG1 的 8-oxoG 切除和β消除活性。此外，使用 NaBH4 进行的捕获测定表明，hRad9、hRad1 和 hHus1 的存在增强了共价交联的 hOGG1-8-oxoG/C 双链复合物的形成。从转化有 hRad9、His 融合 hRad1 或 His 融合 hHus1 表达载体的大肠杆菌细胞中纯化出三聚体人 9-1-1 复合物。与单个人类 9-1-1 亚基相比，它还显示出增强体外 hOGG1 糖苷酶活性的相似活性。使用流式细胞术和分光荧光法检测 HEK293 细胞中的 8-oxoG 表明，hOGG1 或人类 9-1-1 的过表达减少了 H2O2 处理后 8-oxoG 残基的形成。在过表达 hOGG1 和人类 9-1-1 的所有三个亚基的 HEK293 细胞中观察到最高的 8-oxoG 减少。这些表明，单个人类 9-1-1 亚基和人类 9-1-1 复合物几乎具有相同的能力来增强 hOGG1 的体外 8-oxoG 切除活性，但去除 H2O2 处理细胞中 8-oxoG 残基的最大效果来自整个 9-1-1 复合物。
