Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex
Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex
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DOI：
10.1016/j.dnarep.2009.06.004
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发表时间：
2009-10-02
期刊：
影响因子：
3.8
通讯作者：
Han, Ye Sun
中科院分区：
文献类型：
--
作者：
Park, Min Ju;Park, Jong-Hwa;Han, Ye Sun
RAD9-RAD1-HUS1(9-1-1)是一种检查点蛋白复合体，在DNA损伤感知、细胞周期停滞、DNA修复或细胞凋亡等方面发挥重要作用。人8-氧鸟嘌呤DNA糖基酶(HOGG1)是修复8-氧鸟嘌呤(8-oxoG)的主要DNA糖基酶。在这项研究中，我们发现了hOGG1与人类9-1-1之间的一种新的相互作用，并研究了这种相互作用的功能后果。用瞬时转染的HEK293细胞进行免疫共沉淀实验证明hOGG1与9-1-1蛋白之间存在相互作用。随后，利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)进行的GST下拉检测表明，hOGG1直接与人9-1-1复合体的各个亚基相互作用。采用含有8-oxoG/C错配的DNA双链的体外切割实验表明，hRad9、hRad1和hHus1增强了hOGG1的8-oxoG切割和P-消除活性。此外，通过使用NaBH4的捕获分析，hRad9、hRad1和hHus1的存在促进了共价交联的hOGG1-8-oxoG/C双链复合体的形成。用hRad9、His-融合hRad1和His-融合hHus1表达载体，从大肠杆菌细胞中纯化了人9-1-1三聚体复合体。与单独的人9-1-1亚基相比，它在体外也显示出类似的增强hOGG1糖基酶活性的活性。流式细胞仪和荧光分光光度分析检测HEK293细胞中8-oxoG的表达表明，hOGG1或人9-1-1的过表达减少了过氧化氢处理后8-oxoG残基的形成。在高表达hOGG1和人9-1-1的三个亚基的HEK293细胞中，8-oxoG的降解率最高。这表明单独的人9-1-1亚基和人9-1-1复合体在体外增强hOGG1的8-oxoG切割活性的能力几乎相同，但在H_2O_2处理的细胞中，去除8-oxoG残基的最大效果来自于9-1-1复合体。(C)2009爱思唯尔B.V.保留所有权利。
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 P-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 NaBH4. 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 H2O2 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 H2O2-treated cells was derived from the 9-1-1 complex as a whole. (C) 2009 Elsevier B.V. All rights reserved.