title: Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex.
abstract: 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.
authors.affiliation: Department of Advanced Technology Fusion, Konkuk University, Seoul, Republic of Korea.
authors.forename: Min Ju
authors.initials: MJ
authors.lastname: Park
authors.forename: Jong-Hwa
authors.initials: JH
authors.lastname: Park
authors.forename: Soo-Hyun
authors.initials: SH
authors.lastname: Hahm
authors.forename: Sung Il
authors.initials: SI
authors.lastname: Ko
authors.forename: You Ri
authors.initials: YR
authors.lastname: Lee
authors.forename: Ji Hyung
authors.initials: JH
authors.lastname: Chung
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Sohn
authors.forename: Yunje
authors.initials: Y
authors.lastname: Cho
authors.forename: Lin-Woo
authors.initials: LW
authors.lastname: Kang
authors.forename: Ye Sun
authors.initials: YS
authors.lastname: Han
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport
chemical_list: D018797:Cell Cycle Proteins; C549605:HUS1 protein, human; C462949:HUS1B protein, human; D021122:Protein Subunits; C069930:rad9 protein; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D006861:Hydrogen Peroxide; D005092:Exonucleases; C113574:Rad1 protein, human; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human