Enhancement of OGG1 protein AP lyase activity by increase of APEX protein

8-Hydroxyguanine (oh(8)G) is a major form of oxidative DNA damage produced by reactive oxygen species (ROS). The human OGG1 gene encodes a DNA glycosy lase that excises oh(8)G from double-stranded DNA. In this study, we invest igated a mode of interaction between OGG1 and APEX proteins in the repair o f oh(8)G under oxidative stresses. DNA cleavage assay using oh(8)G-containi ng oligonucleotides showed that the phosphodiester bond on the 3'-side of o h(8)G was cleaved by the AP lyase activity of GST-OGG1 protein and the phos phodiester bond on the 5'-side of oh(8)G was cleaved by the DNA 3'-repair d iesterase activity of APEX protein. Gel mobility shift assay showed that th e complex of GST-OGG1 protein and oh(8)G-containing oligonucleotides mostly changed into the complex of APEX protein and oligonucleotides by addition of APEX protein into the reaction mixture, We next analyzed alterations in the amount of 8-hydroxydeoxyguanosine (oh(8)dG) in DNA and the levels of OG G1 and APEX expression in HeLa S3 cells treated with 2 mM hypochlorous acid , a kind of ROS. An approximately four-fold increase in the amount of oh(8) G was detected by the HPLC-ECD method. Reverse transcriptase-polymerase cha in reaction (RT-PCR) and Western blot analyses indicated that the level of APEX expression increased approximately four-fold, whereas the level of OGG 1 expression was unchanged. However, in the DNA cleavage assay, the AP lyas e activity of GST-OGG1 protein was significantly increased in the presence of a molar excess of APEX protein. These results indicate that, under sever e oxidative stresses, OGG1 mRNA is not induced and the amount of OGG1 prote in is not remarkably increased, but the activity of OGG1 protein is enhance d by the increase of APEX protein in the cells. (C) 2001 Elsevier Science B .V. All rights reserved.