Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis.
7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.
Department of Pharmacology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Republic of Korea.
Ja-Eun
JE
Kim
Jin-Won
JW
Hyun
Hiroshi
H
Hayakawa
Seongwon
S
Choi
Jinhee
J
Choi
Myung-Hee
MH
Chung
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D003853:Deoxyribonucleosides / Q000096:biosynthesis; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D009154:Mutation
D017931:DNA Primers; D003853:Deoxyribonucleosides; C024829:8-hydroxyguanine; D006147:Guanine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine