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Ultrasensitive tracking of oxidized guanine incorporation into DNA and RNA via nucleotide salvage with accelerator mass spectrometry

2009년 8월 5일 14시 42분 35초
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금 12시 : 00분
생명화학 - Nanomaterials and Nucleic Acids for Molecular Therapy
저자 및
경희대학교 화학과, Korea
Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-oxodG), which initiates diseases related to aging and carcinogenesis. Insufficient detection sensitivity, however, has hampered measurement of these putative mutagenic products in cells or animals. I report use of a sensitive and precise accelerator mass spectrometry (AMS) assay to track the fate of [14C]8-oxodG incorporation into DNA and RNA of MCF-7 human breast cancer cells. MCF-7 cells were grown in the presence of [14C]8-oxodG and radiocarbon was found to be partitioned into both DNA and RNA, indicating that 8-oxoguanine was detached from the deoxyribose ring and attached covalently to a ribosyl species to form an appropriate nucleotide for incorporation into RNA. I also observed evidence for phosphorylated 8-oxodG derivatives and 8-oxoG derivatives in the nucleotide pool. Moreover, oxidative stressing of the cells by exposure to 17ß-estradiol reduced the rate of 8-oxodG incorporation, indicating induction of a mechanism to limit the triphosphate derivative of 8-oxodG required for incorporation by DNA polymerases. These data suggest that 8-oxodG in the nucleotide pool is an unexpected source of 8-oxoguanine forDNA and RNA incorporation, which may have important consequences for understanding diseases resulting from mutagenesis and altered protein function.