| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 98 === 8-oxo-7,8-dihydroguanine (8-oxoG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA. It is associated with tumorigenesis because its miscoding nature to adenine and it causes G:C to T:A transversion during DNA replication. In mammalian cells, 8-oxodG is primarily removed by human 8-oxoguanine DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are more than 7 alternatively spliced forms of hogg1 gene and α- and β-hogg1 are two major forms in various human tissues. These two hOGG1 proteins have different cell localizations. α-hOGG1 is localized in nucleus and mitochondria; whereas β-hOGG1 is only in mitochondria. Previous studies in our lab have found that β- but not α-hOGG1 protein directly interacts with the mitochondrial protein NADH dehydrogenase 1 beta sub complex 10 (NDUFB10), suggesting that β-hOGG1 is functionally associated with mitochondria. My research goal is to investigate the roles of α- and β-hOGG1 in mitochondrial DNA repair. In the present study, we used specific siRNAs to knock down (KD) the endogenous α- and β-hogg1 expression. My experimental findings showed that the α- and β-hogg1 KD cells were sensitive to oxidative stress induced by menadione treatment, detected by the MTT assays and AnnexinV-FITC apoptosis analyses. By semi-quantitative PCR for mitochondrial DNA (mtDNA), it was found that α- and β-hogg1 KD cells were deficient in removal of oxidative-damaged DNA lesions in mitochondrial genome, suggesting that both α- and β-hOGG1 proteins are involved in mtDNA repair. In addition, by the in vitro BER functional assay it was found that the in vitro BER activity was defective in both α-hOGG1 and β-hOGG1 KD whole-cell extracts (WCE), indicating that α- and β-hOGG1 proteins are both involved in overall oxidative BER in cells. Results of the in vitro BER using the nuclear proteins showed that α- but not β-hogg1 KD cells were deficient in incision activity, indicating that α-hOGG1 mainly contributes to nuclear BER repair. However, in the mitochondrial protein fractions, β-hogg1 KD cells showed a more severe defect in mitochondrial BER activity. In summary, we conclude that both α-hOGG1 and β-hOGG1 were required for mitochondrial DNA repair.
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