역전사 효소에 의한 cDNA 합성시 8-oxo-7, 8-dihydroguanosine과 쌍을 이룬 DNA 염기 분석
Analysis of base pairing of 8-oxo-7,8-dihydroguanosine in cDNA synthesis by reverse transcriptases
디옥시리보 핵산 DNA 역전사 효소;
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Reactive oxygen species are generated in organisms by UV radiation, superoxide, hydrogen peroxide hydroxyl radicals of chemicals biotransformation during cellular metabolism. DNA damages generated by the reaction of DNA with reactive oxygen species is supposed one of the major reasons for mutations and carcinogenesis. The most commonly measured marker of oxdative DNA damage is deoxyribonucleoside (8-oxodG). The guanosine derivative 8-oxodG is a stable compound and early allowed its chemical synthesis and incorporation into DNA. Consequently, a wealth of chemical and biochemical data are now available on this important lesion. Recently, oxidative base damages in RNA strands were also analyzed in the oxidoreduction of Torula yeast RNA, and also revealed oxidatively damaged sites using DNA primer extension assay with AMV-RT. In our previous studies, in order to elucidate the base pairing properties of 8-oxoG and its 2′-O methylation form of 8-oxoG-Me, we identified the incorporated DNA bases opposite 8-oxoG and 8-oxoG-Me in a condition of noncompetition cDNA synthesis. We proved that both the 8-oxoG and 8-oxoG-Me could pair with dC or T and the degrees of pairing are dependent on the origins of reverse transcriptases. In this study, we analyzed DNA bases opposit 8-oxoG or 8-oxoG-Me using Maxam-Gilbert sequencing method in a competition condition of cDNA synthesis by reverse transcriptases. DNA primers of 15mer [5′-d(ATGTCGACACCCAAT-3′)] and 16mer [5′-d(ATGTCGACACCCAATT-3′)] were prepared by phosphoamidite method and were analyzed their purities by RP-HPLC, PAGE and Maldi-Toff. cDNA synthesis was carried with template RNA(5′-UCCAUUUUCAXAAUUGGGUGUCGACAUAGC-3′) containing G, 8-oxoG or 8-oxoG-Me, DNA primer, the same concentrations of mixed dNTPs and reverse transcriptase (AMV-RT and MMLV-RT). The extended 23 base long of cDNAs from ^(32)P labelled DNA primer 16mer by AMV-RT and MMLV-RT were isolated from denaturated PAGE, desalted by a sephadex G-50 and then relabelled. However, any cDNAs was not produced in a reaction with 15 mer DNA primer and RNA templates except unmodified G was contained. In analysis of sequences by Maxam-Gilbert sequencing method, almost the 17^(th) dC was inserted opposit G, 8-oxoG and 8-oxoG-Me by both AMV-RT and MMLV-RT. In conclusion, investigation of the incorporation of dNTPs opposite the 8-oxoG and 8-oxoG-Me during competion or non competition cDNA synthesis, most of dC was paired with G, 8-oxoG and 8-oxoG-Me regardless by reverse transcriptases. Further study will be carried out using different RNA sequences containing 8-oxoG and 8-oxoG-Me with another reverse transcriptases.