| Autor: |
Schmaltz, Lillian F., Myong-Chul Koag, Yi Kou, Louis Zhang, Seongmin Lee |
| Předmět: |
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| Zdroj: |
Biochemical Journal; May2023, Vol. 480 Issue 9, p573-585, 13p |
| Abstrakt: |
Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2' -fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2' -F-N7-alkylG DNA into 2' -F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supFbased colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2' -FN7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5' -GGT(methyl FapyG)G-3' context were single nucleotide deletions at the 5' -G of the lesion. Overall, our results demonstrate that 2' -fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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