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 supF-based 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′-F-N7-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.
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May 2023
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Cover Image
Cover Image
The cover image is a fluorescent microscopy image showing colocalization of HA-GFP -NPT2A Arg495Cys (green) with FLAG-NHERF1 (red) in OK cells. Treatment with PTH does not change NPT2A Arg495Cys-NHERF1 colocalization at the apical membrane. For more information see the article by Sneddon and colleagues (pp. 685–699) in this issue. The image was provided by Tatyana Mamonova.
Research Article|
May 04 2023
Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine
Lillian F. Schmaltz;
Lillian F. Schmaltz
Data curation, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing
From the Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, U.S.A.
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Myong-Chul Koag;
Myong-Chul Koag
Investigation, Visualization, Writing - original draft, Writing - review & editing
From the Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, U.S.A.
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Yi Kou;
Yi Kou
Validation, Investigation, Visualization, Writing - original draft
From the Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, U.S.A.
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Louis Zhang;
Louis Zhang
Investigation, Visualization
From the Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, U.S.A.
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Seongmin Lee
Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Project administration, Writing - review & editing
From the Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, U.S.A.
Correspondence: Seongmin Lee (seongminlee@austin.utexas.edu)
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Publisher: Portland Press Ltd
Received:
August 31 2022
Revision Received:
March 29 2023
Accepted:
April 19 2023
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2023
Biochem J (2023) 480 (9): 573–585.
Article history
Received:
August 31 2022
Revision Received:
March 29 2023
Accepted:
April 19 2023
Citation
Lillian F. Schmaltz, Myong-Chul Koag, Yi Kou, Louis Zhang, Seongmin Lee; Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine. Biochem J 17 May 2023; 480 (9): 573–585. doi: https://doi.org/10.1042/BCJ20220460
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