Methylating agents react with DNA at the N7 position of deoxyguanosine (dG), forming N7-dG adducts. These cationic adducts can undergo a secondary reaction involving the addition of hydroxide to C8, followed by imidazole ring opening to form an N5-methyl-formamidopyrimidine (MeFapy) lesion (Figure 1). MeFapy lesions exist as a mixture of interconverting isomers. The deoxyribose ring can undergo opening and re-closure under acidic conditions, resulting in a mixture of α and β anomers. In addition, rotations can occur around the C5-N5 bond, the formyl group bond, and the glycosidic bond. The aim of this study is to understand which species of MeFapy are present in DNA, in what amounts, and how their presence affects DNA duplex structure. The sequence 5’-d(CAT-(MeFapy-G)- ATGACGCT)-3’ containing 13C-labeled MeFapy was synthesized and characterized using nuclear magnetic resonance (NMR) spectroscopy. 1H-13C heteronuclear single quantum correlation spectra revealed that ten species of MeFapy were present in single-strand DNA and six were present in duplex DNA. For both single-strand and duplex DNA, these peaks were divided into two groups, in a 40:60 ratio and which corresponded to lesions in the α and β configurations. The differences in rotameric distributions between single strand and duplex DNA are interpreted in terms of steric restraints imposed upon rotamer interconversion in duplex DNA. In 1H-1H Nuclear Overhauser effect (NOESY) spectra, the MeFapy lesion in duplex DNA did not show crosspeaks between T03-H1’ and MeFapy-G04-H8 and between MeFapy-G04-H1’ and A05-H8, indicating disruption of the normal duplex structure.

Characterization of the N5-methyl-formamidopyrimidine lesion

253rd National Meeting (2017)

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