Configurational and Conformational Equilibria of N-6-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) Lesion in DNA

摘要

The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N-6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either alpha or beta deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5 -N-5 bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a C-13-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5'-TXT-3' and the dodecamer 5'-CATXATGACGCT-3' (X = C-13-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the CS-N-5 bond to produce R-a and S-a atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. H-1 NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the alpha and beta anomers exist in a 3:7 ratio, favoring the beta anomer. For the beta anomer, with respect to the C5-N-5 bond, the R-a and S-a atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the alpha anomer, the E-S-a isomer is present at 12%, whereas all other isomers are present at 5-7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations responses to the MeFapy-dG lesion.