Synthesis and study of C-nucleotide analogues of formamidopyrimidine lesions in DNA

Abstract

Formamidopyrimidine (Fapy) lesions are produced in DNA through the reaction of purines under oxidative stress conditions. Fapy•dA and Fapy•dG may exist as a mixture of anomers in DNA, and the equilibration between α- and β-anomers has been determined to be a rapid process. Fapy lesions may play an important role in mutagenic events and the onset of genetic diseases such as cancer. Another factor that suggests the importance of Fapy lesions is that they are substrates for the DNA repair protein Formamidopyrimidine DNA glycosylase (Fpg). In order to better understand the potential effects of the individual α- or β-anomers of Fapy lesions on DNA polymerases and repair proteins, non-epimerizable C-nucleotide analogues α-18, β-18 and β-19 were studied in DNA. The use of α-18, β-18 and β-19 as structural probes of Fapy lesions was supported by molecular modeling results. Only minor variations exist between the analogues and the Fapy lesions. UV-melting studies of duplexes containing α-18, β-18 and β-19 do not show the same similarities with Fapy•dA and Fapy•dG and reveal that there is no preference for stabilization opposite any nucleotide. Klenow (exo-) prefers to incorporate the correct dNTP opposite β-18 and β-19. However, misincorporation of dATP is also observed. Comparison of the relative misinsertion frequencies of β-18 and β-19 to Fapy•dA and Fapy•dG respectively reveal similar preferences. This suggests that the β-anomer is the biologically relevant diastereomer of both Fapy lesions. The ability to accommodate the insertion of dATP opposite β-18 or β-19 can be rationalized through the adoption of the syn-conformation. The formamide can present a "T-like" base-pairing pattern that can induce Klenow (exo-) to insert dATP. The interactions of Fpg protein with duplex DNA containing β-18 and β-19 opposite the four native nucleotides are very similar to investigations involving Fapy•dA and Fapy•dG. The Fpg protein binds the duplexes containing the β-18 opposite any of the four nucleotides with a Kd <27 nM. The Fpg protein discriminates between duplexes containing β-19, and only binds to the β-19:dC duplex (Kd = 11.8 nM).