Probing the Configurations of Formamidopyrimidine Lesions Fapy·dA and Fapy·dG in DNA Using Endonuclease IV

Abstract

The formamidopyrimidines Fapy·dA and Fapy·dG are produced in DNA as a result of oxidative stress. These lesions readily epimerize in water, an unusual property for nucleosides. The equilibrium mixture slightly favors the β-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise α-deoxyadenosine was used as a tool to determine the configuration of Fapy·dA and Fapy·dG in DNA. Endo IV incision of the C-nucleoside analogues of Fapy·dA was used to establish selectivity for the α-anomer. Incision of α-C-Fapy·dA follows Michaelis−Menten kinetics (K_m = 144.0 ± 7.5 nM, k_cat = 0.58 ± 0.21 min^-1), but the β-isomer is a poor substrate. Fapy·dA incision is considerably slower than that of α-C-Fapy·dA, and does not proceed to completion. Endo IV incision of Fapy·dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing α-Fapy·dA. The extent of Fapy·dA incision suggests that the lesion exists predominantly (∼90%) as the β-anomer in DNA. Endo IV incises Fapy·dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its β-anomer in DNA.