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 beta-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise alpha-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 alpha-anomer. Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate. Fapy.dA incision is considerably slower than that of alpha-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 alpha-Fapy.dA. The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-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 beta-anomer in DNA.

Selective Incision of the α‐N5‐Methyl‐Formamidopyrimidine Anomer by Escherichia coli Endonuclease IV

“…A subsequent denaturation-reannealing cycle followed by addition of a third aliquot of Endo IV resulted in an additional 14% increase of the incision product (73% total), representing ~34% cleavage of the MeFapy-dG-containing duplex remaining after the prior two rounds of Endo IV treatment (41%). These results are similar to those reported by Patro et al for the incision of the Fapy-dA containing duplex and is consistent with selective incision of the α - MeFapy-dG anomer [29]. …”

“…The heating and annealing cycle is expected to equilibrate the α - and β -anomers of the MeFapy-dG lesion [29]. Additional Endo IV was added and the 5′- 32 P-ACCACGCTAGC-3′ incision product was found to be ~59% (Figure 2(b)); the 23% increase of the incision products represents ~36% cleavage of the MeFapy-dG duplex remaining (64%) after the initial Endo IV incision.…”

“…Endo IV contains three Zn 2+ atoms in its active site and recognizes the damage site by a double nucleotide-flipping mechanism [22–24]. Oligonucleotides containing the α - dA, α - dT, α - dC, α - Fapy-dA, and α - Fapy-dG lesions as well as the configurationally stable N 6 -carbon Fapy analogues α -C-Fapy-dA have been shown to be substrates for Endo IV [25–29]. Oligonucleotides containing MeFapy-dG have also been examined as potential substrates for Endo IV.…”

“…Asagoshi et alreported that a MeFapy-dG-containing oligonucleotide was not a substrate for Endo IV and concluded that this lesion existed as the β -MeFapy-dG anomer [30, 31]. However,Ishchenko et al[32] reported incision of the MeFapy-dG lesion with a catalytic efficiency comparable to that reported for α -C-Fapy-dA [29]. Interestingly, evidence that Endo IV plays a role in the repair of deoxyribosylurea lesions has also been reported [33]; this lesion can also undergo anomerization, and it is likely that the α -anomer is a substrate for Endo IV [34–36].…”

Destabilization of DNA duplexes by oxidative damage at guanine: implications for lesion recognition and repair

“…Unfortunately, the first problem in studying FapydG experimentally is the difficulty in producing pure oligonucleotides containing a b-FapydG lesion, which is assumed to be the naturally occurring anomer in the DNA duplex (Patro et al 2004). Chemically synthesized b-FapydG tends to rapidly anomerize to give a-FapydG under conditions required for DNA synthesis (Haraguchi & Greenberg 2001).…”