Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV.
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.
Patro
Jennifer N
JN
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Haraguchi
Kazuhiro
K
Delaney
Michael O
MO
Greenberg
Marc M
MM
D004249:DNA Damage; D004269:DNA, Bacterial / Q000737:chemistry; D003839:Deoxyadenosines / Q000737:chemistry; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005559:Formamides / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis; D009705:Nucleosides / Q000138:chemical synthesis; D009841:Oligonucleotides / Q000138:chemical synthesis; D011743:Pyrimidines / Q000737:chemistry*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005559:Formamides; D005663:Furans; C475814:N4-(2-deoxypentofuranosyl)-4,6-diamino-5-formamidopyrimidine; C435881:N6-(2-deoxy-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine; D009692:Nucleic Acid Heteroduplexes; D009705:Nucleosides; D009841:Oligonucleotides; D011743:Pyrimidines; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli