PMID:15491146
| Citation |
Patro, JN, Haraguchi, K, Delaney, MO and Greenberg, MM (2004) Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV. Biochemistry 43:13397-403 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi049035s |
| Keywords |
DNA Damage; DNA, Bacterial/chemistry; Deoxyadenosines/chemistry; Deoxyribonuclease IV (Phage T4-Induced)/chemistry; Escherichia coli Proteins/chemistry; Formamides/chemistry; Furans/chemistry; Hydrolysis; Kinetics; Nucleic Acid Heteroduplexes/chemical synthesis; Nucleosides/chemical synthesis; Oligonucleotides/chemical synthesis; Pyrimidines/chemistry; Substrate Specificity |
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