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

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Biochemistry (Impact Factor: 3.02). 11/2004; 43(42):13397-403. DOI: 10.1021/bi049035s
Source: PubMed


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.

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    • "One such example is α-adenosine (α-dA) that thermally destabilizes the parent duplex and is processed by endonuclease IV (65,66). Recent evidence reveals that endo IV processes α-anomers of Fapy lesions including those of Fapy-dG (50) and Me-Fapy-dG (67). Collectively, the existing database of Fapy-dG analogues conforms to the consensus regarding the relative energetic impacts of α- and β-isomers. "
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    ABSTRACT: Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.
    Nucleic Acids Research 03/2011; 39(13):5776-89. DOI:10.1093/nar/gkr082 · 9.11 Impact Factor
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    • "Endo IV contains three Zn2+ 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 N6-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. "
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    ABSTRACT: Formamidopyrimidines (Fapy) lesions result from ring opening of the imidazole portion of purines. Fapy lesions can isomerize from the natural beta-anomeric stereochemistry to the alpha-configuration. We have unambiguously demonstrated that the alpha-methyl-Fapy-dG (MeFapy-dG) lesion is a substrate for Escherichia coli Endonuclease IV (Endo IV). Treatment of a MeFapy-dG-containing 24 mer duplex with Endo IV resulted in 36-40% incision. The catalytic efficiency of the incision was comparable to that of alpha-dG in the same duplex sequence. The alpha- and beta-MeFapy-dG anomers equilibrate to ~21 : 79 ratio over ~3 days. Related studies with a duplex containing the alpha-Fapy-dG lesion derived from aflatoxin B(1) epoxide (alpha-AFB-Fapy-dG) showed only low levels of incision. It is hypothesized that the steric bulk of the aflatoxin moiety interferes with the binding of the substrate to Endo IV and the incision chemistry.
    Journal of nucleic acids 07/2010; 2010. DOI:10.4061/2010/850234
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    ABSTRACT: Proper maintenance of the genome is of great importance. Consequently, damaged nucleotides are repaired through redundant pathways. We considered whether the genome is protected from formamidopyrimidine nucleosides (Fapy*dA, Fapy*dG) via a pathway distinct from the Escherichia coli guanine oxidation system. The formamidopyrimidines are produced in significant quantities in DNA as a result of oxidative stress and are efficiently excised by formamidopyrimidine DNA glycosylase. Previous reports suggest that the formamidopyrimidine nucleosides are substrates for endonucleases III and VIII, enzymes that are typically associated with pyrimidine lesion repair in E.coli. We investigated the possibility that Endo III and/or Endo VIII play a role in formamidopyrimidine nucleoside repair by examining Fapy*dA and Fapy*dG excision opposite all four native 2'-deoxyribonucleotides. Endo VIII excises both lesions more efficiently than does Endo III, but the enzymes exhibit similar selectivity with respect to their action on duplexes containing the formamidopyrimidines opposite native deoxyribonucleotides. Fapy*dA is removed more rapidly than Fapy*dG, and duplexes containing purine nucleotides opposite the lesions are superior substrates compared with those containing formamidopyrimidine-pyrimidine base pairs. This dependence upon opposing nucleotide indicates that Endo III and Endo VIII do not serve as back up enzymes to formamidopyrimidine DNA glycosylase in the repair of formamidopyrimidines. When considered in conjunction with cellular studies [J. O. Blaisdell, Z. Hatahet and S. S. Wallace (1999) J. Bacteriol., 181, 6396-6402], these results also suggest that Endo III and Endo VIII do not protect E.coli against possible mutations attributable to formamidopyrimidine lesions.
    Nucleic Acids Research 02/2005; 33(10):3331-8. DOI:10.1093/nar/gki655 · 9.11 Impact Factor
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