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

Abstract

Formamidopyrimidines (Fapy) lesions result from ring opening of the imidazole portion of purines. Fapy lesions can isomerize from the natural β-anomeric stereochemistry to the α-configuration. We have unambiguously demonstrated that the α-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 α-dG in the same duplex sequence. The α- and β-MeFapy-dG anomers equilibrate to ~21 : 79 ratio over ~3 days. Related studies with a duplex containing the α-Fapy-dG lesion derived from aflatoxin B1 epoxide (α-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.

Synthesis and Characterization of Oligonucleotides Containing a Nitrogen Mustard Formamidopyrimidine Monoadduct of Deoxyguanosine

“…The incision efficiency was approximately half of that of the MeFapy-dGuo lesion and ∼4.3-fold lower than α-dGuo in the same sequence context (Table 2). 44 The k cat values for the three substrates were similar, and differences in their incision efficiency were largely due to K m . An active site pocket of Endo IV is hypothesized to accommodate α-nucleotides.…”

“…Endo IV has been used previously to approximate the α/β ratio of Fapy-dGuo, Fapy-dAdo, and MeFapy-dGuo in duplex DNA by the selective incision of the α-anomer. 44,59−61 We applied this assay to the NM-Fapy-dGuo containing 24-mer to determine the α/β ratio of the lesion. The modified 24-mer ( 10 ) was 5′- 32 P-labeled, annealed to its complement, and incubated with Endo IV (Figure 3).…”

“…This process should re-equilibrate the NM-Fapy-dGuo lesion to its original mixture of anomers. 44,61 Additional Endo IV was added, and after 40 min, gel analysis indicated that the total level of incision was ∼75%; the 25% increase represents 50% of the duplex remaining after the first incision reaction. The reaction mixture was denatured and reannealed again, and a third portion of Endo IV was added.…”

“…44 The labeled NM-Fapy-dGuo containing 24-mer duplex was incubated at 37 °C and pH 7.5. Aliquots were removed after 0.5 h and then approximately every 24 h and were subjected to Endo IV treatment.…”

Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells

“…Importantly, DNA substrate specificity of APE1 is modulated by divalent cations, pH, and ionic strength (20): at low concentrations of Mg 2+ (≤1 mM), APE1 exhibits dramatically increased 3′→5′ exonuclease (25) and NIR-endonuclease activities (20), whereas at higher concentration of Mg 2+ (≤5 mM), APE1 shows increased AP endonuclease and 3′-phosphodiesterase activities (20,26). As we and others have shown, APE1 recognizes an extremely large variety of different DNA substrates including 5,6-dihydropyrimidines, αdN (20), 5-hydroxypyrimidines (27), formamidopyrimidines (28), ethenobases, thymine glycol (29), and bulky lesions such as the 6-4 photoproduct (30) and benzene-derived DNA adducts (31).…”