Selective Incision of the alpha-N-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 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.

Scheme 1

Fapy-dG lesions derived from oxidative or alkylative damage to dG.

Scheme 2

The β- and α-anomers of Fapy-dG.

Figure 1

Structures of the methyl and aflatoxin B₁ Fapy-dG lesions.

Figure 2

Gel electrophoretic analysis of the incision of the MeFapy-dG containing duplex with E. coli Endo IV. (a) Incision after the annealing of the 5′-³²P-labelled MeFapy-dG containing oligonucleotide (24 mer) with its complement. The right lane is a standard of the 5′-³²P-ACCACGCTAGC-3′ incision product. (b) Percentage of the incision product after subsequent denaturation-reannealing and additional Endo IV.

Figure 3

Full-can mass spectrum of the Endo IV incision of the MeFapy-dG containing duplex; m/z 1825.4 [M-4H] and 1460.3 [M-5H] are the 5′-ACCACGCTAGC-(MeFapy-dG)-AGTCCTAACAAC-3′, m/z 1484.9 [M-5H] and 1237.2 [M-6H] are the 5′-GTTGTTAGGACTCGCTAGCGTGGT-3′, m/z 1094.1 [M-3H] and 820.3 [M-4H] are the 5′-ACCACGCTAGC-3′, and m/z 1345.2 [M-3H] and 1009.0 [M-4H]) is the 5′-p(MeFapy-dG)- AGTCCTAACAAC-3′ oligonucleotide.

Figure 4

Steady-state kinetic analysis for the incision of the MeFapy-dG-(a) and α-dG-(b) containing duplexes when the modified base was opposite dC. The Endo IV concentration was 0.08 nM.

Figure 5

Gel electrophoretic analysis of the equilibration of the β- and α-MeFapy-dG anomers in duplex DNA. (a) Anomerization of the β-MeFapy-dG after initial incision with Endo IV. Aliquots were treated with additional Endo IV every 24 h; the increase in the incision product is attributed to equilibration of the β-MeFapy-dG to the α-anomer. (b) Treatment of the MeFapy-dG-containing duplex with Endo IV after incubation at 37°C, pH 7.0 for up to 5 days.

Figure 6

Thermal melting analysis of the 12 mer duplex containing the MeFapy-dG lesion opposite dC after 5 days at 37°C. The T_m for the unmodifed duplex was 59°C.

Figure 7

Gel electrophoretic analysis of the incision of the α-AFB-Fapy-dG-containing 5′-³²P-labeled 24 mer duplex by Endo IV. The right lane is a standard of the 5′-³²P-ACCACTACTAT-3′ incision product.

Figure 8

Conformation of the α-AFB-Fapy-dG-containing 10 mer duplex. The AFB moiety (magenta) was intercalated to the 5′-side of the Fapy-G base, which was paired with its complement (pdb code 2KH3).