DNA polymerase bypass in vitro and in E. coli of a C-nucleotide analogue of Fapy-dG.

Nucleotide analogues are useful mechanistic probes for studying kinetic mechanisms of polymerases and DNA repair enzymes.1–5 Structural studies of protein-DNA complexes are also made possible by nucleotide based substrate analogues.6, 7 There are also a significant number of examples of modified nucleosides that are successful therapeutic agents.8, 9 Although to our knowledge no modified nucleotide that inhibits a base excision repair protein has been used for therapeutic purposes, the potential for inhibitors of such enzymes to function as drugs is recognized.4, 10, 11 C-nucleotides carbocyclic nucleotides are not substrates for glycosylases due to their lower oxidation state. We describe the bypass of a C-nucleotide analogue (β-C-Fapy•dG) of Fapy•dG (N-(2-deoxy-α,β -D-erythropentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine)) by the exonuclease deficient Klenow fragment of DNA polymerase I from E. coli (Klenow exo¯) and its bypass in vivo. The results described herein and those previously reported concerning the properties of β-C-Fapy•dG and its respective nucleotide triphosphate (β-C-Fapy•dGTP) suggest that this nucleotide analogue may be useful as an inhibitor of DNA base excision repair.5, 12 Fapy•dG is produced when DNA is exposed to oxidizing conditions. The lesion is derived from a common intermediate whose reactivity is partitioned between Fapy•dG and 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (OxodG) formation (Scheme 1).13 Consequently, the chemical and biochemical aspects of Fapy•dG are often compared to the respective properties of OxodG, an extremely well studied DNA lesion.14 The two lesions are produced in comparable yields when DNA is exposed to γ-radiolysis under aerobic conditions, but the G-value for Fapy•dG production is approximately twice that of OxodG under anaerobic conditions.15, 16 OxodG and Fapy•dG share many common biochemical traits as well. For instance, both lesions are excised by the bifunctional formamidopyrimidine DNA glycosylase (Fpg) from E. coli selectively when opposite dC, and the repair protein MutY incises dA opposite OxodG or Fapy•dG.5, 17 Furthermore, Fapy•dG and OxodG induce bacterial DNA polymerases to misincorporate dA opposite them in vitro.18, 19 Their effect on DNA polymerase activity is reflected in the outcome of experiments in cells using single stranded vectors containing the lesions at a defined site. Replicative bypass of Fapy•dG in COS-7 cells results in slightly higher G → T transversion frequencies than when genomes containing OxodG are bypassed.20 In contrast, significantly lower mutation frequencies for both lesions are recorded in E. coli using the restriction endonuclease and postlabeling (REAP) assay, but OxodG produces higher G → T transversion frequencies than Fapy•dG in this organism.21 Scheme 1 Carbocyclic (1) and C-nucleotide (β-C-Fapy•dG) analogues of Fapy•dG have been used in structural studies and as biochemical probes.5, 22 The former has proven to be very useful for examining the binding of BER proteins to a formamidopyrimidine via X-ray crystallography.7 DNA containing β-C-Fapy•dG binds the Fpg repair protein with nanomolar affinity. Although MutY deglycosylates an opposing dA opposite β-C-Fapy•dG much more slowly than when Fapy•dG or OxodG are present in the duplex, DNA containing the analogue binds the enzyme ~7-fold more strongly than does undamaged biopolymer.5 Studies on β-C-Fapy•dGTP suggest that low levels of this analogue can be incorporated selectively opposite dC.12 The specificity constant for the incorporation of β-C-Fapy•dG opposite dC is only ~10−4 that of dG incorporation. These data indicate that β-C-Fapy•dG will replace 1 out of every 106 dGs if the concentration of its triphosphate is 1% of dGTP. This incorporation level is comparable to that observed for the production of some lesions in DNA.23 The incorporation efficiency of β-C-Fapy•dGTP is even more significant because the analogue is an extremely poor substrate for MutT, the enzyme that protects the nucleotide triphosphate pool by hydrolyzing OxodGTP.12