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

Abstract

Bypass of the configurationally stable analogue (β-C-Fapy•dG) of the formamidopyrimidine lesion derived from 2'-deoxyguanosine oxidation (Fapy•dG) was studied in vitro and in E. coli. The exonuclease deficient Klenow fragment of E. coli DNA polymerase I (Klenow exo¯) misincorporated dA most frequently opposite β-C-Fapy•dG, but its efficiency was <0.2% of dC insertion. Klenow exo¯ fidelity was enhanced by the enzyme's high selectivity for extending duplexes only when dC was opposite β-C-Fapy•dG. The expectations raised by these in vitro data were realized when β-CFapy•dG replication was studied in E. coli by transfecting M13mp7(L2) bacteriophage DNA containing the nucleotide analogue within the lacZ gene in 4 local sequence contexts. The bypass efficiency of β-C-Fapy•dG varied between 45% and 70% compared to a genome containing only native nucleotides. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C.; Essigmann, J. M. Methods Enzymol. 2006, 408, 1.) The levels of mutations could not be distinguished between those observed when genomes containing native nucleotides were replicated, indicating that the mutagenicity of β-C-Fapy•dG was < 1%. These data and previous reports indicate that β-C-Fapy•dG is a good model of Fapy•dG in E. coli. In addition, these results and the previous report of β-C-Fapy•dG binding to the base excision repair protein formamidopyrimidine glycosylase suggest that this analogue could be useful as a DNA repair inhibitor. KeywordsDNA lesions; DNA damage; DNA replication; modified nucleotides 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 *Corresponding author: Marc M. Greenberg,, E-mail: mgreenberg@jhu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof ...

Error‐prone replication bypass of the imidazole ring‐opened formamidopyrimidine deoxyguanosine adduct

“…To dissect the differences between the α and β forms, several studies utilized stabilized analogs of Fapy‐dG. These investigations demonstrated that the β anomer preferred a dC as the pairing partner in duplex DNA and modestly inhibited DNA synthesis in vitro [Ober et al, ; Weledji et al, ; Büsch et al, ; Gehrke et al, ]. This was in contrast to the α anomer that strongly destabilized DNA structure regardless of the opposing nucleotide, and represented a nearly complete block to all DNA polymerases examined, including low‐fidelity Dpo4 and pol η [Büsch et al, ].…”

“…Collectively, these data may suggest that the α anomer is a cytotoxic lesion, with no or only a minor role in mutagenesis, whereas the β anomer could be primarily responsible for Fapy‐dG‐induced mutations, particularly G → T transversions. However, a conformationally fixed analog of β‐Fapy‐dG was essentially nonmutagenic in E. coli [Weledji et al, ]. It is worth noting that the highest frequency of G → T transversions observed for native Fapy‐dG in E. coli was <2% [Patro et al, ].…”

Site-Specific Synthesis and Characterization of Oligonucleotides Containing an N6-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine Lesion, the Ring-Opened Product from N7-Methylation of Deoxyguanosine

“…Replication studies of Fapy-dGuo and its configurationally stable analogues have also been reported (43, 44). The mutagenicity of the Fapy-dGuo lesion was low in bacteria (55, 56); the mutagenic frequency of Fapy-dGuo was higher in mammalian cell lines and also found to be sequence dependent (55). The major mutation was Gua→Thy transversions in both systems.…”

The Formamidopyrimidines: Purine Lesions Formed in Competition With 8-Oxopurines From Oxidative Stress

“…These configurationally stable Fapy analogues are useful probes, particularly in templates for co-crystallization studies with base excision repair proteins, and are potentially useful as repair enzyme inhibitors. 29–31 …”