Studies on the Replication of the Ring Opened Formamidopyrimidine, Fapy·dG in Escherichia coli

Abstract

Addition of hydroxyl radicals to the C8 position of 2 0-deoxyguanosine generates an 8-hydroxyguanyl radical that can be converted into either 8-oxo-7,8-dihydro-2 0-deoxyguanosine or N-(2-deoxy-D-pento-furanosyl)-N-(2,6-diamino-4-hydroxy-5-formamido-pyrimidine) (Fapy-dG). The Fapy-dG adduct can adopt different conformations and in particular, can exist in an unnatural a anomeric configuration in addition to canonical b configuration. Previous studies reported that in 5 0-TGN-3 0 sequences, Fapy-dG predominantly induced G ! T transversions in both mammalian cells and Escherichia coli, suggesting that mutations could be formed either via insertion of a dA opposite the 5 0 dT due to primer/template mis-alignment or as result of direct miscoding. To address this question, single-stranded vectors containing a site-specific Fapy-dG adduct were generated to vary the identity of the 5 0 nucleotide. Following vector replication in primate cells (COS7), complex mutation spectra were observed that included $3-5% G ! T transversions and $14-21% G ! A transitions. There was no correlation apparent between the identity of the 5 0 nucleotide and spectra of mutations. When conditions for vector preparation were modified to favor the b anomer, frequencies of both G ! T and G ! A substitutions were significantly reduced. Mutation frequencies in wild-type E. coli and a mutant deficient in damage-inducible DNA polymerases were significantly lower than detected in COS7 and spectra were dominated by deletions. Thus, mutagenic bypass of Fapy-dG can proceed via mechanisms that are different from the previously proposed primer/template misalignment or direct misinser-tions of dA or dT opposite to the b anomer of Fapy-dG. Environ. Mol. Mutagen. 58:182-189, 2017. V C 2017 Wiley Periodicals, Inc.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

“…Comparison of the mutagenesis caused by 8-oxoG and FapyG was performed in simian kidney cells (COS-7), and more Gua → Thy transversion mutations were caused by FapyG than by 8-oxoG in several sequence contexts (31). However, a recent study showed that in E. coli, the mutagenicity of 8-oxoG is greater than that of FapyG (32). It is also possible that the reduced mutagenicity we observed in the triple mutant strain is partially due to the further oxidation product of 8-oxoG, Sp, which gives rise to a combination of Gua → Thy and Gua → Cyt transversion mutations (33,34) and can be recognized by MmuNeil3.…”

Consequences and Repair of Oxidative DNA Damage

“…46,[94][95][96][97] In contrast FapyG, like 8-oxoguanine, can be bypassed by Klenow exo À DNA polymerase which inserts adenine opposite 98 and in E. coli leads to G-T transversions. In a single sequence context, FapyG is not as mutagenic in E. coli as it is in simian kidney cells; 99,100 however, recent studies suggest that FapyG is as potent a premutagenic lesion in E. coli as 8-oxoguanine. 101 Similar to guanine, the 8-position on the imidazole ring of adenine is also susceptible to oxidation resulting in 8-oxoadenine.…”

The Fpg/Nei Family of DNA Glycosylases

“…Taken together these data supported the idea that 8-oxoguanine is a biologically important, potentially mutagenic oxidative DNA lesion. However recent studies have shown that unmethylated FapyG is also a good substrate for Fpg (18,19) and like 8-oxoG, A can also be incorporated opposite FapyG (20,21) and the incorporated A can be removed by MutY (22). FapyG, which is formed from the same adduct radical as 8-oxoG (23), appears to be responsible for a substantial number of mutations originally attributed to 8-oxoG and thus is also a biologically relevant substrate (24).…”