Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA.

Abstract

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.

Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation

“…The half-life of 7MeA in DNA in vivo is only 2-3 hours, which is similar to its half-life in vitro at pH 7.2, 37°C (89). Fapy-7MeA is a mutagenic lesion displaying A / G transitions in single-stranded M13mp18 DNA transfected into SOS-induced E.coli (87,90). In these studies, dimethylsulfate-treated DNA was compared before and after treatment with alkali, which hydrolyzed the imidazole rings of N7-methylated adenines and guanines, forming the Fapy derivatives.…”

“…N7-methyladenine N7-methyladenine (7MeA) is a minor lesion formed at a level 40-fold below that of 7MeG, which is typically the most abundant lesion in alkylated DNA (87). Like 7MeG, 7MeA possesses a cationic imidazole ring, which facilitates depurination and, alternatively, can favor hydrolysis of the five-membered ring to form the formamidopyrimidine (Fapy) derivative, Fapy-7MeA; this latter hydrolysis reaction is especially favored for the 7MeA in RNA (88), which has a stabilized glycosidic bond as compared with DNA.…”

“…Whereas this lesion does not cause misparing with deoxyadenosine monophosphate (dAMP) or deoxythymidine monophosphate (dTMP), in vitro experiments using E.coli DNA polymerase I and poly[ 5#-deoxyguanosine-deoxycytidine-3# (dGpC)] templates (102) or Klenow fragment and M13mp18 template DNA (103) show that Fapy-7MeG blocks DNA chain elongation. Fapy-7MeG lesions present on M13mp18 phage template DNA also lead to a 2-to 3-fold increase in G / C and G / T transversions when transfected into SOS-induced E.coli (87). However, DNA polymerase I preferentially incorporates deoxycytidine monophosphate (dCMP) opposite Fapy-7MeG and a Fapy-7MeG:C pair is extended most efficiently compared with other possibilities.…”

A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage

“…Interestingly, we found an increase of the frequency of A > G or T > C mutations in the mutM2 mutant. In addition to OG, E. coli MutM efficiently excised 4,6-diamino-5-formamidopyrimidine (FapyAde) and 8-oxo-A (7, 50), which are both described to lead to A > G or T > C transition mutations (51)(52)(53). Subsequent biochemical studies investigating MutM2 capacity to excise FapyAde and 8-oxo-A might help reveal the physiologic function of this enzyme in nonpathogenic mycobacteria.…”

PEN110 treatment functionally inactivates the PBMNCs present in RBC units: comparison to the effects of exposure to gamma irradiation

“…Abasic sites in nucleic acids represent potent stop signals for DNA and RNA polymerases 11,12 . Covalent adducts of PEN110 with DNA and abasic sites can also result in mutations if they are bypassed during the replication with inappropriate base insertion opposite the lesion 13‐15 . The abasic sites are chemically unstable; therefore, their formation can lead to spontaneous strand breakage 16 .…”