Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1, N ⁶ -ethenoadenine and hypoxanthine but not of 3, N ⁴ -ethenocytosine or 8-oxoguanine

Abstract

It has previously been reported that 1,N 6 -ethenoadenine (A), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N 4 -ethenocytosine (C), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing A and Hx, but retained wild-type levels of activity for C and 8-oxoG. These findings indicate that (i) the previously identified A-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts A and C are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.

Base Excision Repair Intermediates Induce p53-independent Cytotoxic and Genotoxic Responses

“…For example, Roth and Samson found that myeloid progenitor bone marrow cells from Aag (Ϫ/Ϫ) mice were less MMS-sensitive than corresponding cells from wild type animals, whereas other groups found that Aag (Ϫ/Ϫ) ES cells and MEFs were more MMS-sensitive or Me-lex-sensitive than Aag (ϩ/ϩ) counterparts (23,37,48,49). The results described here reveal that the ␤-pol expression status of cells can be a variable in the MMS-sensitivity phenotype upon Aag gene deletion.…”

“…mediated by a similar phenomenon. Clearly, spontaneous generation of Aag substrates does not lead to lethality in mice, since Aag (Ϫ/Ϫ) mice are viable and are without an obvious phenotype (23,37). We therefore bred a colony of ␤-pol (ϩ/Ϫ) / Aag (Ϫ/Ϫ) mice to determine if genetic loss of Aag would rescue the ␤-pol (Ϫ/Ϫ) -associated lethality.…”

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

“…Here, we used molecular beacon-based DNA glycosylase assay to test whether the inhibition of ANPG by ⑀C residues could occur in cultured mammalian cells. ANPG is the only known Hx-DNA glycosylase in mammalian cells (20,45); therefore, inosine-FD oligonucleotides allow us to measure its activity with high specificity. The fluorescent oligonucleotide (35F-control) without a quencher residue, control non-modified oligonucleotide (35FD-control), and inosine-FD were delivered into human HeLa cells (Fig.…”

Repair of exocyclic DNA adducts: rings of complexity

“…(45,46) Using cell-free extracts and synthetic oligonucleotides/modified DNA, ANPG was shown to be the primary glycosylase excising eA, 1,N 2 -eG, 3mA and Hx. (46)(47)(48) Such analysis provides an unambiguous means for the designation of the substrate specificity and for the exploration of backup activities for the missing enzyme. Biologically, however, it is surprising that these knockout mice did not show any overt phenotypic abnormalities (45,46) or significant increase in the spontaneous mutation rate, even increased mutations were observed in the hprt gene of the T lymphocytes of ANPG À/À mice treated with methyl methanesulfonate.…”

“…It is puzzling, though, that the increased levels of adducts were not paralleled by the increased incidence of liver tumors in these mice. (49) Interestingly, even though no other glycosylase activity against eA was detected using the in vitro approach, (46,47) one study reported that there was residual repair of eA adducts in the ANPG À/À mice. (50) Whether this residual activity is from another DNA glycosylase or from other repair pathway(s) remains to be determined.…”