Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

Abstract

ABSTRACT3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N 6 -ethenoadenine (A) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only A DNA glycosylase in liver, testes, and kidney; another A DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag ؊͞؊ mice are alkylation sensitive, indicating that Aag ؊͞؊ mice may be similarly sensitive.In the face of inescapable DNA-damaging agents and inevitable spontaneous DNA degradation, the constant challenge to preserve genomic integrity has been met by the evolution of numerous pathways that protect against the genotoxic effects of DNA-damaging agents. Unless processed properly, DNA damage can be mutagenic, carcinogenic, and teratogenic, and DNA damage also may contribute to aging (1).Alkylating agents are found in our environment, in our food, inside all cells as natural metabolites, and in the clinic as cancer chemotherapeutic agents. Base excision repair (BER) is one of the major pathways for the repair of damaged DNA bases and proceeds through a sequence of reactions requiring several different enzymes. The first step involves excision of the damaged base (for which most cells are known to have several different DNA glycosylases). Base excision by glycosylases is followed by strand cleavage in the vicinity of the abasic site (by AP endonuclease or AP lyase), and preparation of the DNA ends for gap filling and ligation. DNA polymerase fills the gap, and DNA ligase seals the remaining nick, thus completing the BER process (1).Human and rat 3-methyladenine (3MeA) DNA glycosylases, so named because 3MeA was the first substrate identified for this class of enzymes (2), actually display an unexpectedly broad substrate range, including guanines methylated at the N3 or the N7 position (3-6), deaminated adenine [i.e., hypoxanthine (Hx)] (7), oxidized guanine 8-oxoguanine (8oxoG) (8), cyclic etheno adducts on both adenine and guanine (9, 10), and haloethylated purines (unpublished observations). The mouse alkyladenine DNA glycosylase (Aag) has not been assayed for release of all of these substrates, though it has been shown to act on N3 and N7 methylpurines and on 8oxoG (8,11). The precise biological effects of all of the DNA lesions repaired by mammalian 3MeA DNA glycosylases are not yet known for mammals, though there is strong evidence that 3MeA is cytotoxic (12), and other lesions may be mutagenic, namely Hx (13), 8oxoG (14), 1,N 6

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

“…However, only ∼30% of the 3MeA lesions remained, suggesting that there is active removal of 3MeA in Aag -/-cells. Interestingly, previous in vitro studies of Aag -/-cell and tissue extracts did not reveal any residual 3MeA DNA glycosylase activity (12,21,40). This may have been the case because the in vitro conditions were incombatible with the unknown repair system, such as NER.…”

Repair of exocyclic DNA adducts: rings of complexity

“…(44) Part of the above biochemical results on substrate specificity was verified when ANPG À/À knockout mice were generated independently by the groups of Elder and Samson. (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.…”

“…(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.…”

“…(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. (45) When the same mice were challenged with vinyl carbamate (49) or ethyl carbamate, (50) levels of eA were significantly higher and persisted longer in DNA from ANPG À/À mice than wild-type mice, indicating the cellular removal of eA by ANPG.…”

“…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.…”