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

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA targeting anticancer compounds. The basis for glycosylase specificity toward N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here, we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched, and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing the first illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how AlkD’s HEAT-repeats distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

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DNA-damaging agents produce a plethora of cellular responses that include p53 induction, cell cycle arrest, and apoptosis. It is generally assumed that it is the DNA damage produced by these agents that triggers such responses, but there is limited direct evidence to support this assumption. Here, we used DNA alkylation repair proficient and deficient isogenic mouse cell lines to demonstrate that the signal to trigger p53 induction, cell cycle arrest, and apoptosis in response to alkylating agents does emanate from DNA damage. Moreover, we established that 3-methyladenine, a relatively minor DNA lesion produced by most methylating agents (which form mainly 7-methylguanine), can specifically induce sister chromatid exchange, chromatid and chromosome gaps and breaks, S phase arrest, the accumulation of p53, and apoptosis. This study was made possible by the generation of 3-methyladenine DNA glycosylase null mutant cells by targeted homologous recombination and by the chemical synthesis of a methylating agent that almost exclusively produces 3-methyladenine DNA lesions. The combined use of these two experimental tools has defined the biological consequences of 3-methyladenine, a DNA lesion produced by endogenous cellular metabolites, environmental carcinogens, and chemotherapeutic alkylating agents.

A series of sulfonate esters that are attached to a noncationic minor-groove-binding N-methylpyrrole dipeptide (Lex) related to netrospin have been synthesized. The compounds prepared differ in two respects: (1) the length [(CH2)2 vs (CH2)8] of the tether between the DNA affinity binding portion of the molecule and the sulfonate ester and (2) whether a methyl group [MeOSO2(CH2)n-Lex] or the dipeptide including the aliphatic tether [MeSO2O(CH2)n-Lex] is covalently transferred to the DNA. The DNA-cleavage patterns of these bimolecular alkylating compounds have been mapped in 32P-end-labeled restriction fragments using neutral thermal hydrolysis and alkali treatment to expose single-strand breaks at bases with thermally labile modifications. In contrast to the alkylation of DNA by simple alkyl alkanesulfonate esters, that predominantly yield major-groove alkylation at N7-guanine, the modification of DNA by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex occurs primarily at N3-adenine residues associated with previously footprinted Lex DNA affinity binding regions. The ratio for the formation of N3-methyladenine (minor groove) to N7-methylguanine (major groove) in calf thymus DNA is 1:7 for dimethyl sulfate, while only the former adenine product is observed with MeSO2O(CH2)n-Lex indicating the change in groove specificity. DNA cleavage by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex is efficiently inhibited by the coaddition of distamycin; however, only the DNA damage generated by the latter is blocked by NaCl. As expected, increasing the length of the (CH2)n tether from n = 2 to n = 8 moves the alkylation site by 1-2 base pairs further from the affinity binding domain. Finally, a comparison of the methylation patterns of MeOSO2(CH2)n-Lex as a function of tether length provides an insight into Lex sequence and orientational preferences.

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2′-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2–8 kcal mol−1 over a 16–116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5′ of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

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