A cDNA expression library from a human cell line was introduced into an E. coli strain deficient in the repair of 3-meAde bases in DNA. E. coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents. A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate. Crude extracts of the E. coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA. The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1. The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells.

Single-stranded M13mp18 phage DNA was methylated with dimethylsulfate (DMS), and further treated with alkali to ring-open N7-methylguanine residues and yield 2-6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) residues. Nucleotide incorporation during in vitro DNA synthesis on methylated template using E. coli DNA polymerase Klenow fragment (Kf polymerase) was reduced compared to the unmethylated template. Additional treatment of the methylated template with NaOH to generate Fapy residues, further reduced in vitro DNA synthesis compared to the synthesis on methylated templates, which suggested that Fapy residues were a block to in vitro DNA synthesis. Analysis of the termination products on sequencing gels, assuming that synthesis stops one base before a blocking lesion, indicated that arrest of DNA synthesis upon direct alkylation of single-stranded DNA occurred 1 base 3' to template adenine residues in the case of Kf polymerase and 1 base 3' to adenine and cystosine residues for T4 polymerase. When the alkylated templates were treated with NaOH to produce a template which converted all the N7-methylguanine residues to Fapy residues, the blocks to DNA synthesis were still observed one base before adenine residues. In addition to the stops previously observed for the methylated templates, however, new stops occurred one base 3' to template guanine residues for synthesis using both Kf polymerase and T4 polymerase. Fapy residues, therefore, represent a potential lethal lesion which may also arrest in vivo DNA synthesis if not repaired.

Nitrogen mustards are among the DNA alkylating agents most widely used in chemotherapy. The homogeneous Escherichia coli AlkA protein (3-methyladenine-DNA glycosylase II) is shown to excise damaged guanine and adenine bases from DNA modified by mechlorethamine, uracil mustard, phenylalanine mustard and chlorambucil, and less efficiently acridine mustard adducts. Homogeneous recombinant human and rat 3-methyladenine-DNA glycosylases excise adducts formed by nitrogen mustards less efficiently than the AlkA protein. In addition to the in vitro excision of adducts, the AlkA protein eliminates cytotoxic mechlorethamine adducts from DNA in vivo.

The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.