The promutagenic and genotoxic exocyclic DNA adduct 1,N 2 -ethenoguanine (1,N 2 -⑀G) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatchspecific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N 2 -⑀G from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N 2 -⑀G lesion more efficiently (Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N 6 -ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N 2 -⑀G-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N 2 -⑀G when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N 2 -⑀G when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N 2 -⑀G-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N 2 -⑀G residues in vivo.

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(⑀)-adducts. E-bases have been implicated in vinyl chlorideinduced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N 4 -ethenocytosine (⑀C) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to ⑀C when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the ⑀C⅐ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the ⑀C⅐ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that ⑀C could be more genotoxic than 1,N 6 -ethenoadenine (⑀A) residues in vivo. The proposed model of ANPG-mediated genotoxicity of ⑀C provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.

No abstract