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

Abstract

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.

DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice

“…Moreover, inflammationinduced εA, εC, and 8oxoG increased more dramatically in the Aag-deficient animals, consistent with the ability of Aag to excise both εA and 8oxoG. Interestingly, although Aag binds tightly to εC, it does so without catalyzing its excision (38); it seems likely that the binding of Aag to εC in vivo facilitates its repair via an alternative repair pathway. In summary, the accumulation of DNA base damage in Aag -/-mice in response to DSS provides a causal link between repair deficiency and carcinogenesis.…”

“…Of the base lesions found at higher levels with DSS treatment and Aag deficiency, εA induces A:T to G:C and A:T to T:A mutations, εC induces C:G to A:T and C:G to T:A mutations, and 8oxoG induces G:C to T:A transversions (39)(40)(41). In addition, both εC and εA are replication-blocking lesions (38,42). It has been reported that DSS promotion of AOM-or dimethylhydrazine-initiated tumors (AOM is a proximal carcinogen of dimethylhydrazine) results in tumors with predominantly G:C to A:T transitions in Ctnnb1, consistent with the mutational specificity of these carcinogens (43,44).…”

Damage of DNA and proteins by major lipid peroxidation products in genome stability

“…ANPG binds strongly to DNA containing εC, and although does not excise this modified base, does not allow its excision by the proper εC glycosylase, like TDG [95]. This may explain observation that the level of εC measured in different human tissues was constantly higher than that of εA [96].…”

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

“…With regard to the etheno-base adducts (see Fig. 1 -ethenocytosine lesion (33). MYH (MUTYH) is unusual in that it recognizes the 8-oxodG A base pair but removes the normal DNA base adenosine (Table 2), among other lesions.…”

“…-ethenocytosine lesion in DNA but not excise the lesion, effectively ''hijacking'' the repair protein (33). -ethenocytidine (K) (15,19,74).…”