Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct.
Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced 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 (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC 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 epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.
Gros
Laurent
L
Groupe "Réparation de l'ADN," CNRS Unité Mixte de Recherche 8113/LBPA-ENS Cachan, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France.
Maksimenko
Andrei V
AV
Privezentzev
Cyril V
CV
Laval
Jacques
J
Saparbaev
Murat K
MK
D000818:Animals; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D018736:DNA Adducts; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D015227:Lipid Peroxidation; D051379:Mice; D009153:Mutagens; D041681:NIH 3T3 Cells; D009841:Oligonucleotides / Q000737:chemistry; D020033:Protein Isoforms; D020349:Surface Plasmon Resonance; D013997:Time Factors
C018289:3,N(4)-ethenocytosine; D018736:DNA Adducts; D017931:DNA Primers; D004277:DNA, Single-Stranded; D009153:Mutagens; D009841:Oligonucleotides; D020033:Protein Isoforms; D003596:Cytosine; D004247:DNA; D004256:DNA Polymerase I; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II