1,N(2)-ethenoguanine, a mutagenic DNA adduct, is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase.
The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) 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 mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG 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)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)). 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)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG 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)-epsilonG-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)-epsilonG residues in vivo.
Saparbaev
Murat
M
Groupe Réparation de l'ADN, Unité Mixte de Recherche 8532 CNRS, Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée-Ecole Normale Supérieure Cachan, Institut Gustave Roussy, 94805 Villejuif Cedex, France. smurat@igr.fr
Langouët
Sophie
S
Privezentzev
Cyril V
CV
Guengerich
F Peter
FP
Cai
Hongliang
H
Elder
Rhoderick H
RH
Laval
Jacques
J
D000818:Animals; D002474:Cell-Free System; D018736:DNA Adducts; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides / Q000494:pharmacology; D011485:Protein Binding; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D018736:DNA Adducts; D009841:Oligonucleotides; C085534:1,(N2)-ethenoguanine; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C076131:DNA-3-methyladenine glycosidase II