TY - JOUR
T1 - 1,N2-ethenoguanine, a mutagenic DNA adduct, is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase
AU - Elder, Rhoderick
AU - Saparbaev, Murat
AU - Langouët, Sophie
AU - Privezentzev, Cyril V.
AU - Peter Guengerich, F.
AU - Cai, Hongliang
AU - Elder, Rhoderick H.
AU - Laval, Jacques
PY - 2002/7/26
Y1 - 2002/7/26
N2 - The promutagenic and genotoxic exocyclic DNA adduct 1,N2-ethenoguanine (1,N2-εG) 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,N2-εG 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,N2-εG lesion more efficiently (kcat/Km 0.95 × 10-3 min-1 nM-1) than the ANPG protein (kcat/Km = 0.1 × 10-3 nM-1). Additionally, while the non-conserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N6-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N2-εG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N2-εG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N2-εG 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,N2-εG-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,N2-εG residues in vivo.
AB - The promutagenic and genotoxic exocyclic DNA adduct 1,N2-ethenoguanine (1,N2-εG) 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,N2-εG 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,N2-εG lesion more efficiently (kcat/Km 0.95 × 10-3 min-1 nM-1) than the ANPG protein (kcat/Km = 0.1 × 10-3 nM-1). Additionally, while the non-conserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N6-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N2-εG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N2-εG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N2-εG 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,N2-εG-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,N2-εG residues in vivo.
U2 - 10.1074/jbc.M111100200
DO - 10.1074/jbc.M111100200
M3 - Article
SN - 1083-351X
VL - 277
SP - 26987
EP - 26993
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 30
ER -