Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".
Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.
Oh
H K
HK
Chemical Carcinogenesis Laboratory, National University of Singapore, Republic of Singapore.
Teo
A K
AK
Ali
R B
RB
Lim
A
A
Ayi
T C
TC
Yarosh
D B
DB
Li
B F
BF
D000477:Alkylating Agents / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004260:DNA Repair; D018604:Epitope Mapping; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006847:Hydrocarbons, Iodinated / Q000378:metabolism / Q000494:pharmacology; D008780:Methyltransferases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017354:Point Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D006847:Hydrocarbons, Iodinated; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; C014055:methyl iodide; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003545:Cysteine