Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.
Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical. Our hypothesis also predicts that the linkage between DNA and MGMT is 1-(guan-1-yl)-2-(cystein-S-yl)ethane. This notion was tested by forming the complex with 35S-labeled recombinant human MGMT and a chloroethylnitrosourea-treated oligodeoxynucleotide. After degradation by depurination and proteolytic digestion, the identity of the [35S]cysteine-guanine linkage was confirmed by comparison with the synthetic marker compound using high performance liquid chromatography and UV spectrometry. These results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor. The data also suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA-MGMT complex formation may constitute a significant secondary lesion.
Gonzaga
P E
PE
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Potter
P M
PM
Niu
T Q
TQ
Yu
D
D
Ludlum
D B
DB
Rafferty
J A
JA
Margison
G P
GP
Brent
T P
TP
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase
D000970:Antineoplastic Agents; C017139:1-(2-chloroethyl)-1-nitrosourea; D006147:Guanine; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005038:Ethylnitrosourea