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.