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Identification of the Cross-Link between Human O⁶-Methylguanine-DNA Methyltransferase and Chloroethylnitrosourea-treated DNA¹

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [P. E. G., P. M. P., T. P. B.]; Department of Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 [T. N., D. Y., D. B. L.]; Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, United Kingdom [J. A. R., G. P. M.]; and Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163 [T. P. B.]

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.

¹ This work was supported by NIH Grants CA14799, CA36888, and CA44499, by the North Atlantic Treaty Organization, by the American Lebanese Syrian Associated Charities, and by the Cancer Research Campaign (United Kingdom).

² To whom requests for reprints should be addressed, at Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.

Received 5/12/92. Accepted 8/24/92.

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