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Prevention of 1-(3-Deoxycytidyl),2-(1-deoxyguanosinyl)ethane Cross-Link Formation in DNA by Rat Liver O⁶-Alkylguanine-DNA Alkyltransferase¹

Division of Oncology, Department of Medicine, Albany Medical College, Albany, New York 12208

Previous studies have shown that the haloethylnitrosoureas introduce the cross-link 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane into DNA. This structure is evidently formed by the following sequence of events: an initial attack of a haloethyl group on the O⁶ position of guanine, formation of the reactive intermediate, 1,O⁶-ethanoguanine, and reaction of this intermediate with deoxycytidine in the opposite DNA strand. To investigate the role of O⁶-alkylguanine-DNA alkyltransferase in preventing the formation of this cross-link, a DNA substrate containing O⁶-fluoroethylguanine has been prepared by reacting DNA with N-2-fluoroethyl-N'-cyclohexyl-N-nitrosourea. The O⁶-fluoroethylguanine content of this substrate decreases when it is incubated at 37°C and pH 7.8 in the absence of repair factors because of the chemical instability of O⁶-fluoroethylguanine; however, this loss is accelerated by the addition of rat liver O⁶-alkylguanine-DNA alkyltransferase, indicating that this repair factor recognizes and repairs O⁶-fluoroethylguanine in DNA; furthermore, by using [chloroethyl-¹⁴C]N-chloroethyl-N'-cyclohexyl-N-nitrosourea, it can be shown directly that the addition of rat liver O⁶-alkylguanine-DNA alkyltransferase prevents 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane formation. These studies link the presence of repair activity to the prevention of a specific cytotoxic lesion in DNA.

¹ Supported by USPHS Grants CA 32171 and CA 32446 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.

² To whom requests for reprints should be addressed, at the Division of Oncology, Department of Medicine, Albany Medical College, Albany, NY 12208.

³ Present address: Department of Surgery, Albany Medical College, Albany, NY 12208.

⁴ Present address: Vermont Regional Cancer Center, University of Vermont, Burlington, VT 05405.

Received 9/16/85. Revised 1/ 2/86. Revised 3/26/86. Accepted 3/31/86.