Covalent capture of a human O-6-alkylguanine alkyltransferase-DNA complex using N-1,O-6-ethanoxanthosine, a mechanism-based crosslinker

The DNA repair protein O-6-alkylguanine alkyltransferase (AGT) is responsib le for removing promutagenic alkyl lesions from exocyclic oxygens located i n the major groove of DNA, i.e. the O-6 and O-4 positions of guanine and th ymine. The protein carries out this repair reaction by transferring the alk yl group to an active site cysteine and in doing so directly repairs the pr emutagenic lesion in a reaction that inactivates the protein. In order to t rap a covalent AGT-DNA complex, oligodeoxyribonucleotides containing the no vel nucleoside N-1,O-6-ethanoxanthosine (X-e) have been prepared. The eX nu cleoside was prepared by deamination of 3 ' ,5 ' -protected O-6-hydroxyethy l-2 ' -deoxyguanosine followed by cyclization to produce 3 ' ,5 ' -protecte d N-1, O-6-ethano-2 ' -deoxyxanthosine, which was converted to the nucleosi de phosphoramidite and used in the preparation of oligodeoxyribonucleotides . Incubation of human AGT with a DNA duplex containing X-e resulted in the formation of a covalent protein-DNA complex. Formation of this complex was dependent on both active human AGT and X-e and could be prevented by chemic al inactivation of the AGT with O-6-benzyIguanine. The crosslinking of AGT to DNA using OX occurs with high yield and the resulting complex appears to be well suited for further biochemical and biophysical characterization.