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 responsible for removing promutagenic alkyl lesions from exocyclic oxygens located in the major groove of DNA, i.e. the O(6) and O(4) positions of guanine and thymine. The protein carries out this repair reaction by transferring the alkyl group to an active site cysteine and in doing so directly repairs the premutagenic lesion in a reaction that inactivates the protein. In order to trap a covalent AGT-DNA complex, oligodeoxyribonucleotides containing the novel nucleoside N(1),O(6)-ethanoxanthosine ((e)X) have been prepared. The (e)X nucleoside was prepared by deamination of 3',5'-protected O(6)-hydroxyethyl-2'-deoxyguanosine followed by cyclization to produce 3',5'-protected N(1),O(6)-ethano-2'-deoxyxanthosine, which was converted to the nucleoside phosphoramidite and used in the preparation of oligodeoxyribonucleotides. Incubation of human AGT with a DNA duplex containing (e)X resulted in the formation of a covalent protein-DNA complex. Formation of this complex was dependent on both active human AGT and (e)X and could be prevented by chemical inactivation of the AGT with O(6)-benzylguanine. The crosslinking of AGT to DNA using (e)X occurs with high yield and the resulting complex appears to be well suited for further biochemical and biophysical characterization.
Noll
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DM
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. dmndawit@jhmi.edu
Clarke
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ND
D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008956:Models, Chemical; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D012263:Ribonucleosides / Q000138:chemical synthesis / Q000737:chemistry*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C437719:N1,N6-ethanoxanthosine; D009838:Oligodeoxyribonucleotides; D012263:Ribonucleosides; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase