Nucleic Acids Research, 2001, Vol. 29, No. 19 4025-4034
© 2001 Oxford University Press
Covalent capture of a human O6-alkylguanine alkyltransferase–DNA complex using N1,O6-ethanoxanthosine, a mechanism-based crosslinker
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
The DNA repair protein O6-alkylguanine alkyltransferase (AGT) is responsible for removing promutagenic alkyl lesions from exocyclic oxygens located in the major groove of DNA, i.e. the O6 and O4 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 N1,O6-ethanoxanthosine (eX) have been prepared. The eX nucleoside was prepared by deamination of 3',5'-protected O6-hydroxyethyl-2'-deoxyguanosine followed by cyclization to produce 3',5'-protected N1,O6-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 eX resulted in the formation of a covalent protein–DNA complex. Formation of this complex was dependent on both active human AGT and eX and could be prevented by chemical inactivation of the AGT with O6-benzylguanine. The crosslinking of AGT to DNA using eX occurs with high yield and the resulting complex appears to be well suited for further biochemical and biophysical characterization.
* To whom correspondence should be addressed. Tel: +1 410 614 1760; Fax: +1 410 502 6910; Email: dmndawit{at}jhmi.edu
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Alzeer and O. D. Scharer A modified thymine for the synthesis of site-specific thymine-guanine DNA interstrand crosslinks Nucleic Acids Res., September 11, 2006; 34(16): 4458 - 4466. [Abstract] [Full Text] [PDF]
|
|