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Uracil as an Alternative to 5-Fluorocytosine
in Addressable Protein Targeting

J.A. Wendel and S.S. Smith*

    City of Hope National Medical Center
1500 E. Duarte Rd.
Duarte, CA 91010-3000
email:
ssmith@coh.org

This is an abstract for a talk to be given at the
Fifth Foresight Conference on Molecular Nanotechnology.
The full paper is now available.

 

Bacterial DNA methyltransferases offer an approach to addressable protein targeting in macromolecular assembly that may permit the construction of a variety of usefully ordered protein arrays (Smith 1995, Smith et al. 1997, Smith et al. 1997a). In this system for macromolecular assembly, the natural recognition specificity of the bacterial DNA cytosine methyltransferases is used to target fusion proteins to preselected sites on a DNA scaffold through the formation of a stable covalent attachment with the mechanism-based inhibitor, 5-Fluorocytosine. Current evidence on the mechanism of action of bacterial DNA methyltransferases suggests that proton circulation during catalysis can facilitate substrate binding, nucleophilic attack and product release. Ab initio modeling of the reaction has been used to analyze the mechanism of inhibition for commonly studied inhibitors of the reaction in order to determine whether or not they can substitute for 5-Fluorocytosine in DNA as covalent targets for the enzymes. The ab initio data suggest that in addition to the well known thermodynamic barrier to b-elimination that blocks the release of the methyltransferase from the 5-fluoro, 5-methyl-dihydrocytosine formed with 5-fluorocytosine, each of the these inhibitors creates a significant kinetic barrier to methyl transfer by lowering the energy of the HOMO (Highest Occupied Molecular Orbital) of the activated pyrimidine relative to that of activated cytosine. This kinetic barrier causes methyltransferases to stall after covalent attachment to C6 of any of the cytosine analogs studied. Subsequent methyltransfer to produce the 5-methyldihydropyrimidine complex is slow for each of these compounds with the relative reaction rates consistent with the calculated values of the HOMO for five inhibitors studied. Of these inhibitors, dUra is readily attacked and stable complexes are formed in the absence of significant methyltransfer. The data suggest that dUra is a valuable and cost effective substitute for 5FdCyt in the production of ordered macromolecular assemblies.

*Supported by grant N00014-94-1-1116 from the Office of Naval Research

References
[1] Smith, S. S., (1995) Nucleoprotein-Based Nanoscale Fabrication. In: Biological and Biomedical Science and Technology Division 1995 Programs, ed. E. Eisenstadt, (U.S. Navy Publication ONR 34196-3), pp. 161-162.
[2] Smith, S.S., Niu, L., Baker, D.J., Wendel, J.A., Kane, S. E. and Joy, D.S. (1997) Nanoscale Addressing in Macromolecular Assembly. Miami BiotechnologyShort Reports, Vol. 8. F.Ahmad et al., eds. IRL at Oxford University Press, p.13.
[3] Smith, S.S., Niu, L. Baker, D.J., Wendel, J.A. Kane, S.E.,and Joy, D.S (1997a) Nucleoprotein Based Nanoscale Assembly. Proc. Natl. Acad. Sci. U.S.A. 94:2162-2167.


*Corresponding Address:
Steven Smith, City of Hope National Medical Center, 1500 E. Duarte Rd, Duarte, CA 91010-3000, ph: 818-301-8316, fax: 818-301-8972, email: ssmith@coh.org or jaw@131.215.33.4



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