DIRECT MEASUREMENT OF THE SUBSTRATE PREFERENCE OF URACIL-DNA GLYCOSYLASE

Site-directed mutants of the herpes simplex virus type 1 uracil-DNA gl ycosylase lacking catalytic activity have been used to probe the subst rate recognition of this highly conserved and ubiquitous class of DNA repair enzyme utilizing surface plasmon resonance. The residues aspart ic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1 995) Nature 373, 487-493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87-92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis, The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single-an d double-stranded oligonucleotides, with or without uracil, was monito red by real-time biomolecular interaction analysis using surface plasm on resonance. Both mutants exhibited comparable rates of binding and d issociation on the same uracil-containing substrates, Interaction with single-stranded uracil-DNA was found to be stronger than with double- stranded uracil-DNA, and the binding to Gua:Ura mismatches was signifi cantly stronger than that to Ade:Ura base pairs suggesting that the st ability of the base pair determines the efficiency of interaction. Als o, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sit es, These results suggest that it is uracil in the DNA, rather than DN A itself, that is recognized by the uracil-DNA glycosylases.