A HIGH RESOLUTION STRUCTURE OF AN EcoRV-DNA COMPLEX. Mark P.Thomas, E.Louise Hancox, Stephen E.Halford & R.Leo Brady.

Department of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

Structures of EcoRV complexed to substrate and product DNA have previously been refined to 2Å (Kostrewa & Winkler, 1995). On the basis of these structures and kinetic data (Vipond et al, 1995, Baldwin et al, 1995) a model of the transition state with two metals bound to the scissile phosphorane group has been proposed.

We are now studying EcoRV complexed with a series of altered DNA substrates. Studies of the influence of the phosphate backbone on recognition and hydrolysis of DNA by EcoRV have utilised diastereoisomeric phosphorothioate DNA analogues (Thorogood et al, 1996). The rate of hydrolysis is dependent on the position of the phosphorothioate moiety in the recognition sequence and on the diastereoisomer. By determining crystal structures of EcoRV co-crystallised with each of these phosphorothioate DNA analogues we hope to provide a firmer structural basis to explanations of the data pertaining to reaction kinetics and substrate recognition and specificity.

Baldwin, G.S., Vipond, I.B. & Halford, S.E. (1995) Biochemistry, 34, 705-714.

Kostrewa, D. & Winkler, F.K. (1995) Biochemistry, 34, 683-696.

Vipond, I.P., Baldwin, G.S. & Halford, S.E. (1995) Biochemistry, 34, 697-704.

Thorogood, H., Grasby, J.A., Connolly, B.A. (1996) J.Biol.Chem. in press.