ROLE OF PROTEIN-INDUCED BENDING IN THE SPECIFICITY OF DNA RECOGNITION- CRYSTAL-STRUCTURE OF ECORV ENDONUCLEASE COMPLEXED WITH D(AAAGAT)+D(ATCTT)

The crystal structure of EcoRV endonuclease has been determined at 2.1 Angstrom resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous targe t site are not essential to enzyme-induced DNA bending, even when thes e constraints are removed directly at the locus of the bend. The sciss ile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like confor mation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate s ites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynami c considerations suggest that the sequence-dependent energy cost of DN A bending is likely to Flay an important role in determining the speci ficity of EcoRV. This differential cost is manifested at the binding s tep for bent non-cognate sequences and at the catalytic step for unben t non-specific sequences. (C) 1998 Academic Press Limited.