The Energetics of the Interaction of BamHI Endonuclease with its Recognition Site GGATCC Lisa E. Engler 1 , Paul Sapienza 1 , Lydia F. Dorner 2 , Rebecca Kucera 2 , Ira Schildkraut 2 and Linda Jen-Jacobson 1 * 1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh PA 15260, USA 2 New England Biolabs, Beverly MA 01915, USA The interaction of BamHI endonuclease with DNA has been studied crys- tallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding af®nity (in the absence of the catalytic cofactor Mg 2 ) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) ¯anking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by in¯uencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg 2 , ®rst- order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by sub- strate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca 2 at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repul- sion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage. # 2001 Academic Press Keywords: BamHI endonuclease; protein-DNA recognition; ¯anking sequence; electrostatic repulsion; phosphate contacts *Corresponding author Introduction Restriction endonucleases have generated signi®- cant interest because of their biological function in host-controlled restriction 1 and their well-known uses in recombinant DNA technologies. Their highly speci®c interactions with their DNA recog- nition sites also present an attractive set of models for site-speci®c protein-DNA interactions. The endonucleases discriminate against incorrect DNA sites much more stringently than do other site- speci®c DNA-binding proteins, 2 so that one might expect these enzymes to exemplify the most rigor- ous rules and constraints that govern speci®city. The structures of a substantial number of restric- tion endonucleases in complex with their DNA rec- ognition sites have now been solved 3±9 and for EcoRV, BamHI and EcoRI, multiple high-resolution structures (e.g. cognate and non-speci®c com- plexes, with or without divalent metals) are available 6,10-15 (J. M. Rosenberg, unpublished struc- tures deposited in RCSB Protein Data Bank). Despite these evident advantages, the study of the solution thermodynamics of these protein-DNA interactions has lagged behind the structural characterization. Extensive thermodynamic and pre-steady-state kinetic data were ®rst obtained for EcoRI endonuclease, 16-20 which demonstrates high binding speci®city in the absence of the divalent cation (Mg 2 ) required for catalysis, as does its iso- schizomer RsrI. 21 The progress of similar studies on EcoRV endonuclease has generated some con- troversy because some reports claim little or no DNA-binding speci®city in the absence of divalent cation, 22-24 whereas other studies suggest that sub- stantial speci®c binding can be detected, 25 and is E-mail address of the corresponding author: LJEN@pitt.edu doi:10.1006/jmbi.2000.4428 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 619±636 0022-2836/01/020619±18 $35.00/0 # 2001 Academic Press