The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC

The interaction of BamHI endonuclease with DNA has been studied crystallogr aphically, but has not been characterized rigorously in solution. The enzym e binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence o f the catalytic cofactor Mg2+) increases 5400-fold as oligonucleotide lengt h increases from 10 to 14 bp. Binding is modulated by sequence context outs ide the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects bi nding by influencing the free energy levels of the complexes rather than th at of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate conta cts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg2+, first- order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate l ength in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca2+ at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative c harges at the catalytic site contributes at least 3-4 kcal/mol of unfavorab le binding free energy. This electrostatic repulsion destabilizes the enzym e-DNA complex and favors metal ion binding and progression to the transitio n state for cleavage. (C) 2001 Academic Press.