The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC.
The interaction of BamHI endonuclease with DNA has been studied crystallographically, 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 affinity (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) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing 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+), 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 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 repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.
Engler
L E
LE
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Sapienza
P
P
Dorner
L F
LF
Kucera
R
R
Schildkraut
I
I
Jen-Jacobson
L
L
D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D015245:Deoxyribonuclease BamHI / Q000737:chemistry / Q000378:metabolism*; D004734:Energy Metabolism; D007700:Kinetics; D015335:Molecular Probes; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D012996:Solutions; D055672:Static Electricity; D013816:Thermodynamics
D002413:Cations, Divalent; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D012996:Solutions; D004247:DNA; D015245:Deoxyribonuclease BamHI