Journal of Molecular Biology
Regular articleThe energetics of the interaction of BamHI endonuclease with its recognition site GGATCC1
Introduction
Restriction endonucleases have generated significant interest because of their biological function in host-controlled restriction1 and their well-known uses in recombinant DNA technologies. Their highly specific interactions with their DNA recognition sites also present an attractive set of models for site-specific protein-DNA interactions. The endonucleases discriminate against incorrect DNA sites much more stringently than do other site-specific DNA-binding proteins,2 so that one might expect these enzymes to exemplify the most rigorous rules and constraints that govern specificity.
The structures of a substantial number of restriction endonucleases in complex with their DNA recognition sites have now been solved3, 4, 5, 6, 7, 8, 9 and for EcoRV,BamHI and EcoRI, multiple high-resolution structures (e.g. cognate and non-specific complexes, with or without divalent metals) are available6, 10, 11, 12, 13, 14, 15 (J. M. Rosenberg, unpublished structures deposited in RCSB Protein Data Bank).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 first obtained for EcoRI endonuclease,16, 17, 18, 19, 20 which demonstrates high binding specificity in the absence of the divalent cation (Mg2+) required for catalysis, as does its isoschizomer RsrI.21 The progress of similar studies on EcoRV endonuclease has generated some controversy because some reports claim little or no DNA-binding specificity in the absence of divalent cation,22, 23, 24 whereas other studies suggest that substantial specific binding can be detected,25 and is only enhanced25, 26, 27 by Ca2+, which does not support catalysis. Similarly, the TaqI,28 Cfr9I,29 PvuII,30 Cfr10I31and MunI32 endonucleases are reported to have very weak or no binding specificity in the absence of divalent cation. Specific binding by these enzymes is enhanced by several factors which may be related: the presence of Ca2+ (PvuII,30 MunI,32 Cfr10I31), lower pH (MunI,32 EcoRV25) or mutations that eliminate active-site carboxylate residues.33 These are not unique properties of the latter group, however, since EcoRI binding is also enhanced by the same factors16 (G. Bosco and L.J.-J., unpublished results). It is thus important, in the interest of further thermodynamic studies to illuminate structure-function relationships, that we understand whether there are truly two distinct classes of restriction endonucleases which differ in their dependence on divalent cation to “trigger” or “confer” specificity33 or, alternatively, whether these various enzymes represent a continuum of specific-binding affinities which are only enhanced in various degrees by the binding of divalent cations at their active sites.
The earlier experience with EcoRI and EcoRV endonucleases suggests another potentially obscuring issue: it is easy to be misled by arbitrary choices of DNA substrates, conditions and/or methods for studying protein-DNA interactions. For example, it was shown for EcoRV endonuclease25 that the widely used gel-retardation method yielded anomalously low values of the equilibrium association constant under suboptimal conditions, but gave excellent agreement with the membrane-filter-binding method if scrupulous attention was paid to conditions such as pH and salt concentration. Furthermore, it has long been known34 that EcoRI cleavage is sensitive to DNA base sequence surrounding the recognition site, and much later shown for both EcoRI35, 36, 37 and EcoRV25 endonucleases that this effect represents a surprisingly large influence on protein-DNA binding. This factor should be explored systematically rather than preselected for some arbitrary reason such as resemblance to a plasmid sequence of dubious biological relevance.
Here, we introduce BamHI endonuclease as a new model system. BamHI endonuclease has been well studied crystallographically: structures are available for the apoenzyme,38 the specific recognition complex without6 and with11 divalent metals, for the post-cleavage complex11 and for the non-specific complex.15 These structures demonstrate that BamHI and EcoRI endonucleases have a striking structural homology in the spatial arrangement of the α/β-core that includes the recognition α-helices and the active site residues, even though there is little or no homology between these enzymes at the level of primary sequence.39 The BamHI recognition sequence GGATCC is related to that of EcoRI (GAATTC) by two symmetrical A·T to G·C changes; both the similarities and the differences in these sites provide potentially useful points of comparison. We report here the fundamental features of the BamHI endonuclease equilibrium interaction with DNA in solution, as well as some elementary aspects of the pre-steady-state cleavage kinetics. We show that protein-DNA binding is strongly sensitive to both the length of a DNA oligonucleotide and the base sequence surrounding the GGATCC site. We provide data showing that flanking-sequence preferences are dominated by the immediately abutted three base-pairs on both sides of the cognate site, and that preferences do not simply correlate with thermal stability of the DNA duplex.40 We also show that a cluster of negative charges at the BamHI catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy.
Section snippets
Oligomeric state of BamHI endonuclease in solution
The intracellular concentration of BamHI has been estimated to be slightly less than 1 μM.41 In the absence of DNA, the protein demonstrates a dimer-tetramer equilibrium at micromolar concentrations42 forming tetramers below 0.3 M NaCl and dimers at 0.45 M NaCl or higher. Since most in vitro studies are carried out at relatively low salt concentrations (0.1-0.2 M), we directly determined the oligomeric state of the active (DNA-binding) form of BamHI endonuclease by gel-permeation chromatography
Conclusions: principal features of the BamHI model system
Our data show that the interaction of BamHI endonuclease with DNA has significant similarities with those of EcoRI and EcoRV endonucleases, and also significant differences. All three enzymes exhibit localized, site-specific DNA binding in the absence of divalent metal, although binding is enhanced by divalent metals (Table 3). Divalent metal ions do not “trigger” binding specificity for EcoRI or BamHI, since site-specific complexes in the absence of divalent metal show “thermodynamic
BamHI endonucleases
Wild-type BamHI and mutant E113K endonucleases were overexpressed and purified as described.41 A gene encoding mutant E111A was cloned between the NdeI and SapI sites downstream of the T7 promoter in the pTYB1 vector of the IMPACT-CN system (New England Biolabs) such that the fusion order was BamHI endonuclease-intein-chitin binding domain. The E111A mutant protein was then purified as described66 by chitin-affinity chromatography; cleavage of the intein to release the protein leaves no
Acknowledgements
This work was supported in part by grant GM-29207 from the National Institutes of Health to L.J.-J. We are grateful to Lewis Jacobson for many insightful discussions and critical reading of the manuscript.
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Edited by R. Ebright