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The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC1

https://doi.org/10.1006/jmbi.2000.4428Get rights and content

Abstract

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 Mg2+) 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 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 length 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 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.

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.

References (74)

  • J. Zebala et al.

    Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease

    J. Biol. Chem.

    (1992)
  • H.G. Nastri et al.

    Catalytic and DNA binding properties of PvuII restriction endonuclease mutants

    J. Biol. Chem.

    (1997)
  • M. Thomas et al.

    Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

    J. Mol. Biol.

    (1975)
  • L. Jen-Jacobson et al.

    Structural and thermodynamic strategies for site-specific DNA binding proteins

    Structure

    (2000)
  • M. Newman et al.

    Structure of restriction endonuclease BamHI phased at 1.95 Å resolution by MAD analysis

    Structure

    (1994)
  • A.A. Travers

    DNA bending and kinking-sequence dependence and function

    Curr. Opin. Struct. Biol.

    (1991)
  • A.A. Gorin et al.

    B-DNA twisting correlates with base-pair morphology

    J. Mol. Biol.

    (1995)
  • M.A. El Hassan et al.

    Propeller-twisting of base-pairs and the conformational mobility of dinucleotide steps in DNA

    J. Mol. Biol.

    (1996)
  • M.J. Packer et al.

    Sequence-dependent DNA structuredinucleotide conformational maps

    J. Mol. Biol.

    (2000)
  • N.C. Horton et al.

    Role of protein-induced bending in the specificity of DNA recognitioncrystal structure of EcoRV endonuclease complexed with d(AAAGAT)+d(ATACTT)

    J. Mol. Biol.

    (1998)
  • L. Jen-Jacobson

    Structural-perturbation approaches to thermodynamics of site-specific protein-DNA interactions

    Methods Enzymol.

    (1995)
  • J.M. Rosenberg

    Structure and function of restriction endonucleases

    Curr. Opin. Struct. Biol.

    (1991)
  • D.R. Lesser et al.

    Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex

    J. Biol. Chem.

    (1992)
  • S.Y. Xu et al.

    Isolation of BamHI variants with reduced cleavage activities

    J. Biol. Chem.

    (1991)
  • F.K. Winkler

    Structure and function of restriction endonucleases

    Curr. Opin. Struct. Biol.

    (1992)
  • K.J. Breslauer

    Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order-disorder transitions

    Methods Enzymol.

    (1995)
  • M.T. Record et al.

    Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between protein and helical DNA

    Methods Enzymol.

    (1991)
  • A.D. Riggs et al.

    The lac repressor operator interaction. III. Kinetic studies

    J. Mol. Biol.

    (1970)
  • R.J. Roberts et al.

    Type II restriction endonucleases

  • Y.C. Kim et al.

    Refinement of EcoRI endonuclease crystal structurea revised protein chain tracing

    Science

    (1990)
  • F.K. Winkler et al.

    The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments

    EMBO J.

    (1993)
  • X. Cheng et al.

    Structure of PvuII endonuclease with cognate DNA

    EMBO J.

    (1994)
  • M. Newman et al.

    Structure of BamHI endonuclease bound to DNApartial folding and unfolding on DNA binding

    Science

    (1995)
  • M. Newman et al.

    Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence

    EMBO J.

    (1998)
  • C.M. Lukacs et al.

    Understanding the immutability of restriction enzymescrystal structure of BglII and its DNA substrate at 1.5 Å resolution

    Nature Struct. Biol.

    (2000)
  • M. Deibert et al.

    Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA

    Nature Struct. Biol.

    (2000)
  • D. Kostrewa et al.

    Mg2+ binding to the active site of EcoRV endonucleasea crystallographic study of complexes with substrate and product DNA at 2 Å resolution

    Biochemistry

    (1995)
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