High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.

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The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu 2þ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu 2þ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu 2þ -ion based long-range distance constraints. Double electron-electron resonance data yield Cu 2þ -Cu 2þ and Cu 2þ -nitroxide distances that are uniquely consistent with one Cu 2þ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu 2þ ions bind per complex. Unexpectedly, Mg 2þ -catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu 2þ binding at these hitherto unknown sites, 13 Å away from the Mg 2þ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu 2þ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu 2þ , the Mg 2þ -dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable doublestrand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg 2þ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg 2þ . Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.T he biochemical basis of specificity in the interaction of proteins with DNA sites is a major problem of modern molecular genetics. Studies of many protein-DNA complexes by crystallography have elucidated the intermolecular recognition contacts (1), but it is clear that point-to-point contacts cannot fully explain specificity. Solution biochemical and computational studies have shown that a comprehensive view of specificity determination must also include factors such as shape recognition (2), mutual accommodation of the macromolecules through DNA distortion or conformational selection (1, 3-6), and/or DNA-induced protein folding (5, 7). Thermodynamic studies have revealed that specific protein-DNA association is often driven primarily by the favorable entropy increase provided by desolvation of the apposed complementary surfaces (8-10).For those DNA-binding proteins that are also DNA-modifying enzymes (nucleases, methylases, recombinases, repair enzymes, etc.) a key question is the relationship between DNA-binding specificity and catalytic specificity. One exemplary system for addressing specificity determination is the EcoRI endonuclease (3, 11), a 62 kDa homodimer that recognizes the DNA site 5′-GAATTC-3′ and binds as much as 90,000-fold better (3, 12) than a...

We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an intersrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.The highly selective recognition of the DNA sequence GAATTC by EcoRI endonuclease involves a number of interdependent contributions to specificity. The structure of endonuclease-DNA cocrystalline complexes (1, 2) shows that the endonuclease makes hydrogen bonds or nonpolar contacts with nearly all major-groove functional groups ofthe bases. The protein also makes symmetrical contacts to DNA phosphates on both strands at pNpGAApTTC. These contacts are indispensable to recognition of the canonical site (3, 4), and a subset (pNGAApTTC) contributes to discrimination against sites with one incorrect base pair (EcoRI* sites) by "adapting" in a stereotypical pattern (3), suggesting that formation of the canonical set of protein-phosphate contacts is sequence dependent.Taking into account the favorable energetic contributions of the protein-base and protein-phosphate interactions and the large favorable contribution (5) of the "hydrophobic effect," we proposed (3) that the energetic cost of distorting the DNA in the complex makes a significant unfavorable contribution to the interaction with the correct DNA site and an even more unfavorable contribution to interaction with incorrect natural DNA sites.In the canonical complex, the DNA adopts a pronounced torsional kink (2) in the middle of the recognition site that unwinds the DNA and widens the major groove to permit insertion of the recognition elements of the endonuclease (1, 2). This kinked geometry differs from that ofthe free DNA (6) in an exaggerated negative roll angle between the central base pairs (-57°; compared with free DNA -6°; J. M. Rosenberg, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.personal communication) such that these base pairs are completely unstacked; this alone may cost +8 kcal/mol (7). Other import...

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