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.

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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The "UV footprinting" technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure ofthe EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.By itself, the DNA double helix is genetically inactive. The binding of sequence-specific proteins to the DNA helix is necessary to accurately transcribe, replicate, modify, rearrange, and repair genetic messages. The ability to detect and monitor sequence-specific protein-DNA interactions thus provides a powerful tool for elucidating the molecular mechanisms responsible for rendering the genetic message biologically active, stable, and inheritable.Becker and Wang (1) described the development of a "UV footprinting" technique that can detect sequence-specific protein-DNA interactions in vivo. Protein contacts can inhibit or enhance UV-photoproduct formation by affecting the ability of DNA to adopt a geometry necessary for the formation of a UV photoproduct. Thus, differences in the strand-breakage patterns of protein-free and protein-bound DNA can be used to detect protein-DNA contacts at singlenucleotide resolution.Sequence-specific contacts between DNA and regulatory proteins, such as the lac repressor and Xenopus transcription factor IIIA, strongly inhibit the ability of bound DNA to be damaged by UV light (1,2). In contrast, proteins that do not make sequence-specific contact with the DNA helix, such as nucleosomes or histone H1, only weakly alter the UV photoreactivity of DNA (2).In this paper, the ability of UV light to detect intimate contacts between the EcoRI endonuclease and its recognition sequence is examined in detail. Because a high-resolution x-ray crystal structure of the endonuclease-DNA complex is available (3), we are able to show that strong inhibition of UV photoreactivity at particular bases occurs when the mobility of the photoreactive base is restricted by protein contact. We also demonstrate that kinking of the DNA phosphate backbone by the endonuclease greatly enhances the UV photoreactivity of DNA at the site of the kink. MATERIALS AND METHODSEnzyme and DNA Preparation. EcoRI endonuclease, the EcoRI derivative DELN29, and oligonucleotide ( Fig. 1) were prepared and purified as described (4).Ethylation-Interference ...

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