title: The energetic basis of specificity in the Eco RI endonuclease--DNA interaction.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, PA 15260.
authors.forename: D R
authors.initials: DR
authors.lastname: Lesser
authors.forename: M R
authors.initials: MR
authors.lastname: Kurpiewski
authors.forename: L
authors.initials: L
authors.lastname: Jen-Jacobson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010710:Phosphates; D004247:DNA; D015246:Deoxyribonuclease EcoRI