title: Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.
abstract: We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
authors.forename: D R
authors.initials: DR
authors.lastname: Lesser
authors.forename: A
authors.initials: A
authors.lastname: Grajkowski
authors.forename: M R
authors.initials: MR
authors.lastname: Kurpiewski
authors.forename: M
authors.initials: M
authors.lastname: Koziolkiewicz
authors.forename: W J
authors.initials: WJ
authors.lastname: Stec
authors.forename: L
authors.initials: L
authors.lastname: Jen-Jacobson
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D063086:Organothiophosphates / Q000378:metabolism*; D011487:Protein Conformation; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009838:Oligodeoxyribonucleotides; D063086:Organothiophosphates; D004247:DNA; D015246:Deoxyribonuclease EcoRI