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

• Published in: The Journal of biological chemistry 1992-12, Vol.267 (34), p.24810-24818
• Main Authors: LESSER, D. R, GRAJKOWSKI, A, KURPIEWSKI, M. R, KOZIOLKIEWICZ, M, STEC, W. J, JEN-JACOBSON, L
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Deoxyribonuclease EcoRI - chemistry; Deoxyribonuclease EcoRI - metabolism; DNA - chemistry; DNA - metabolism; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemical synthesis; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - metabolism; Organothiophosphates - metabolism; Protein Conformation; Stereoisomerism; Structure-Activity Relationship; Substrate Specificity; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)35836-8

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.