Chiral Phosphorothioates as Probes of Protein Interactions with Individual DNA Phosphoryl Oxygens:  Essential Interactions of EcoRI Endonuclease with the Phosphate at pGAATTC

• Published in: Biochemistry (Easton) 1996-07, Vol.35 (27), p.8846-8854
• Main Authors: Kurpiewski, Michael R, Koziolkiewicz, Maria, Wilk, Andrzej, Stec, Wojciech J, Jen-Jacobson, Linda
• Format: Article
• Language: English
• Subjects: Base Sequence; Deoxyribonuclease EcoRI - metabolism; DNA - metabolism; DNA-Binding Proteins - metabolism; Escherichia coli; Kinetics; Models, Molecular; Molecular Sequence Data; Oligodeoxyribonucleotides - metabolism; Oligonucleotide Probes - metabolism; Protein Binding - drug effects; Sodium Chloride - pharmacology; Stereoisomerism; Substrate Specificity; Thionucleotides - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960261e

The contact between EcoRI endonuclease and the “primary clamp” phosphate of its recognition site pGAATTC is absolutely required for recognition of the canonical and all variant DNA sites. We have probed this contact using oligonucleotides containing single stereospecific (R P)− or (S P)−phosphorothioates (Ps). At the GAApTTC position, where the endonuclease interacts with only one phosphoryl oxygen at the central DNA kink, R P-Ps inhibits and S P-Ps stimulates binding and cleavage [Lesser et al. (1992) J. Biol. Chem. 267, 24810−24818]; in contrast, at the pGAATTC position both diastereomers inhibit binding. For single-strand substitution, the penalty in binding free energy (ΔΔG°bind) is slightly greater for S P-Ps (+0.9 kcal/mol) than for R P-Ps (+0.7 kcal/mol). Binding penalties are approximately additive for double-strand substitution (R P,R P-Ps or S P,S P-Ps). Neither Ps diastereomer in one DNA strand affects the first-order rate constants for cleavage in the unmodified DNA strand, and only S P-Ps inhibits the cleavage rate constant (3-fold) in the modified DNA strand. Thus, the second-order cleavage rate (including binding and catalysis) is inhibited 14-fold by S P-Ps and 45-fold by S P,S P-Ps. In the canonical complex, the phosphate at pGAATTC is completely surrounded by protein and each nonbridging phosphoryl oxygen receives two hydrogen bonds from the endonuclease, such that in either orientation the increased bond length of P−S- inhibits binding. However, the pro-S P oxygen interacts with residues that are connected (by proximity or inter-side-chain hydrogen bonding) to side chains with essential roles in catalysis, so cleavage is preferentially inhibited when these side chains are slightly displaced by the S P-Ps diastereomer.