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Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATTC.

Abstract：

: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 (Rp)- or (Sp)- phosphorothioates (Ps). At the GAApTTC position, where the endonuclease interacts with only one phosphoryl oxygen at the central DNA kink, Rp-Ps inhibits and Sp-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 (delta delta G0bind) is slightly greater for Sp-Ps (+ 0.9 kcal/mol) than for Rp-Ps (+ 0.7 kcal/mol). Binding penalties are approximately additive for double-strand substitution (Rp,Rp-Ps or Sp,Sp-Ps). Neither Ps diastereomer in one DNA strand affects the first-order rate constants for cleavage in the unmodified DNA strand, and only Sp-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 Sp-Ps and 45-fold by Sp,Sp-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-Sp 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 Sp-Ps diastereomer.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Kurpiewski MR,Koziolkiewicz M,Wilk A,Stec WJ,Jen-Jacobson L

doi

10.1021/bi960261e

subject

Has Abstract

pub_date

1996-07-09 00:00:00

pages

8846-54

issue

eissn

0006-2960

issn

1520-4995

pii

bi960261e

journal_volume

pub_type

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Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATTC.