CHIRAL PHOSPHOROTHIOATES AS PROBES OF PROTEIN INTERACTIONS WITH INDIVIDUAL DNA PHOSPHORYL OXYGENS - ESSENTIAL INTERACTIONS OF ECORI ENDONUCLEASE WITH THE PHOSPHATE AT PGAATTC
Mr. Kurpiewski et al., CHIRAL PHOSPHOROTHIOATES AS PROBES OF PROTEIN INTERACTIONS WITH INDIVIDUAL DNA PHOSPHORYL OXYGENS - ESSENTIAL INTERACTIONS OF ECORI ENDONUCLEASE WITH THE PHOSPHATE AT PGAATTC, Biochemistry, 35(27), 1996, pp. 8846-8854
The contact between EcoRI endonuclease and the ''primary clamp'' phosp
hate of its recognition site pGAATTC is absolutely required for recogn
ition 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 er 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
G degrees(bind)) is slightly greater for S-p-Ps (+0.9 kcal/mol) than f
or R(p)-Ps (+0.7 kcal/mol). Binding penalties are approximately additi
ve for double-strand substitution (R(p),R(p)-Ps Or S-p,S-p-Ps). Neithe
r Ps diastereomer in one DNA strand affects the first-order rate const
ants for cleavage in the unmodified DNA strand, and only S-p-Ps inhibi
ts the cleavage rate constant (3-fold) in the modified DNA strand. Thu
s, 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 canonic
al complex, the phosphate at pGAATTC is completely surrounded by prote
in and each nonbridging phosphoryl oxygen receives two hydrogen bonds
from the endonuclease, such that in either orientation the increased b
ond length of P-S- inhibits binding. However, the pro-S-p oxygen inter
acts with residues that are connected (by proximity or inter-side-chai
n hydrogen bonding) to side chains with essential roles in catalysis,
so cleavage is preferentially inhibited when these side chains are sli
ghtly displaced by the S-p-Ps diastereomer.