CHIRAL PHOSPHOROTHIOATES AS PROBES OF PROTEIN INTERACTIONS WITH INDIVIDUAL DNA PHOSPHORYL OXYGENS - ESSENTIAL INTERACTIONS OF ECORI ENDONUCLEASE WITH THE PHOSPHATE AT PGAATTC

Citation
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
Citations number
38
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
27
Year of publication
1996
Pages
8846 - 8854
Database
ISI
SICI code
0006-2960(1996)35:27<8846:CPAPOP>2.0.ZU;2-6
Abstract
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.