Linked Life Data

8688420

Source:http://linkedlifedata.com/resource/pubmed/id/8688420

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
27
pubmed:dateCreated
1996-8-26
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8846-54
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATTC.
pubmed:affiliation
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.