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Titolo:
Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: Separating their roles in specific and non-specific binding
Autore:
Sidorova, NY; Rau, DC;
Indirizzi:
NICHHD, Lab Phys & Struct Biol, NIH, Bethesda, MD 20892 USA NICHHD Bethesda MD USA 20892 s & Struct Biol, NIH, Bethesda, MD 20892 USA
Titolo Testata:
JOURNAL OF MOLECULAR BIOLOGY
fascicolo: 4, volume: 310, anno: 2001,
pagine: 801 - 816
SICI:
0022-2836(20010720)310:4<801:LOEDFI>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
OSMOTIC-STRESS; RESTRICTION ENDONUCLEASES; LINEAR DIFFUSION; RI ENDONUCLEASE; PROTEIN; CLEAVAGE; SEQUENCES; COMPLEX;
Keywords:
protein-DNA interactions; osmotic stress; EcoRI restriction endonuclease; dissociation rate; bound water;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
35
Recensione:
Indirizzi per estratti:
Indirizzo: Rau, DC NICHHD, Lab Phys & Struct Biol, NIH, Bethesda, MD 20892 USA NICHHD Bethesda MD USA 20892 ct Biol, NIH, Bethesda, MD 20892 USA
Citazione:
N.Y. Sidorova e D.C. Rau, "Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: Separating their roles in specific and non-specific binding", J MOL BIOL, 310(4), 2001, pp. 801-816

Abstract

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in orderto distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along nonspecific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and thedissociation of non-specifically bound protein. We demonstrated previouslythat the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and nonspecific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratioof specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependenceon viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.

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Documento generato il 29/09/20 alle ore 17:29:45