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Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress
Nina Y Sidorova1, Donald C Rau
1Laboratory of Physical and Structural Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biophysical Journal
|September 30, 2004
Summary
Restriction endonuclease EcoRI binding to DNA is highly specific. High osmotic pressure can remove water from EcoRI "star" complexes, affecting DNA binding and cleavage activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The restriction endonuclease EcoRI exhibits high specificity for its DNA recognition sequence (GAATTC).
- Deviations from the recognition sequence, known as "star" sequences (e.g., TAATTC, CAATTC), significantly reduce EcoRI binding affinity.
- The role of water molecules in mediating protein-DNA interactions, particularly for specific vs. nonspecific binding, remains an area of investigation.
Purpose of the Study:
- To investigate the role of sequestered water molecules in the binding of EcoRI to specific and "star" DNA sequences.
- To determine how osmotic pressure affects the dissociation rates and water content of EcoRI-DNA complexes.
- To correlate water removal from "star" complexes with EcoRI cleavage activity.
Main Methods:
- Measurement of binding free energy dependence on water activity.
- Determination of water molecule sequestration in protein-DNA complexes.
- Novel measurements of dissociation rates for noncognate sequence complexes.
- Competition equilibrium experiments under varying osmotic pressures.
Main Results:
- At low osmotic pressures, the water sequestered by "star" sequence complexes is indistinguishable from nonspecific complexes (approx. 110 water molecules).
- High osmotic pressure (5 Osm) effectively removes water from the TAATTC "star" sequence complex (nearly 90%), but less so from the CAATTC complex.
- Nonspecific complexes do not readily lose sequestered water even at high osmotic pressures.
- The sequence-dependent water loss from "star" complexes correlates with known "star" cleavage activities.
Conclusions:
- Sequestered water plays a crucial role in stabilizing EcoRI binding to specific and "star" DNA sequences.
- Osmotic pressure can modulate the hydration shell of EcoRI-DNA complexes, influencing binding stability.
- The differential removal of water from "star" complexes provides insights into the mechanism of sequence-specific recognition and cleavage by EcoRI.