Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress

Biophysical Journal
Nina Y Sidorova, Donald C Rau

Abstract

The binding of the restriction endonuclease EcoRI to DNA is exceptionally specific. Even a single basepair change ("star" sequence) from the recognition sequence, GAATTC, decreases the binding free energy of EcoRI to values nearly indistinguishable from nonspecific binding. The difference in the number of waters sequestered by the protein-DNA complexes of the "star" sequences TAATTC and CAATTC and by the specific sequence complex determined from the dependence of binding free energy on water activity is also practically indistinguishable at low osmotic pressures from the 110 water molecules sequestered by nonspecific sequence complexes. Novel measurements of the dissociation rates of noncognate sequence complexes and competition equilibrium show that sequestered water can be removed from "star" sequence complexes by high osmotic pressure, but not from a nonspecific complex. By 5 Osm, the TAATTC "star" sequence complex has lost almost 90 of the approximately 110 waters initially present. It is more difficult to remove water from the CAATTC "star" sequence complex. The sequence dependence of water loss correlates with the known sequence dependence of "star" cleavage activity.

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Citations

Aug 19, 2007·Nature Protocols·Lance M Hellman, Michael G Fried
Apr 23, 2013·Nucleic Acids Research·Nick Kamps-HughesEric A Johnson
May 14, 2009·PloS One·Nilesh K Banavali, Alexander D Mackerell
Oct 31, 2009·Journal of Molecular Biology·Donald C Rau, Nina Y Sidorova
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Jan 29, 2010·Electrophoresis·Nina Y SidorovaDonald C Rau
Aug 30, 2013·Lab on a Chip·Marcus OttenHermann E Gaub

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