Specific binding by EcoRV endonuclease to its DNA recognition site GATATC.

Journal of Molecular Biology
L E EnglerL Jen-Jacobson

Abstract

Restriction endonuclease EcoRV has been reported to be unable to distinguish its specific DNA site, GATATC, from non-specific DNA sites in the absence of the catalytic cofactor Mg2+, and thus to exercise sequence specificity solely in the catalytic step. In contrast, we show here that under appropriate conditions of pH and salt concentration, specific complexes with oligonucleotides containing the GATATC site can be detected by either filter-binding or gel-retardation. Equilibrium binding constants (K(A)) are easily measured by both direct equilibrium and equilibrium-competition methods. The preference for "specific" over "non-specific" binding at pH 7 in the absence of divalent cations is about 1000-fold (per mole of oligonucleotide) or 12,000-fold (per mole of binding sites). Ethylation-interference footprinting shows that the "specific" complex includes strong contacts to the phosphate groups GpApTpApTC. Specific DNA binding is strongly pH-dependent, decreasing about 15-fold for each increase of one pH unit above pH 6, but non-specific binding is not; thus, binding specificity decreases with increasing pH. Gel retardation and filter-binding at pH < or = 7 yield essentially identical values of K(A) for specific-site binding, ...Continue Reading

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Citations

Oct 8, 2008·Proceedings of the National Academy of Sciences of the United States of America·B van den BroekG J L Wuite
Jan 15, 2000·Journal of Biomolecular Structure & Dynamics·J R Wenner, V A Bloomfield
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