Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Specific versus Nonspecific DNA Binding of the Restriction Endonuclease EcoRV Measured by Self-Cleavage Assay
The type II restriction endonucleases binding to DNA is a paradigm for the specific recognition. Usually restriction endonucleases can distinguish between cognate and nonspecific DNA sequences quite efficiently in the absence of divalent cofactor that is required for cleavage. There are, however, many conflicting results in literature regarding ability of the EcoRV restriction endonuclease to distinguish between specific and nonspecific DNA sequences in the absence of divalent ions. One group only has demonstrated significant specificity. The majority of researchers do not see meaningful preferential binding, typically less than a 10-fold difference between the recognition sequence and nonspecific DNA. The x-ray structures for specific and non-cognate DNA-EcoRV complexes are, however, noticeably different in the absence of metal co-factors suggesting it is probable that EcoRV specific and nonspecific binding free energies should differ substantially. The total surface area buried on complex formation is about 1800 square angstroms larger in the case of cognate DNA binding suggesting that there should be also significant difference in hydration between two complexes. We have applied the self-cleavage assay developed by us previously to measure EcoRV-DNA solution binding. This technique does not have the limitations of more commonly used assays as gel mobility shift, filter binding, and anisotropy of fluorescently labeled complexes. Our results indicate significant EcoRV binding specificity in the absence of divalent ions. We confirm that EcoRV binding specificity is strongly pH dependent. We have also uncovered an unusual slow transition between specific binding modes that may account for the discrepancies seen in the literature.
Additionally, using the osmotic stress technique combined with a self-cleavage assay we measure differences in hydration between specific and nonspecific DNA-EcoRV complexes. We find that specificity of the EcoRV binding to DNA is strongly promoted by the presence of neutral solutes used to set water activity.
Laboratory of Physical and Structural Biology