Abstract
In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186-2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (pi), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during site-specific DNA recognition.
Highlights
The BamHI endonuclease binds the DNA recognition sequence 5Ј-GGATCC-3Ј with remarkable specificity
Previous studies that investigated the effects of increased osmotic stress on endonuclease specificity for both BamHI and EcoRI reveal that slight perturbations in water activity could cause the enzymes to lose specificity for their cognate DNA recognition sequences (4 – 6)
The stringent molecular recognition of the endonucleases was restored by the application of hydrostatic pressure under star conditions, presumably through the replacement of waters released at elevated osmotic stress levels
Summary
The BamHI endonuclease binds the DNA recognition sequence 5Ј-GGATCC-3Ј with remarkable specificity. In the presence of cosolvents, BamHI and EcoRI have a propensity to cleave at non-cognate or “star” sites which are characterized by base pair (bp) changes within the hexameric cognate recognition sequence In these studies which employed added solutes to modulate the various colligative properties of the reaction solution, increase in the observed star activity was only correlated with osmotic pressure () and was not correlated with other solution properties, such as dielectric constant, viscosity, and mole fraction of water [4, 6]. The stringent molecular recognition of the endonucleases was restored by the application of hydrostatic pressure under star conditions, presumably through the replacement of waters released at elevated osmotic stress levels This led to the conclusion that the observed star activity was because of increased osmotic stress from the added solutes, apparently through the removal of key water molecules or the hydration-linked conformational changes that are central to site-specific recognition. Several biological reactions, such as the opening/ closing of membrane-bound channels [17], electron transfer of
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