Multiple metal ions drive DNA association by PvuII endonuclease.
Restriction enzymes serve as important model systems for understanding the role of metal ions in phosphodiester hydrolysis. To this end, a number of laboratories have reported dramatic differences between the metal ion-dependent and metal ion-independent DNA binding behaviors of these systems. In an effort to illuminate the underlying mechanistic details which give rise to these differences, we have quantitatively dissected these equilibrium behaviors into component association and dissociation rates for the representative PvuII endonuclease and use these data to assess the stoichiometry of metal ion involvement in the binding process. The dependence of PvuII cognate DNA on Ca(II) concentration binding appears to be cooperative, exhibiting half-saturation at 0.6 mM metal ion and yielding an n(H) of 3.5 +/- 0.2 per enzyme homodimer. Using both nitrocellulose filter binding and fluorescence assays, we observe that the cognate DNA dissociation rate (k(-)(1) or k(off)) is very slow (10(-)(3) s(-)(1)) and exhibits a shallow dependence on metal ion concentration. DNA trap cleavage experiments with Mg(II) confirm the general irreversibility of DNA binding relative to cleavage, even at low metal ion concentrations. More dramatically, the association rate (k(1) or k(on)) also appears to be cooperative, increasing more than 100-fold between 0.2 and 10 mM Ca(II), with an optimum value of 2.7 x 10(7) M(-)(1) s (-)(1). Hill analysis of the metal ion dependence of k(on) indicates an n(H) of 3.6 +/- 0.2 per enzyme dimer. This value is consistent with the involvement in DNA association of two metal ions per subunit active site, a result which lends new strength to arguments for two-metal ion mechanisms in restriction enzymes.
Conlan
Lori H
LH
Department of Chemistry and Biochemistry, University of Missouri St. Louis, 63121, USA.
Dupureur
Cynthia M
CM
D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D003101:Collodion; D004269:DNA, Bacterial / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005374:Filtration / Q000295:instrumentation; D005454:Fluorescence Polarization; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D004269:DNA, Bacterial; D003101:Collodion; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium