The Effect of Active Site Mutations on the Homodimeric Behavior of the PvuII Restriction Endonuclease

by Papadakos, Grigorios A

The Effect of Active Site Mutations on the Homodimeric Behavior of the PvuII Restriction Endonuclease (August 2008) Grigorios A. Papadakos, B.S., University of Athens, Greece Chair of Committee: Dr. C. M. Dupureur The PvuII restriction endonuclease, a homodimer of two 18 kDa subunits, belongs to the type II family of restriction enzymes. As a part of the Proteus vulgaris RM system, it specifically cleaves the 5’-CAG|CTG-3’ sequence in the presence of Mg 2+ ions. Located in the active site of PvuII, Tyrosine 94 has previously been shown to be involved in the metal ion binding by the enzyme. The profile of the Ca2+ dependence of the DNA binding to the Y94F variant is shown to be clearly biphasic. The application of a sequential binding model yielded two weak binding constants in the upper phase with a coupling energy (?Gocoop) at -0.3, while two tight binding constants are shown for the lower phase with -1.4 kcal/mole interaction energy. The similar metal binding pattern between the Y94F and the WT PvuII for Mg 2+, Ca2+, Tb 3+ and Eu3+ in the absence of DNA is also shown. The application of 1H-15N HSQC spectroscopy in the presence of Ca2+ and DNA and the chemical denaturation of the Y94F variant confirm the conformational impact of Tyr94. It is concluded that the removal of the aromatic ii hydroxyl group of Tyr94 slightly repositions the metal ions in the active site of PvuII affecting the intra and/or inter-subunit interactions among the metal binding sites. The single chain (SC) PvuII bearing a covalent linker between the two subunits is utilized in the exploration of the modes of cooperativity among the metal binding sites. The heterodimeric WT|E68A-SC PvuII was prepared and studied in parallel to the WT- SC homodimer. Global analysis of DNA binding isotherms at different Ca2+ concentrations for the WT|E68A-SC variant returned an intra-subunit ?Gocoop at +1.6 and +1.0 kcal/mole in the absence and presence of DNA, respectively. Combined with similar analysis for the WT-SC variant, the corresponding values for the inter-subunit ?Gocoop are shown at -2.8 and -1.1 kcal/mole for the occupation of two sites simultaneously. The sequential binding of metal ions in the absence and presence of DNA is overall unfavorable with significant negative interaction being observed between the metal sites. It is shown that the effect of Ca2+ ions on DNA binding is greater than the effect of the DNA on the affinity for metal ions. The cleavage of plasmid DNA under single turnover conditions reveals a similar dependence of the nicking and linearization rates on the concentration of Mg 2+ ions for the WT-SC and the WT|E68A-SC PvuII. The series of events leading to the linear product (DNA association, nicking, release of the intermediate, re-association and linearization) in the presence of metal ions in one PvuII subunit is not significantly slower than the synchronized double strand cleavage in the presence of metal ions in both PvuII subunits. iii ????? ?????????µ????? µ?? ?????? ??? ??????? ????????, ?????, ??????? ??? ??????? iv