Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

7-25-2008

Graduate Advisor

Cynthia Dupureur, PhD

Committee

Robert Calsyn, Ph.D.

James S. Chickos

Wendy M. Olivas

Abstract

The PvuII restriction endonuclease, a homodimer of two 18 kDa subunits, belongs to the type II family of restriction enzymes. 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 a coupling energy (ΔGcoop) at -0.54 for the upper phase and -1.15 kcal/mole for the lower phase. The similar metal binding pattern between the Y94F and the WT PvuII for Mg2+, Ca2+, Tb3+ and Eu3+ in the absence of DNA is also shown. Through 1H-15N HSQC spectroscopy and chemical denaturation of the Y94F variant the conformational impact of Tyr94 is confirmed. The Y94F 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 ΔGcoop at ?1.7 and -2.3 kcal/mole in the absence and presence of DNA, respectively. Combined with similar analysis for the WT-SC variant, the inter-subunit ΔGcoop values are shown at -1.1 and -3.1 kcal/mole. It is shown that the effect of Ca2+ ions on DNA binding is greater than the effect of the DNA on the affinity for Ca2+ ions. The cleavage of plasmid DNA under single turnover conditions reveals a similar dependence of the nicking and linearization rates on the concentration of Mg2+ 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) with Mg2+ ions in one PvuII subunit is not slower than the synchronized double strand cleavage with both PvuII subunits bearing Mg2+ ions.

Included in

Chemistry Commons

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