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Townsend, Jacqueline E (2011) ECORI ENDONUCLEASE-DNA COMPLEXES STUDIED BY THERMODYNAMICS AND ELECTRON SPIN RESONANCE SPECTROSCOPY. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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This work focuses on adducing general principles applicable to site-specific protein-DNA interactions by linking function to structural, thermodynamic and dynamic properties. The interaction of EcoRI endonuclease with specific, miscognate, and nonspecific DNA sequences is used as a model for protein-DNA interactions. We use four pulse Double Electron-Electron Resonance (DEER) Electron Spin Resonance (ESR) experiments to map distances and distance distributions between nitroxide spin labels placed at positions within the ‘arms' and the main domain of the EcoRI homodimer. These experiments show that the DNA occupies a similar binding cleft and is enfolded by the arms of the enzyme in all three classes of EcoRI-DNA complex. Additionally, changes in dynamics of main domain and arm residues within the three complexes were explored using Continuous Wave (CW) ESR spectroscopy. A position adjacent to a protein-phosphate contact shows decreased mobility relative to other arm residues that are not at the protein-DNA interface. Signal from this position shows the largest amount of an immobile component in the specific complex, progressively less immobile in the miscognate and nonspecific complexes. This fits with distribution breadths from DEER-ESR spectra and biochemical evidence that the nearby phosphate contact is made only in the specific complex. Residues at other positions show mobilities that are in agreement with our hypothesis that residues in the arms would be relatively more mobile than those in the main domain Using Electron Spin Echo Envelope Modulation (ESEEM) ESR we show that the paramagnetic Cu2+ ion is coordinated by an imidazole nitrogen. These experiments thus reveal a novel metal ion binding site. DEER measurements of distances between Cu2+ ions and Cu2+-nitroxide distances in the homodimeric EcoRI-DNA complex establish that the Cu2-coordinating residue is histidine 114, which is proximal to but not at the active site. This is consistent with our biochemical studies that show that Cu2+ cannot replace Mg2+ as a catalytic cofactor but instead completely inhibits EcoRI cleavage. We also use isothermal titration calorimetry (ITC) to directly determine a stoichiometry of two Cu2+ ions bound per homodimeric EcoRI-DNA complex; that is, each histidine 114 coordinates one Cu2+ ion.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Townsend, Jacqueline Ejet14@pitt.eduJET14
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairJen-Jacobson, Linda ljen@pitt.eduLJEN
Committee MemberPeebles, Craig cpeebles@pitt.eduCPEEBLES
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.eduJBRODSKY
Committee MemberRosenberg, Johnjmr@jmr3.xtal.pitt.eduROSENBRG
Committee MemberSaxena , Sunilsksaxena@pitt.eduSKSAXENA
Date: 30 September 2011
Date Type: Completion
Defense Date: 28 July 2011
Approval Date: 30 September 2011
Submission Date: 6 August 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: copper coordination; DNA recognition; noncognate complexes; protein dynamics; site-directed-spin-labeling; protein-DNA interaction; restriction enzymes
Other ID:, etd-08062011-130345
Date Deposited: 10 Nov 2011 19:57
Last Modified: 15 Nov 2016 13:48


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