Our long-term objective is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in DNA-protein interactions. Our previous work with EcoRI, BamHI and EcoRV endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the free energy changes in protein-DNA interactions, including the contributions of direct protein- base and protein-phosphate contacts and the energy required to distort the DNA. For the next project period, we propose the following interrelated aims: 1. To determine and dissect the entropic and enthalpic contributions to the formation of """"""""canonical"""""""" protein-DNA complexes, using titration- calorimetric, van't Hoff and osmotic-stress methods. To combine this analysis with structure-perturbation methods to determine how flanking- sequence or intra-site conformational determinants affect the conformational and vibrational entropy changes (DeltaDeltaS(o) conf and DeltaDelta(o) vib) and to determine the thermodynamic characteristics of the """"""""adaptive"""""""" complexes formed with sites differing by one base-pair (""""""""star"""""""" sites) and with sites containing base-analogs that perturb base- phosphate networks. 2. To determine how particular sequence contexts (i.e., outside a recognition site) influence site-specific interactions of three restriction endonucleases (EcoRI, BamHI, EcoRV) that distort their DNA sites in different ways and have different patterns of phosphate contacts immediately flanking the recognition site. 3. To complete our systematic study of the structural features within the DNA recognition site that govern the energetic contribution of DNA distortability to the EcoRI endonuclease-DNA interaction, and to extend this analysis to BamHI endonuclease. 4. To use existing """"""""promiscuous"""""""" mutants of EcoRI endonuclease to determine how the endonuclease-DNA interface and the energetics of the interaction may be modified by the introduction of new favorable interactions or the elimination of unfavorable interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029207-19
Application #
6179465
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Chin, Jean
Project Start
1981-04-01
Project End
2001-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
19
Fiscal Year
2000
Total Cost
$267,346
Indirect Cost
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Sinha, Kaustubh; Sangani, Sahil S; Kehr, Andrew D et al. (2016) Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex. Biochemistry 55:6115-6132
Hancock, Stephen P; Hiller, David A; Perona, John J et al. (2011) The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein. J Mol Biol 406:285-312
VanderVeen, Laurie A; Harris, Thomas M; Jen-Jacobson, Linda et al. (2008) Formation of DNA-protein cross-links between gamma-hydroxypropanodeoxyguanosine and EcoRI. Chem Res Toxicol 21:1733-8
Engler, L E; Sapienza, P; Dorner, L F et al. (2001) The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC. J Mol Biol 307:619-36
Watrob, H; Liu, W; Chen, Y et al. (2001) Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor. Biochemistry 40:683-92
Connolly, B A; Liu, H H; Parry, D et al. (2001) Assay of restriction endonucleases using oligonucleotides. Methods Mol Biol 148:465-90
Jen-Jacobson, L; Engler, L E; Jacobson, L A (2000) Structural and thermodynamic strategies for site-specific DNA binding proteins. Structure 8:1015-23
Liu, W; Chen, Y; Watrob, H et al. (1998) N-termini of EcoRI restriction endonuclease dimer are in close proximity on the protein surface. Biochemistry 37:15457-65
Engler, L E; Welch, K K; Jen-Jacobson, L (1997) Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. J Mol Biol 269:82-101
Jen-Jacobson, L (1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44:153-80

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