Protein recognition of specific base sequences in double-helical DNA lies at the heart of many biological processes, including the regulation and expression of genetic information and site-specific recombination. The long-term objective of this project is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in site-specific DNA-protein interactions. Previous work with EcoRI and BamHI endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the changes in free energy (deltaGdegree), enthalpy (deltaHdegree) and entropy (deltaSdegree) in formation of protein-DNA complexes. The approach is aimed at uniting structural and energetic perspectives on specificity and at testing the generality of the principles adduced. It is now proposed to determine how molecular strain (DNA bending in the EcoRV complex; electrostatic repulsion in the BamHI complex) affects thermodynamic parameters (deltaHdegree, deltaSdegree and deltaCdegreep), and to determine how particular sequence contexts (i.e., outside a DNA recognition site) act to modulate specificity and to adjust molecular strain in the DNA complexes of EcoRI and EcoRV endonucleases. Existing and newly-engineered mutants of EcoRl endonuclease will be used to determine how the introduction of new favorable interactions or the elimination of unfavorable interactions relaxes specificity by modifying the structure of the protein-DNA interface, the thermodynamics of binding and/or the cleavage kinetics of the system. The BamHI and EcoRV endonucleases will be used for quantitative calibration of the excluded-cosolute method of evaluating H20 release during protein-DNA association, and to compare experimental results with computational predictions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM029207-24
Application #
6879975
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1981-04-01
Project End
2006-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
24
Fiscal Year
2005
Total Cost
$376,995
Indirect Cost
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
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
Sapienza, Paul J; Niu, Tianyi; Kurpiewski, Michael R et al. (2014) Thermodynamic and structural basis for relaxation of specificity in protein-DNA recognition. J Mol Biol 426:84-104
Sinha, Kaustubh; Jen-Jacobson, Linda; Rule, Gordon S (2013) Divide and conquer is always best: sensitivity of methyl correlation experiments. J Biomol NMR 56:331-5
Ruthstein, Sharon; Ji, Ming; Mehta, Preeti et al. (2013) Sensitive Cu2+-Cu2+ distance measurements in a protein-DNA complex by double-quantum coherence ESR. J Phys Chem B 117:6227-30
Lin, Hsiang-Kai; Chase, Susan F; Laue, Thomas M et al. (2012) Differential temperature-dependent multimeric assemblies of replication and repair polymerases on DNA increase processivity. Biochemistry 51:7367-82
Yang, Zhongyu; Kurpiewski, Michael R; Ji, Ming et al. (2012) ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity. Proc Natl Acad Sci U S A 109:E993-1000
Sarver, Jessica L; Townsend, Jacqueline E; Rajapakse, Gayathri et al. (2012) Simulating the dynamics and orientations of spin-labeled side chains in a protein-DNA complex. J Phys Chem B 116:4024-33
Sinha, Kaustubh; Jen-Jacobson, Linda; Rule, Gordon S (2011) Specific labeling of threonine methyl groups for NMR studies of protein-nucleic acid complexes. Biochemistry 50:10189-91
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
Dylla-Spears, Rebecca; Townsend, Jacqueline E; Jen-Jacobson, Linda et al. (2010) Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point. Lab Chip 10:1543-9

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