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 EcoRl endonuclease has shown that both thermodynamic and kinetic factors govern selectivity between DNA sites. We have demonstrated and quantified the importance not only of direct protein-base contacts, but also of sequence-specific formation of particular phosphate contacts and the energy required to distort the DNA into a particular conformation in the complex. In the next project period, we propose thermodynamic and kinetic studies: 1. To identify structural features that may govern the energetic contribution of DNA distortability to the EcoRI endonuclease-DNA interaction, by systematically varying major- and minor-groove functional groups with base analogue replacements. 2. To delineate the functional roles of interactions with DNA phosphates at pNGApATTC in both the enzyme-substrate complex and the transition state complex, by stereospecific substitutions with chiral phosphorothioates and methylphosphonates. 3. 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. 4. To test the generality of the principles deduced from Eco RI endonuclease, by comparing particular properties of BamHI and EcoRV endonucleases. For BamHI endonuclease, we will determine by base analogue studies the relative contributions to the interaction made by direct protein-base contacts and DNA conformational properties. For EcoRV endonuclease, we will determine (using novel noncleavable phosphorothiolate oligonucleotides) if binding discrimination or protein-phosphate contacts are affected by Mg++ and whether flanking sequence has unusually strong effects on binding discrimination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029207-13
Application #
2175429
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1981-04-01
Project End
1997-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
13
Fiscal Year
1994
Total Cost
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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