We propose to use the highly specific interaction between EcoRI endonuclease and oligonucleotides containing the normal (GAATTC) or variant recognition sites as a model system for the study of sequence-specific DNA-protein interactions. We have demonstrated an unexpected role for electrostatic interactions at GAATpTC in DNA cleavage. We have used data on equilibrium binding and cleavage kinetics to formulate a refined model which correctly orders the effects of base substitutions in the recognition site. We have also demonstrated that contacts between the protein and particular DNA phosphates are altered when the enzyme binds to recognition sites containing a single base substitution, and that the phosphate contact at pXGAATTC is dependent upon flanking sequence. Here we propose to build upon this foundation by: a) Determining whether and how hydrogen bonding interactions to bases in the recognition site depend upon each other; b) Assessing the functional roles and interdependences of interactions between the protein and individual DNA phosphates; c) Systematically investigating the basis for the influence of flanking base sequence on phosphate interactions; and d) Identifying a carboxyl residue whose chemical modification inactivates the endonuclease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029207-07
Application #
3276734
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1981-04-01
Project End
1993-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
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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