The immediate goals of this research are to use X-ray diffraction techniques to determine the following structures: (A) a co-crystalline complex between a cognate oligo nucleotide and EcoRI endonuclease; (B) the structure of this restriction enzyme in the absence of DNA, and (C) DNA-EcoRI endonuclease complexes in which the DNA-protein interface is perturbed by the introduction of specific alterations in the sequences the DNA and/or endonuclease. The first goal (A) is very close to realization because a 3 angstrom electron density map of the DNA-protein complex has been obtained. This map is revealing the molecular architecture of the DNA-protein interface and the first priority of the proposed research is to extend this image to the diffraction limit of the crystals (at least 2.6 angstroms). A comparison of the DNA-protein complex with the structure obtained in the absence of DNA (B) should yield information on conformational changes associated with DNA binding. Biochemical evidence from several laboratories has demonstrated that the DNA-EcoRI endonuclease complexes can form even when the DNA contains substitutions in the canonical recognition sequence. The perturbed structures (C) should yield information on the structural basis of this plasticity. Additionally, crystallization conditions will be explored for other restriction enzymes, modification methylases and other enzymes which interact with nucleic acids. The ultimate objective of this research is to use these enzymes as model systems to study sequence specific recognition of DNA by proteins such as that which occurs during protein mediated gene regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025671-10
Application #
3273224
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1978-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
10
Fiscal Year
1987
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
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