Genetic Engineering of Taqi Restriction Endonuclease
{'Code': '08070100', 'Directorate': {'Abbreviation': 'BIO', 'LongName': 'Direct For Biological Sciences'}, 'Division': {'Abbreviation': 'MCB', 'LongName': 'Div Of Molecular and Cellular Bioscience'}}
{'SignBlockName': 'Philip Harriman', 'PO_EMAI': None, 'PO_PHON': None}
This project will employ a new approach to understanding protein                function and design.  Protein secondary structure elements fold                 into precise functional regions (domains), which interact in a                  three dimensional array to provide biological activity.                         Insertion of two amino acids into a protein causes a small                      perturbation which has a certain radius of influence within the                 three dimensional structure.  By introducing two-amino acid                     insertions into the linear sequence, one can scan the length of a               protein and determine which regions might comprise functional                   domains and which regions act as the hinges that connect the                    domains.                                                                                                                                                        This proposal presents a general method for constructing such                   insertions in proteins by operating at the level of the DNA                     sequence that codes for the TagI restriction endonuclease.  The                 aim is to understand the specificity of DNA sequence recognition                by TagI endonuclease, and to engineer mutants with altered                      sequence recognition.  Wild type endonuclease, a so called                      "allosteric activation mutant", and a "canonical site nicking                   mutant" will be overproduced, purified, and characterized in                    vitro with respect to sequence specific binding, dissociation,                  facilitated diffusion, and cleavage, using a variety of DNA                     substrates.  Using a unique TagI endonuclease clone, additional                 two-codon insertion mutations will be introduced into the enzyme,               and characterized by both in vitro and in vitro assays.  These                  studies will help to define the sequence specificity domains, and               allow for further targeted saturation mutagenesis of those                      domains.  The new recognition sequence of these mutant proteins                 will be determined using a uniquely labeled DNA substrate.                                                                                                      In collaboration with Dr. John Anderson, purified TagI will be                  co-crystalized with its cognate oligonucleotide for determination               of its three dimensional structure.
{'FirstName': 'Francis', 'LastName': 'Barany', 'PI_MID_INIT': None, 'PI_SUFX_NAME': None, 'PI_FULL_NAME': 'Francis Barany', 'EmailAddress': None, 'NSF_ID': '000123367', 'StartDate': '05/01/1988', 'EndDate': None, 'RoleCode': 'Principal Investigator'}
{'Name': 'Joan and Sanford I. Weill Medical College of Cornell University', 'CityName': 'NEW YORK', 'ZipCode': '100226145', 'PhoneNumber': '6469628290', 'StreetAddress': '575 LEXINGTON AVE FL 9', 'StreetAddress2': None, 'CountryName': 'United States', 'StateName': 'New York', 'StateCode': 'NY', 'CONGRESSDISTRICT': '12', 'CONGRESS_DISTRICT_ORG': 'NY12', 'ORG_UEI_NUM': 'YNT8TCJH8FQ8', 'ORG_LGL_BUS_NAME': 'WEILL MEDICAL COLLEGE OF CORNELL UNIVERSITY', 'ORG_PRNT_UEI_NUM': 'QV1RJ11H58C4'}
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{'Code': '1112', 'Text': 'Genetic Mechanisms'}
{'url': 'https://www.nsf.gov/awardsearch/download?DownloadFileName=1987&All=true', 'xml': '8714352.xml'}