The RecA protein is a multifunctional, oligomeric enzyme which is the central component of the processes of recombinational repair and homologous genetic recombination in E. coli. The long term goal of the research is to understand the functional organization and structural dynamics of RecA in order to determine how the many activities catalyzed by this enzyme are coordinated in carrying out the overall process of recombination. Dr. Knight will begin this work by identifying regions of the protein that are involved in particular functions, e.g. DNA binding, ATP binding and hydrolysis and oligomeric contacts by performing protein covalent modification experiments. Also, in collaboration with Dr. Thomas Steitz, Dr. Knight will use the recently solved X-ray crystal structure of RecA to aid in defining important functional and structural areas of the protein. Once regions of the protein have been identified as critical for function and/or structure by cross-linking studies, he will use a variety of mutagenic techniques to alter specific amino acids in that region. A number of genetic selections and screens will be used to differentiate between those mutant proteins which maintain catalytic abilities and those with different recombination activities. These mutant proteins will be analyzed for their ability to undergo ligand-induced conformational changes. Dr. Knight has previously demonstrated that the RecA protein undergoes an ATP-induced conformational change, and subsequent electron microscopic studies suggest that this structural change is critical to RecA function. Further genetic analysis of the relationship between protein structure and function will be performed by using a number of the recombination deficient mutant proteins obtained in this study to select intragenic second site revertants. These revertants will be analyzed to define further the domain structure of RecA protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM044772-01A1
Application #
3304040
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1991-09-01
Project End
1996-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Sage, Jay M; Knight, Kendall L (2013) Human Rad51 promotes mitochondrial DNA synthesis under conditions of increased replication stress. Mitochondrion 13:350-6
Sage, Jay M; Gildemeister, Otto S; Knight, Kendall L (2010) Discovery of a novel function for human Rad51: maintenance of the mitochondrial genome. J Biol Chem 285:18984-90
Bakhlanova, Irina V; Dudkina, Alexandra V; Baitin, Dima M et al. (2010) Modulating cellular recombination potential through alterations in RecA structure and regulation. Mol Microbiol 78:1523-38
Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L (2009) Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C. J Biol Chem 284:31945-52
Bennett, Brian T; Bewersdorf, Jorg; Knight, Kendall L (2009) Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy. Methods 48:63-71
Forget, Anthony L; Kudron, Michelle M; McGrew, Dharia A et al. (2006) RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments. Biochemistry 45:13537-42
Forget, Anthony L; Bennett, Brian T; Knight, Kendall L (2004) Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51. J Cell Biochem 93:429-36
Nastri, H G; Knight, K L (1994) Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities. J Biol Chem 269:26311-22
Konola, J T; Logan, K M; Knight, K L (1994) Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site. J Mol Biol 237:20-34
Skiba, M C; Knight, K L (1994) Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli. J Biol Chem 269:3823-8

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