This project has two long-term goals: First, to characterize the biochemical properties of the LexA protein of Escherichia coli, and second, to identify and characterize other cellular proteins which interact with activated RecA protein. The LexA protein is a repressor of a set of genes, often termed the LexA regulon, which is controlled by the SOS regulatory system. This system is activated when cellular DNA is damaged. The key event in controlling the SOS response is a specific proteolysis of LexA protein. Cleavage inactivates the repressor. In vitro, LexA protein also has an intrinsic protease activity (""""""""autodigestion"""""""") which cleaves the molecule in an intramolecular reaction. Autodigestion and RecA-dependent cleavage appear to be closely related. The work is aimed at characterizing these cleavage reactions and the relationsip between them, using a combination of genetic and biochemical techniques. Mutants will be isolated and sequenced which impair or enhance the reaction. Mutant proteins will be isolated and their ability to take part in the cleavage reactions will be determined. Pseudorevertants of these mutants will be characterized. In addition, the structure of LexA protein will be studied, using recombinant DNA techniques to alter its structure. Its binding to its specific operators will be further characterized, methods will be developed to assess accurately the relative affinities for different binding sites, and co-operative binding to adjacent operators will be studied. Interaction of LexA protein with activated RecA protein will be characterized. Efforts will be made to identify and characterize other cellular proteins which also interact with activated RecA protein, an important goal because the mutagenesis process induced by the SOS system required activated RecA protein. These experiments thus will test the model that activated RecA plays its role in mutagenesis by binding to other proteins, perhaps those involved in DNA replication, and modifying their function. This work is important for two diverse reasons: First, characterizing LexA protein should give insights into protein structure and function, as well as specific DNA binding. Second, mutagenesis is important in carcinogenesis and a biochemical understanding of this process in bacteria offers the best approach to the problem, since we can integrate genetic and biochemical approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024178-13
Application #
3272082
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-08-01
Project End
1991-03-31
Budget Start
1989-08-01
Budget End
1991-03-31
Support Year
13
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Medicine
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85722
Michalowski, Christine B; Little, John W (2013) Role of cis-acting sites in stimulation of the phage ? P(RM) promoter by CI-mediated looping. J Bacteriol 195:3401-11
Little, John W; Michalowski, Christine B (2010) Stability and instability in the lysogenic state of phage lambda. J Bacteriol 192:6064-76
Little, John W (2010) Evolution of complex gene regulatory circuits by addition of refinements. Curr Biol 20:R724-34
Degnan, Patrick H; Michalowski, Christine B; Babic, Andrea C et al. (2007) Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage lambda. Mol Microbiol 64:232-44
Babic, Andrea C; Little, John W (2007) Cooperative DNA binding by CI repressor is dispensable in a phage lambda variant. Proc Natl Acad Sci U S A 104:17741-6
Atsumi, Shota; Little, John W (2006) A synthetic phage lambda regulatory circuit. Proc Natl Acad Sci U S A 103:19045-50
Atsumi, Shota; Little, John W (2006) Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach. Proc Natl Acad Sci U S A 103:4558-63
Michalowski, Christine B; Little, John W (2005) Positive autoregulation of cI is a dispensable feature of the phage lambda gene regulatory circuitry. J Bacteriol 187:6430-42
Atsumi, Shota; Little, John W (2004) Regulatory circuit design and evolution using phage lambda. Genes Dev 18:2086-94
Michalowski, Christine B; Short, Megan D; Little, John W (2004) Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry. J Bacteriol 186:7988-99

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