The goals of this research proposal are to elucidate and characterize fundamental interactions that are important for the activities of the RecA protein of Escherichia coli. This protein is required for homology dependent recombination, for regulating a set of genes that participate in a DNA damage stress response (SOS response) and for participating directly in the biochemical process leading to mutagenesis. The basic interactions that will be investigated are those involved in i) RecA protein subunit recognition, ii) ATP binding and hydrolysis, and iii) recognition and binding to other proteins such as lambdoid repressors, the cellular LexA repressor and the umuD protein which is needed for a major pathway of chemical mutagenesis. The sequence(s) within the first 90 residues of RecA protein required for subunit binding will be investigated using affinity chromatography and fusion proteins between RecA and beta-lactamase. Binding of synthetic peptides corresponding to the regions of interest will be studied. Two substitution mutations (Tyr264-Phe264 and Tyr264-Ser264) in the ATP binding site have been constructed by site-directed mutagenesis and will be fully characterized with respect to ATP hydrolysis, DNA binding and the ability to carry out DNA pairing (annealing, strand transfer). Second site suppressor mutations will be isolated and characterized in order to identify other important elements of the ATP binding domain. In addition, site-directed mutagenesis will be used to alter residues with a conserved sequence in TecA protein that is found in a number of nucleotide binding proteins. The properties of the proteins will be characterized in vitro. The interaction of RecA protein-polynucleotide complexes with other cellular proteins will be investigated, in particular, the role of Gly204 in LexA protein binding will be examined. Finally, the role of RecA-like activity in homologous recombination and DNA repair will be investigated by constructing yeast strains lacking the gene encoding this function. These latter experiments will provide additional insight into the mechanisms of recombination in eurkaryotic cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029558-10
Application #
3277235
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-07-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Tang, M; Bruck, I; Eritja, R et al. (1998) Biochemical basis of SOS-induced mutagenesis in Escherichia coli: reconstitution of in vitro lesion bypass dependent on the UmuD'2C mutagenic complex and RecA protein. Proc Natl Acad Sci U S A 95:9755-60
Cai, H; Yu, H; McEntee, K et al. (1995) Purification and properties of DNA polymerase II from Escherichia coli. Methods Enzymol 262:13-21
Cai, H; Yu, H; McEntee, K et al. (1995) Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli. J Biol Chem 270:15327-35
Escarceller, M; Hicks, J; Gudmundsson, G et al. (1994) Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation. J Bacteriol 176:6221-8
Freitag, N E; McEntee, K (1991) Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation. J Biol Chem 266:7058-66
Zhao, X J; McEntee, K (1990) DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r. Mol Gen Genet 222:369-76
Bonner, C A; Hays, S; McEntee, K et al. (1990) DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli. Proc Natl Acad Sci U S A 87:7663-7
Freitag, N; McEntee, K (1989) ""Activated""-RecA protein affinity chromatography of LexA repressor and other SOS-regulated proteins. Proc Natl Acad Sci U S A 86:8363-7
Halbrook, J; McEntee, K (1989) Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae. J Biol Chem 264:21403-12
Freitag, N; McEntee, K (1988) Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns. J Biol Chem 263:19525-34

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