Our goals are to elucidate the molecular features of the structure, of the E. coli recA protein and relate these structural details to the enzymatic properties of the protein. The approaches to be taken include a combination of biochemical modification studies, recombinant DNA techniques and 'classical' microbial genetics. Biochemical investigations of recA protein include the direct photolinking of DNA to the polynucleotide binding site of recA protein. Identification of the regions of recA protein covalently crosslinked to DNA will be accomplished using tryptic digestion, HPLC and amino acid analyses. Using a similar approach the ATP binding site of recA protein has been identified and we now wish to identify the amino acid residue(s) to which ATP is covalently linked. This will be accomplished by further proteolytic digestion of the modified peptide and amino acid sequence analysis. Using photoaffinity labeling and tryptic mapping techniques we will investigate the conservation of this ATP binding domain among five heterologous recA proteins derived from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The genes for these heterologous proteins have been cloned into E. coli K12 and are expressed at high levels in appropriate genetic backgrounds. This comparative analysis will provide valuable information on the nature of recA protein ATP interaction. The information gained by biochemical modification studies of domains of recA protein will permit a more detailed functional analysis through the application of site-directed mutagenesis techniques to the appropriate region of the recA structural gene. Both Ba131 deletion and oligonucleotide-directed mutagenesis will be employed to modify those regions of recA protein that are important for substrate binding. We will then analyze the effects of these modifications or the functions of recA protein both in vitro and in vivo. Additional information on the domains of recA will be obtained by DNA sequence analysis of the cloned recA genes from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The ability of the cloned genes to complement defects of an E. coli K12 recA- mutation indicates conservation of important functional domains among these heterologous proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029558-04A1
Application #
3277229
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-07-01
Project End
1989-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
4
Fiscal Year
1985
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
Knight, K L; Hess, R M; McEntee, K (1988) Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r. J Bacteriol 170:2427-32

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