The RecA protein of E. coli, which is both a recombinease and a so-called protease, has a central role in repair of damaged DNA; both activities participate in repair. The protease function of the RecA protein involves at least three functional sites, namely a binding site for the substrate whose cleavage is thereby enhanced, and two effector-binding sites whose states determine whether or not the binding site is active.
One aim of this project is to determine the location, structure, and interaction of the three sites of RecA protease function and to determine how they are related to the sites for recombinase function. A mutational approach is being used. Mutations called recA (Prt-c) confer constitutive protease activity on RecA protein without the usual need for DNA damaging agents. In vitro studies of the effector- and substrate- binding properties of various recA (Prt-c Rec+) and recA (Prt-c Rec-) mutants will be carried out to determine where on the RecA polypeptide the active sites are located and how their functional coupling is accomplished. New recA mutants, including suppressors and double mutants, will be isolated or constructed and their genes sequenced, which will also eanble one to determie which reigons of the RecA polypeptide are functionally coupled. A sequence analysis of new recA mutants that alter substrate recognition will identify the substrate binding site and aid in explaining how the binding is coupled functionally to the effector- binding sites. The differences between the direct role of the RecA protein in spontaneous and UV-induced mutagenesis and its indirect role in enhancing proteolyis of repressors will be studied and the role of putative cryptic lesions in spontaneous mutation will be tested. These experiments are a continuation of our general aim to provide a framework for a definitive understanding of the function of the RecA protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035850-03
Application #
3289156
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-01-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Tessman, I; Kennedy, M A; Liu, S K (1994) Unusual kinetics of uracil formation in single and double-stranded DNA by deamination of cytosine in cyclobutane pyrimidine dimers. J Mol Biol 235:807-12
Mohammad, T; Tessman, I; Morrison, H et al. (1994) Photosensitized inactivation of infectious DNA by urocanic acid, indoleacrylic acid and rhodium complexes. Photochem Photobiol 59:189-96
Tessman, I; Kennedy, M A (1994) DNA polymerase II of Escherichia coli in the bypass of abasic sites in vivo. Genetics 136:439-48
Liu, S K; Eisen, J A; Hanawalt, P C et al. (1993) recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities. J Bacteriol 175:6518-29
Kuan, C T; Tessman, I (1992) Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins. J Bacteriol 174:6872-7
Tessman, I; Liu, S K; Kennedy, M A (1992) Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine. Proc Natl Acad Sci U S A 89:1159-63
Kuan, C T; Tessman, I (1991) LexA protein of Escherichia coli represses expression of the Tn5 transposase gene. J Bacteriol 173:6406-10
Tessman, I; Kennedy, M A (1991) The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation. Mol Gen Genet 227:144-8
Kuan, C T; Liu, S K; Tessman, I (1991) Excision and transposition of Tn5 as an SOS activity in Escherichia coli. Genetics 128:45-57
Liu, S K; Tessman, I (1990) Error-prone SOS repair can be error-free. J Mol Biol 216:803-7

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