The long-term goal of the proposed research is to understand fully the mutagenic activity (SOS mutagenesis) induced in E. coli in response to radiations and carcinogenic chemicals as part of a more complex response (the SOS response) to genetic damage. LexA protein is the common repressor of at least 17 SOS genes, which are derepressed when RecA protein is activated by DNA damage to a form (RecA*) that can cause proteolytic cleavage of LexA. Activated RecA* has an additional essential role in SOS mutagenesis, distinct from cleavage of LexA or any other repressor. The additional activity could be either proteolytic processing of one or more other proteins or a mechanistic function possible only in the activated state. As an approach to identification of this unknown RecA* role, a mutant allele, recA730, that causes constitutive hyperactivity of RecA*, will be cloned, RecA730 protein purified, and its activity in an in vitro assay for cleavage of LexA compared with that of RecA+ and other mutant RecA proteins. Another mutant allele, recA718, which causes UV sensitivity and UV hypermutability, will also be cloned and its protein product purified and partially characterized. Both RecA730 and RecA718 will be made available to laboratories investigating RecA protein activities in various in vitro systems. The following additional investigations, all relating to aspects of SOS mutagenesis not well understood, are also proposed: 1) determination of the basis of the mutator activity caused by plasmid pKM101 that differs from SOS mutator activity in several ways, e.g., it is expressed in lexA(Ind-) strains and is enhanced by low temperatures; 2) testing of the hypothesis that SOS mutator activity involves inhibition of the proofreading activity of DNA polymerase III, by examining the interaction of mutD(dnaQ) alleles (which are known to inhibit Pol III proofreading) with recA730, which causes constitutive SOS mutator activity; 3) determination of the basis of the high frequency of novel recA alleles recovered in crosses of K12 recA441 donors x B/r recA+ recipients, and 4) reexamination of the phenomenon known as """"""""mutation frequency decline"""""""" (MFD) to test the hypothesis that it is caused by medium-dependent changes in DNA conformation that affect repair of UV photoproducts at specific sites in certain tRNA genes. A deeper understanding of the events leading from DNA damage to mutation in bacteria may provide new avenues of approach to human carcinogenesis, since at least some activities analogous to components of the SOS response have been demonstrated in human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI010778-14
Application #
3124808
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1975-01-01
Project End
1989-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
14
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
Schools of Arts and Sciences
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
Witkin, E M (1994) Mutation frequency decline revisited. Bioessays 16:437-44
Witkin, E M; Roegner-Maniscalco, V (1992) Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I. J Bacteriol 174:4166-8
Kirchner, J; Lim, D; Witkin, E M et al. (1992) An SOS-inducible defective retronphage (phi R86) in Escherichia coli strain B. Mol Microbiol 6:2815-24
Witkin, E M (1991) RecA protein in the SOS response: milestones and mysteries. Biochimie 73:133-41
Sweasy, J B; Witkin, E M (1991) Novel SOS phenotypes caused by second-site mutations in the recA430 gene of Escherichia coli. Biochimie 73:437-48
Sweasy, J B; Witkin, E M; Sinha, N et al. (1990) RecA protein of Escherichia coli has a third essential role in SOS mutator activity. J Bacteriol 172:3030-6
Witkin, E M (1989) Ultraviolet mutagenesis and the SOS response in Escherichia coli: a personal perspective. Environ Mol Mutagen 14 Suppl 16:30-4
Garvey, N; Witkin, E M; Brash, D E (1989) Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to ""mutation frequency decline"". Mol Gen Genet 219:359-64
McCall, J O; Witkin, E M; Kogoma, T et al. (1987) Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein. J Bacteriol 169:728-34
Witkin, E M; Roegner-Maniscalco, V; Sweasy, J B et al. (1987) Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli. Proc Natl Acad Sci U S A 84:6805-9

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