The long term goal of this project is to understand the molecular basis of induction of the E. coli SOS response and the resulting mutagenesis. First, we will identify additional genes in the SOS regulon by cloning their regulatory regions upstream from a galK reporter gene in low and high copy number plasmids. LexA binding sites will be located by footprinting and genetic experiments. Since recent experiments have suggested that LexA protein may recognize a second type of operator, the interaction of LexA protein with such operators will be studied using methods previously established for the recA operator. Second, with the coming availability of a crystal structure for RecA protein, we will attempt to define domains which specify RecA functions in the SOS response by using information from recA mutants. This project will involve production of a database of sequence and functional information of already existing mutants. To add to this database, we will measure the cleavage of LexA and UmuD proteins in vivo in uninduced and UV-induced recA80, recA432 and recA433 mutants and analyze the cleavage properties of the purified mutant RecA proteins. Third, we will continue analysis of the signalling mechanism which induces the SOS response by activating RecA protein. We will map fragments of chromosomal DNA which prevent induction of the SOS response when these fragments are present on high copy number plasmids. We will identify the functions of the genes which are present as regulators of the SOS induction pathway. Finally, the relationship between induction of the SOS regulon and enhanced excision of pyrimidine dimers from the transcribed DNA strand will be investigated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024496-16
Application #
2174281
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-01-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1994-12-31
Support Year
16
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Arizona
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Lewis, L K; Harlow, G R; Gregg-Jolly, L A et al. (1994) Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli. J Mol Biol 241:507-23
Peterson, K R; Mount, D W (1993) Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants. J Bacteriol 175:7505-8
Ennis, D G; Little, J W; Mount, D W (1993) Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction. J Bacteriol 175:7373-82
Lewis, L K; Jenkins, M E; Mount, D W (1992) Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor. J Bacteriol 174:3377-85
Mustard, J A; Thliveris, A T; Mount, D W (1992) Sequence of the Salmonella typhimurium LT2 lexA gene and its regulatory region. Nucleic Acids Res 20:1813
Thliveris, A T; Mount, D W (1992) Genetic identification of the DNA binding domain of Escherichia coli LexA protein. Proc Natl Acad Sci U S A 89:4500-4
Lewis, L K; Mount, D W (1992) Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene. J Bacteriol 174:5110-6
Dubnick, M; Thliveris, A T; Mount, D W (1991) Mixed oligo designer (MOD), a computer program to aid planning of automated, mixed oligodeoxyribonucleotide synthesis for mutagenesis experiments. Gene 105:1-7
Thliveris, A T; Little, J W; Mount, D W (1991) Repression of the E coli recA gene requires at least two LexA protein monomers. Biochimie 73:449-56
Dubnick, M; Mount, D W (1990) SPLICE, a computer program for automated extraction of information from GenBank sequence entries. Comput Appl Biosci 6:343-6

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