The goal is to analyze in greater detail the molecular interactions which regulate the SOS response in E. coli. First, specific contacts between amino acids in LexA protein and basepairs in the recA operator will be determined by isolating and sequencing mutant repressors which recognize a symmetrically altered, mutant operator sequence but not the wildtype sequence. Additionally, the role of various basepaires in the two binding sites in the Co1E1 operator on the binding of repressor will be analyzed using a rapid method for isolating and sequencing operator mutants in phage M13mp8/P. Second, we will attempt to obtain evidence for an interaction between LexA and RecA proteins that triggers repressor cleavage. A battery of mutant repressors which is liikely to be deficient in such an interaction is becoming available. An attempt will be made to isolate mutant RecA proteins which promote cleavage of these mutant repressors. The sequence changes in both the lexA and recA mutants would be determined to reveal amino acid residues in the proteins that interact and other possible compensating effects. Third, the direct role which activated Rec A protein plays in SOS mutagenesis of phage Lambda will be analysed through measurement of mutagenesis in various recA mutants and attempted isolation of new mutants based upon several current hypotheses. The molecular mechanism of mutagenesis will be analyzed by direct biophysical methods to determine if DNA synthesis plays a role. Fourth, the importance of various functions in the SOS response will be studied through analysis of mutant LexA proteins which allow constitutive expression of some SOS genes while continuing to repress the others. Finally, mutants of E. coli, which show either defective or constituteive induction of SOS functions will be isolated and analyzed to assist in identifying signal molecules and regions in the RecA protein which are involved in recognizing the inducing signal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM024496-09
Application #
3272353
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-01-01
Project End
1990-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
9
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85722
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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