Biochemical and genetic analyses on DNA binding and cleavage of LexA repressor.
Committee ChairLittle, John W.
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PublisherThe University of Arizona.
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AbstractLexA is an Escherichia coli repressor that controls the expression of about 20 SOS target genes in response to DNA damage. Two biological activities of LexA, DNA binding and proteolytic self-cleavage, play important roles in regulation of SOS response. Upon DNA damage, the repressor function of LexA is inactivated by the cleavage reaction, and the expression of the SOS target genes is derepressed, which results in DNA damage repair. LexA binds to sites with dyad symmetry. However, unlike most repressors, LexA dimerizes very poorly in solution. This poor dimerization could be a disadvantage for tight binding to DNA if the DNA binding species of LexA is a dimer form as observed in phage λ repressor. An alternative DNA binding pathway, the monomer binding pathway, was tested. In this pathway, two LexA monomers bind individually to DNA and then dimerize on the DNA. This monomer binding pathway predicts that LexA should bind to a half-site of the symmetric recA operator sequence. Since binding of LexA monomer to a half-site operator was observed, the monomer binding pathway was favored for the LexA DNA binding pathway. The calculated degree of cooperativity between two LexA monomers was 10⁶. This high degree of cooperativity is generated by protein-protein interaction in the C-terminal potential dimerization domain. The second part of the study focused on the cleavage of LexA. In the course of a screen for defective repressor mutants, an Indˢ mutant, LP89, was isolated. This mutant protein autodigested about 50 times faster than wild-type LexA under physiological conditions in vitro. A LexA combination mutant that contains three Indˢ mutations can undergo the self-cleavage extremely rapidly under these conditions in vivo (t₁/₂ = ≈ 10 second). This multiple mutant, LP89-QW92-EA152, was suggested to overcome most of the rate-limiting steps of the cleavage reaction. Finally, an intermolecular trans-cleavage reaction was developed. In this reaction one molecule of LexA C-terminal fragment containing the active site could cleave many molecules of a LexA substrate containing the cleavage site. This trans-cleavage reaction was used to investigate several aspects of the self-cleavage mechanism of not only LexA but also phage λ CI repressor. First, possible functional roles of three Indˢ mutations were assessed by this trans-cleavage reaction. Second, demonstration of various types of trans-cleavage reaction suggested that both cleavage site and active site of LexA are exposed to the surface of the protein. Third, this reaction was used to study how phage λ CI autodigests much slower than LexA. Since the active sites of both proteins have the same enzymatic activity and substrate specificity, it was strongly suggested that E. coli LexA and phage λ CI repressor have evolved such different cleavage rates by modulating the interaction between their cleavage site and active site.