Use of genetic and computer modeling approaches to identify DNA damage-inducible genes in Escherichia coli.
AuthorLewis, Lysle Kevin.
AdvisorMount, David W.
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe SOS regulon of Escherichia coli consists of more than two dozen genes which are induced by damage to the host chromosome. Most of the products of these genes are active in DNA repair and/or DNA replication processes. Expression of the SOS genes is negatively controlled by a repressor protein encoded by the lexA gene. The present work has used two very different experimental approaches to provide evidence that the SOS regulatory network is larger than previously recognized. The first method employed a novel genetic screening procedure to isolate new SOS genes as promoter fusions on high copy number plasmids. Three genes, dinG, dinH, and polB (dinA), were isolated and characterized. dinG, dinH, and polB were mapped to 17.8, 19.8, and 1.2 minutes on the E. coli chromosome, respectively. The sequences of the dinH and polB regulatory regions contained typical binding sites for LexA protein and were shown to bind LexA protein specifically in vitro. The LexA-responsive sequence element within the dinG regulatory region was defined experimentally by isolating and characterizing operator-constitutive mutations. Determination of the sequence of a 2,417 bp DNA fragment yielded the complete coding sequence of the dinG gene. Additional DNA sequencing revealed that the predicted amino acid sequence of DinH is extremely proline- and glutamine-rich and is homologous to a family of eucaryotic DNA damage-inducible proteins that are similarly rich in these amino acids. The second approach to the identification of new SOS genes employed a combination of mathematical and biochemical analysis methods. Mathematical modeling of the highly conserved 20 bp binding sites for LexA repressor permitted the design of a search method to identify new sites. Six new sites on the E. coli chromosome and four prospective bacteriophage elements were identified in a search of the GenBank database. In vitro binding studies indicated that DNA fragments containing at least six of the ten new sites bound LexA specifically. The locations of several of the putative new operators relative to known genes and large uncharacterized open reading frames strongly suggest roles for most of them in regulation of previously unrecognized SOS genes.
Degree ProgramMolecular and Cellular Biology