The long-term objective of this research program is to understand the regulation of the SOS response in E. coli. The SOS response is an inducible network of functions promoting DNA repair and cellular survival which is activated when E. coli is treated with agents that damage cellular DNA or block its replication. Inducible responses to DNA damage exist in a number of other bacteria and apparently in a variety of eucarytic systems as well. Genetic and biochemical data suggest that activation of the SOS response results upon derepression of a class of genes following the specific proteolytic cleavage of their common repressor, the LexA repressor, by RecA protein. At least 17 different genes, necessary for functions as diverse as mutagenesis, DNA repair, recombination, cell division and prophage induction, have been identified as under LexA repressor control. In particular, this research program aims to understand the physical forces that determine the ability of LexA repressor to recognize and to bind to its operators in the promoter regions of the various genes within this regulon. Such information should increase understanding of the general energetic principles that may determine specificity of DNA recognition in other protein-nucleic acid binding systems. Recognition of specific DNA sequences by proteins is involved in the control of many cellular processes (eg, replication, transcription and translation). Therefore, in addition to understanding the mechanism by which E. coli copes with DNA damage, such studies are potentially relevant to understanding the molecular origin of and developing therapeutic strategies for control of diseases involving defects in any of these fundamental cellular processes. The specific experimental goals for this grant period are: 1) to investigate the thermodynamics of the nonspecific binding interactions of LexA repressor with single-stranded and double-stranded DNA. Equilibrium binding constants will be characterized as a function of the concentrations and type of electrolyte ions, pH and temperature; 2) to investigate the thermodynamics of the site-specific interaction of LexA repressor with the reca and uvrA operator sites on duplex DNA restriction fragments as a function of solution conditions; 3) to investigate the dependence of the self-association reaction of LexA repressor on solution conditions parallel to those used in the studies of the equilibrium binding of LexA repressor to DNA; 4) to initiate kinetic studies of the interaction of LexA repressor with the recA operator, 5) to initiate thermodynamic studies of the equilibrium binding of LexA repressor to the dual operator sites located at the regulatory region of the lexa gene using quantitative DNA footprinting.
