Genetic and Biochemical Analysis of Specific LexA Cleavage

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9305092 Little Treatment of E. coli by agents that damage DNA or inhibit replication triggers the SOS response, a global response to the damage that promotes cell survival by a variety of means. This response involves the action of two regulatory proteins: LexA, which represses about 20 SOS genes during normal growth, and RecA, which is activated by inducing treatments to a form that catalyzes the specific cleavage of LexA repressor, inactivating it and leading to induction of the SOS genes. Hence, specific cleavage plays a crucial role in the SOS response. In addition to this central regulatory role of LexA cleavage, this reaction is of considerable biochemical interest. Its mechanism is unusual, in that LeA cleaves itself in an intramolecular reaction termed autodigestion, and activated RecA acts to stimulate LexA self-cleavage under physiological conditions. In these conditions, LexA cleavage is designed to proceed slowly, but at the same time to be capable of large rate increases. This view raises two related questions: What is the mechanism of LexA autodigestion, and how can RecA protein facilitate this reaction? Three complementary approaches are proposed to address these questions. The first is to continue to isolate and characterize mutations that influence the rate of LexA cleavage. The second approach is to continue to develop the bimolecular LexA cleavage reaction, in which one molecule of LexA or lambda repressor acts as an enzyme to cleave other molecules of LexA. The third approach is to look for physical evidence that LexA can exist in two different conformational states. %%% An increasingly large number of biologically important reactions are being discovered that are intramolecular. These include self- splicing RNA introns, autophosphorylation and autophosphatase activity of chemotaxis proteins, and autophosphorylation of oncogene proteins, growth factor receptors, and other important proteins. The proposed work is of general signifi cance for the insights it is expected to yield regarding the properties of intramolecular self-processing reactions, and the means by which the rated of these reactions can be regulated by interactions with other effector molecules. ***