Mutant LexA proteins with an increased rate of in vivo cleavage.

Abstract

LexA repressor of Escherichia coi is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo. This cleavage reaction can proceed in vitro hi the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH. Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in Wivo, compared with wild-type LexA. Efficient in vivo cleavage of these mutant proteins also took place without RecA. Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion. These mutations changed Gln-92, which lies near the cleavage site, to tyroine, phenylalanine, or tryptophan. The latter mutation increased the rate of cleavage =500-fold. These fndings imply that the rate of wild-tyie LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNAdamagin treatments. Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.The SOS regulon ofEscherichia coli is a set ofgenes involved in the cellular response to DNA damage (1, 2). This regulon is controlled primarily by the action of LexA LexA cleavage also occurs in vitro under two different conditions: (i) RecA-mediated cleavage can occur at neutral pH and requires RecA and two types of cofactors that activate the RecA (3); (ii) at alkaline pH an intramolecular and RecA-independent cleavage, termed autodigestion, takes place (4-6). Both reactions cleave a specific peptide bond between Ala-84 and Gly-85 of LexA (202 amino acids in size), and both are impaired by many mutations, termed lexA (Ind-), which we will refer to here as Ind-, that were isolated on the basis of defects in in vivo 7,8). These and other findings strongly suggest that the two cleavage reactions have a common mechanism, at least in part, and that activated RecA stimulates autodigestion. Mechanistic considerations have also led to the identification of two residues (Ser-119 and Lys-156) that are essential for the chemistry of cleavage (6).These studies also suggest that LexA contains several separable sites related to cleavage: the cleavage site, analogous to the substrate in an enzyme-substrate interaction; the active site, composed both of groups that carry out the actual chemistry of cleavage and of additional groups that interact with the cleavage site and position it optimally with respect to the catalytic center; and a RecA interaction site, where RecA binds to play its role in RecA-mediated cleavage. We expect that any intramolecular reaction would involve sites of at least the first two types; for example, in the best-studied such reaction, self-splicing of Tetrahymena rRNA, the splice sites are known, and the active site has been localized by a combination of genetic and biochemical analysis (9). Understanding the mechanism of LexA cleavage should give insights into this entire class of reactions, which includes such diverse re...

Analysis of Escherichia coli RecA Interactions with LexA, λ CI, and UmuD by Site-Directed Mutagenesis of recA

“…5. This is referred to as "basal cleavage" and has been seen in many other studies (19,26,29,45,50,52). For wild-type recA, after the addition of the inducing agent, the rate of LexAEK45 cleavage increased substantially (Fig.…”

“…Strains derived from JL2460 and JAM267 were used for analysis of UmuD and CI cleavage, respectively. The Western blotting protocol was as described previously with minor changes (52). Anti-rabbit immunoglobulin G-POD from Boehringer Mannheim was used as a secondary antibody.…”

The bacterial LexA transcriptional repressor

“…The circles indicate positions of substitutions in hypercleavable LexA mutants (solid symbols) or noncleavable mutants (open symbols) [36,37,39,40]. The (damage-inducible gene) [29] controls a regulon of 63 genes [25].…”

LexA cleavage and other self-processing reactions

“…This model was suggested by the properties of a new class of1exA mutations, termed hypercleavable or Inds mutations, that confer increases in the rate of specific cleavage (34,41 (18). A truncated X CI substrate is not cleaved by either enzyme.…”

“…We reasoned (34,41) that analysis of mutant LexA proteins with an increased rate of cleavage might help identify the rate-limiting step or steps in cleavage. The rate of any chemical reaction is determined by the free energy difference between the ground state and the transition state.…”