Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11528-11533, October 1996 Biochemistry Mutant LexA proteins with specific defects in autodigestion (self-processing reactions/LexA repressor/specific cleavage/proteolysis/RecA-mediated cleavage) DONALD P. SHEPLEYt AND JOHN W. LITrLEt#§ Departments of tBiochemistry and tMolecular and Cellular Biology, University of Arizona, Tucson, AZ 85721 Communicated by Sydney Kustu, University of California, Berkeley, CA, July 24, 1996 (received for review April 4, 1996) ABSTRACT In self-processing biochemical reactions, a pro- are under control of two regulatory proteins: the LexA repressor, tein or RNA molecule specifically modifies its own structure. which represses the SOS genes during normal growth; and the Many such reactions are regulated in response to the needs ofthe RecA protein, which is quiescent during normal growth but is cell by an interaction with another effector molecule. In the promptly activated upon inducing treatments to a form that can system we study here, specific cleavage of the Escherichia coli facilitate the specific cleavage of LexA. Cleavage inactivates LexA repressor, LexA cleaves itself in vitro at a slow rate, but in LexA, leading to derepression of the SOS regulon. Although vivo cleavage requires interaction with an activated form ofRecA RecA serves as a catalyst in vivo for LexA cleavage, RecA serves protein. RecA acts indirectly as a coprotease to stimulate LexA an indirect role in stimulating cleavage, and hence we term this autodigestion. We describe here a new class of ex4 mutants, exA function of RecA its coprotease activity. (Adg-; for autodigestion-defective) mutants, termed Adg- for As with any self-processing molecule, LexA has an active site brevity. Adg- mutants specifically interfered with the ability of and a substrate site. The cleavage site is analogous to the LexA to autodigest but left intact its ability to undergo RecA- substrate in an enzyme-catalyzed reaction; the active site is mediated cleavage. The data are consistent with a conforma- composed of a catalytic center, which carries out the chemistry tional model in which RecA favors a reactive conformation of bond breakage, and of a binding pocket, which binds the capable of undergoing cleavage. To our knowledge, this is the cleavage site and positions the peptide bond relative to the first example of a mutation in a regulated self-processing reac- catalytic center. LexA (202 aa) has the following domain tion that impairs the rate of self-processing without markedly structure: the N-terminal domain is the DNA-binding domain; affecting the stimulated reaction. Had wild-type lex4 carried the C-terminal domain allows dimerization and contains the such a substitution, discovery of its self-processing would have active site for cleavage; and the cleavage site (Ala-84/Gly-85) been difficult; we suggest that, in other systems, a slow rate of lies between the two domains. At a mechanistic level, LexA is self-processing has prevented recognition that a reaction is of almost certainly a serine protease; Ser-1 19 in LexA reacts with this nature. a serine protease inhibitor (4), and changes of Ser-119 to Ala completely block cleavage (5). Unlike classical serine pro- teases, however, the serine appears to be activated by a Self-processing biochemical reactions are those in which a deprotonated lysine residue, Lysl56 in LexA. It seems likely protein or RNA molecule carries out a specific covalent that RecA acts by somehow reducing the pKa of Lys-156, modification of its own structure (1). In principle, the rates of allowing cleavage to proceed at neutral pH. self-processing reactions can be very high, because all the A model for the action of RecA was recently developed (6) reactants are present at high local concentrations. Nonethe- based on the properties of a new type of lexA mutation, termed less, in some cases, the rates of self-processing reactions are lexA (Inds) and here referred to as Inds. Inds mutant proteins slow and are greatly stimulated by an external agent, implying display greatly increased rates of specific cleavage, both in vivo that these self-processing molecules are designed to undergo a and in vitro (6-8). Biochemical analysis of the cleavage reaction slow intrinsic reaction but to be capable of large rate increases. for three of these proteins, all changing Gln-92 in LexA to an Examples of regulated reactions include many reactions in aromatic amino acid, led to the conclusion that these mutations two-component regulatory systems; LexA repressor self- mimic, to some extent, the role of RecA in promoting cleavage. cleavage, which is mediated by an activated form of RecA; and Based on the properties of these mutant proteins, we developed an "honorary" self-processing reaction, the GTPase activity of a model (Scheme I) for the specific cleavage reaction. eukaryotic Ras proteins, which is stimulated by interactions with GTPase-activating proteins. K L* k It is of interest in such systems to understand the relation- , ship between the self-processing reaction and its stimulated L*_H L p counterpart. Are the mechanisms of these reactions the same? That is, does the effector increase the rate of the reaction, or does the stimulated reaction follow a different pathway? In the tj Kconf present system, we have explored the properties of mutant L-H . L proteins to address the relationship between self-cleavage and K its RecA-stimulated counterpart. In this work, we describe a L new type of mutant: one that affects the rate of a self- Scheme I processing reaction but not that of the reaction catalyzed by the outside effector. In Scheme I, P represents the products of the cleavage The self-processing reaction we study here plays a central role reaction. According to this model, LexA can exist in two forms: in the SOS regulatory system of Escherichia coli (1-3). In this the L form cannot autodigest, while the L* form autodigests system, a set of '20 genes is derepressed following conditions with a first-order rate constant kref. L and L* interconvert, and that damage DNA or inhibit DNA replication. These SOS genes the [L*]/[L] ratio is defined by the equilibrium constant Kconf. The publication costs of this article were defrayed in part by page charge Abbreviations: X-Gal, 5-bromo-4-chloro-3-indolyl ,B-D-galactoside. payment. This article must therefore be hereby marked "advertisement" in §To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 11528 Downloaded by guest on October 1, 2021 Biochemistry: Shepley and Little Proc. Natl. Acad. Sci. USA 93 (1996) 11529 Both L and L* can be protonated on Lys-156, but the pKa for isolates of pJWL228 derivatives were maintained in JL468, protonation is considerably lower for L* than for L; pKL* is <6, which lacks T7 RNA polymerase, to avoid selective pressure while pKL is 10. for loss of LexA function. One important tenet of this model is that the Inds mutations Assay of Cleavage Rate. Reaction conditions for LexA increase the value of Kc.nf and that RecA has the same effect, autodigestion and RecA-mediated cleavage were measured as but to a far greater extent. If Kc.nf >> 1, the equilibrium described (9). Autodigestion was carried out at pH 10.0 or, between LH+ and L* is shifted toward L*, reducing the where indicated, pH 8.7. In RecA-mediated cleavage reac- apparent pKa of LexA. Activated RecA greatly stabilizes the tions, activated RecA was at 1 ,M and LexA was at 4 ,AM. L* form, even at neutral pH. We omit RecA from this diagram, Rates were estimated by visual inspection of Coomassie- since there may be multiple equilibria involving RecA and stained gels, and have an uncertainty of "20%. In some RecA:LexA complexes, and we do not know which species of experiments, we used Tricine gels (13), which give better LexA bind to RecA. resolution of the cleavage fragments than the Laemmli system. The model predicts that it may be possible to isolate LexA We estimated from mixing experiments that a sample con- mutations that have specific defects in autodigestion but are taining 70% fragments and 30% intact LexA gave about largely unaffected for RecA-mediated cleavage. These muta- equally intense staining for the intact protein and the C- tions would be those that specifically reduce the value ofKco,f. terminal fragment. If RecA could overcome the effect of these mutations, it might In Vivo Assay for Repressor Function. Derivatives of still be able to exert its effect on cleavage, so that it could pJWL184 were introduced into strain JL1752, which is recA- stabilize the L* form well enough to promote cleavage at lexA (Def), so that the only LexA function was provided by the we expect a mutation with a neutral pH. Accordingly, would plasmid, and cleavage could not result from the action of reduced Kconf to be much more severely affected for autodi- than for RecA-dependent cleavage; the difference RecA. This strain also carried a sulA::lacZ operon fusion as a gestion and levels would depend on the extent to which RecA could overcome reporter of LexA repressor function, 13-galactosidase Finally, the the defect. Hence, we term this type of mutation a lexA (Adg-) were detected on MacConkey indicator plates. mutation, for autodigestion-defective, and refer to it here as strain had an F' with a lacIqIS mutation, rendering the Lac Adg- for the sake of brevity. We report the isolation of several repressor insensitive to the presence of the inducer, lactose, in Adg- mutants. the indicator plates, ensuring that LexA was expressed at the basal level from the lac promoter.