Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

Abstract

LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.Escherichia coli LexA and X cI repressors are inactivated in vivo by a specific cleavage reaction that cuts a conserved Ala-Gly bond near the center of the polypeptide chain (1, 2). Cleavage occurs following treatments that damage DNA or inhibit replication (1-3). Inactivation of LexA is rapid and results in derepression of a set of genes, the SOS regulon, largely responsible for DNA repair. Cleavage of the X repressor is much slower than that of LexA, and more severe treatments are required to induce the prophage. Repressor cleavage in vivo requires RecA function; RecA protein is quiescent during normal cell growth but is activated by the inducing treatment to a form that participates in cleavage.Early in vitro studies showed that the role of RecA protein in cleavage is relatively direct (4). At physiological pH, cleavage is dependent on the presence of RecA protein and two cofactors-a nucleoside triphosphate and single-stranded DNA-that activate RecA by forming a ternary complex. For a time, it was tacitly assumed that activated RecA was a conventional protease with a highly specific active site. This view was challenged by the finding (5) that, under different conditions, cleavage of LexA and the X repressor proceeds spontaneously at the same Ala-Gly bond in an intramolecular reaction, termed "autodigestion," that is stimulated by alkaline pH and does not require RecA. Both RecA-dependent cleavage and autodigestion of LexA are inhibited in a mutant protein, LexA3, that also cannot be cleaved in vivo.It was, therefore, proposed that RecA protein is not itself a protease, but rather that it acts indirectly by stimulating the autodigestion reaction. According to this hypothesis, activated RecA is a positive ef...

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

“…Control plasmids in this strain behaved as expected (Table 2): a strain lacking LexA function gave a high level; a strain carrying a noncleavable LexA protein (SA119) gave a very low level; and a strain with wild-type LexA gave an intermediate level, due to the presence of a low basal rate of cleavage, as observed (6,7). Substitution of Gln-92 with any of the three aromatic residues tyrosine, phenylalanine, and tryptophan resulted in decreased repression relative to the wild type, whereas most other substitutions gave repression levels comparable to wild type (Table 2).…”

“…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).…”

“…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)(5)(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 RecA-mediated cleavage (4,7,8).…”

“…pJWL383 is a derivative of pJWL184 with a deletion of codons for amino acid residues 4-180 of LexA; it will be described elsewhere. pSNS117 carries a fusion of lacP to the lexA (Ind-) mutation SA119 (6).…”

Mutagenic DNA repair in enterobacteria

“…After exposure to DNA replicationinhibiting agents such as UV light, the autocatalytic cleavage of LexA results in the inactivation of repressor function and leads to derepression of SOS-controlled genes. In vitro this reaction occurs at alkaline pH or, at more physiological conditions, after interaction with a ternary complex of RecA protein, single-stranded DNA, and ATP (19, 42,43). Elevated levels of the UmuDC proteins are not sufficient to promote mutagenesis.…”