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Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli. In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction. I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein. This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reac tion. Several lines of evidence argued that it was not due to a contaminating protease activity. Autodigestion was stimulated by alkaline pH. It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+. The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular. Purified phage X repressor also broke down under similar conditions to yield products like those resulting from recA protein action. Phage X repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo. This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway. These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease. The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.The SOS regulatory system of Escherichia coli is controlled in part by the interplay of two proteins-the lexA protein, which represses a set of unlinked genes during normal cell growth, and the recA protein, which is required in vivo for inactivation of lexA protein after treatments that derepress the system by damaging DNA or altering its metabolism (reviewed in ref. 1). lexA protein is inactivated by specific cleavage at an alanine-glycine bond near its center (2). This reaction requires recA function in vivo; in vitro, recA protein also participates in cleavage of lexA protein (2-4) and, at a slower rate, a few prophage repressors such as phage X repressor (5, 6).The recA-dependent cleavage reaction has two striking and related features that place it at the heart of the SOS regulatory system. First, recA protein is only conditionally active in the reaction; it must be activated by interaction with one or more effectors before it can support the cleavage reaction. Moreover, activation is a reversible process. These properties allow recA protein to act as a sensor of aberrant DNA metabolism or DNA damage, ...

Complex interacting systems exhibit system behavior that is often not predictable from the properties of the component parts. We have tested a particular system property, that of robustness. The behavior of a system is termed robust if that behavior is qualitatively normal in the face of substantial changes to the system components. Here we test whether the behavior of the phage λ gene regulatory circuitry is robust. This circuitry can exist in two alternative patterns of gene expression, and can switch from one regulatory state to the other. These states are stabilized by the action at the O R region of two regulatory proteins, CI and Cro, which bind with differential affinities to the O R 1 and O R 3 sites, such that each represses the synthesis of the other one. In this work, this pattern of binding was altered by making three mutant phages in which O R 1 and O R 3 were identical. These variants had the same qualitative in vivo patterns of gene expression as wild type. We conclude that the behavior of the λ circuitry is highly robust. Based on these and other results, we propose a two-step pathway, in which robustness plays a key role, for evolution of complex regulatory circuitry.

ABSTRACr The recA and 1exA proteins of Eseherichia coli are involved in a complex regulatory circuit that allows the exression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the A repressor as substrate, is Escherichia coli exhibits a complex response to agents that damage DNA or inhibit DNA replication (1). A number of new cellular processes, including mutagenesis, prophage induction, and new DNA repair capacity, are expressed. These are sometimes called "SOS functions" because they ark believed to aid cell survival. Extensive genetic evidence indicates that expression of these processes is controlled by a regulatory system that involves the products of at least two unlinked genes, recA and lexA. The biochemical analysis of the recA and 1exA proteins has begun, and in this communication we report evidence for a direct interaction between the two proteins.The recA protein has recently been purified and extensively characterized in vitro. It exhibits at least two distinct sets of properties. First, it binds to single-or double-stranded DNA and to double-stranded DNA possessing internal or terminal single-stranded regions; it catalyzes assimilation of single-stranded DNA fragments into homologous duplex DNA, and when bound to single-stranded DNA it promotes unwinding of double-stranded homologous or nonhomologous DNA (2-7). These functions are believed to be important in DNA repair and in genetic recombination. It also exhibits DNA-dependent ATPase activity, which is probably involved with its DNAThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3225 binding activities (2,8,9). Second, the recA protein is a highly specific protease (9-13). It cleaves the phage A repressor, inactivating its function, in a reaction requiring ATP and single-stranded DNA. A mutant form of the recA protein, the product of the tif-1 allele, is several-fold more active than is the wild-type protease in this reaction. In contrast, the biochemical function of the lexA protein is not yet known. Genetic evidence suggests a role in the regulation of the recA gene, perhaps as a simple repressor (14-16). We have recently identified the lexA3 mutant protein by radiolabeling techniques and studies with mutants (17) and have also shown that the wild-type lexA + protein is probably a protein with a mobility slightly different from that of the mutant protein in our electrophores...

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