Autodigestion of lexA and phage lambda repressors.

Abstract

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, ...

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

“…This polymorphic behavior of P RM regulations by a single protein enables the lysogen to maintain a steady but very low level of free CI in a λ lysogen. This low level may allow fast degradation of free CI by SOS, which is induced by RecA, a coprotease (54,55), and thus easy switching of the prophage to a lytic state. It is interesting to notice that the maintenance of a biologically critical level of CI involves formation and interconversion of two topographically different forms of a loop.…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…This damage tolerance mechanism is thought to be dependent upon the bacterial SOS response, in which single-strand DNA (ssDNA) is recognized and bound by the RecA protein [10], which promotes two parallel pathways. Activated RecA (RecA*) induces the self-cleavage of the LexA repressor that in turn allows the transcription of various genes required for DNA repair and cell survival [11]. SOS induces the expression of both DinB and the umuDC operon encoding two polymerases, PolIV and PolV, respectively, for translesion synthesis (TLS).…”

Characterization of DinR, the Bacillus subtilis SOS repressor

“…This region of dyad symmetry is commonly known as the SOS box and is located upstream of most SOS genes or SOS operons (9,15). Following DNA damage, RecA achieves an activated state (RecA*) which mediates the autocatalytic cleavage of LexA (17). After cleavage, LexA no longer binds to the operator, thus allowing expression of the regulated gene.…”