Cleavage Of Repressor Research Articles

Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.

Intracellular d-Serine Accumulation Promotes Genetic Diversity via Modulated Induction of RecA in Enterohemorrhagic Escherichia coli.

The SOS response is a global stress network that is triggered by the presence of DNA damage due to breakage or stalled replication forks. Activation of the SOS response can trigger the replication of lytic bacteriophages and promote genetic diversification through error-prone polymerases. We have demonstrated that the host-associated metabolite d-serine contributes to Escherichia coli niche specification and accumulates inside cells that cannot catabolize it. This results in a modulated activation of the SOS antirepressor RecA that is insufficient to trigger lytic bacteriophage but capable of increasing the SOS-associated mutation frequency. These findings describe how relevant signals not normally associated with DNA damage can hijack the SOS response, promoting diversity as E. coli strains adapt while avoiding unwanted phage lysis.

MicroRNAs Databases: Developmental Methodologies, Structural and Functional Annotations.

microRNA (miRNA) is an endogenous and evolutionary conserved non-coding RNA, involved in post-transcriptional process as gene repressor and mRNA cleavage through RNA-induced silencing complex (RISC) formation. In RISC, miRNA binds in complementary base pair with targeted mRNA along with Argonaut proteins complex, causes gene repression or endonucleolytic cleavage of mRNAs and results in many diseases and syndromes. After the discovery of miRNA lin-4 and let-7, subsequently large numbers of miRNAs were discovered by low-throughput and high-throughput experimental techniques along with computational process in various biological and metabolic processes. The miRNAs are important non-coding RNA for understanding the complex biological phenomena of organism because it controls the gene regulation. This paper reviews miRNA databases with structural and functional annotations developed by various researchers. These databases contain structural and functional information of animal, plant and virus miRNAs including miRNAs-associated diseases, stress resistance in plant, miRNAs take part in various biological processes, effect of miRNAs interaction on drugs and environment, effect of variance on miRNAs, miRNAs gene expression analysis, sequence of miRNAs, structure of miRNAs. This review focuses on the developmental methodology of miRNA databases such as computational tools and methods used for extraction of miRNAs annotation from different resources or through experiment. This study also discusses the efficiency of user interface design of every database along with current entry and annotations of miRNA (pathways, gene ontology, disease ontology, etc.). Here, an integrated schematic diagram of construction process for databases is also drawn along with tabular and graphical comparison of various types of entries in different databases. Aim of this paper is to present the importance of miRNAs-related resources at a single place.

Suppression of the E. coli SOS response by dNTP pool changes.

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30+-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.

No SOS needed: a second bacterial checkpoint system stops cell division.

Cell division must be tightly coordinated with DNA replication so that a complete genome ends up in each daughter cell. That coordination is especially critical when DNA is damaged, as lengthy delays may be required to repair broken strands. To prevent premature division, cells detect the presence of broken DNA with dedicated sensor proteins, which inhibit division while they promote repair. In bacteria, this “checkpoint” system was originally thought to be the sole responsibility of the so-called SOS response, in which damaged DNA leads to the cleavage of a transcriptional repressor, causing synthesis of both repair enzymes and a cell division inhibitor. However, multiple experiments have shown that even when the SOS response is disabled, some bacteria are able to sense damage and delay division. In this issue of PLOS Biology, Joshua Modell, Michael Laub, and colleagues show that a second, independent system works alongside the SOS system, one that is sensitive to a specific type of DNA damage and that induces its own suite of response proteins. The authors began their search for the mystery inhibitor by inducing DNA damage in cells of the model bacterium Caulobacter crescentus in which the SOS system had been inactivated. They found six genes whose up-regulation could not be explained by the actions of known SOS regulators. Through a series of experiments, one gene emerged that bore all the characteristics of a cell division inhibitor and consequently was christened damage-induced cell division inhibitor A, or didA. Overexpression of the didA gene strongly inhibited division (Figure 1) in the absence of SOS proteins, indicating its independence from that system. Conversely, treatment with a DNA toxin in the absence of both didA and the SOS system's inhibitor sidA led most cells to divide despite the DNA damage, reducing viability. Figure 1 Caulobacter cells producing DidA cannot divide as DidA localizes to and blocks the activity of cell division proteins at midcell (DidA-YFP with cell boundaries outlined in white). The presence of both SOS and non-SOS systems introduces redundancy, surely a benefit in such a critical cellular control response. However, the two systems were not responsive to precisely the same insults, the authors found. Both systems responded to a toxin that formed crosslinks between the two strands. The SOS system, but not the didA system, was especially responsive to depletion of the nucleotide pool, while the didA system, but not the SOS system, responded strongly to creation of double-strand breaks. A key step in the Escherichia coli SOS system is the inhibition of polymerization of the structural protein, FtsZ, which forms a ring at the site of constriction, which is critical for positioning of the division machinery (the “divisome”). In contrast to many bacterial division inhibitors, the authors showed that DidA did not interact with FtsZ. Instead, they found evidence that it most likely binds to a late-arriving member of the divisome, FtsN. However, this interaction, they showed, did not disrupt the divisome or prevent the localization of other members of the complex. The actual mechanism, they suggest, involves a complex formed among three divisome proteins: FtsN, FtsI, and FtsW. Excess production of DidA normally would shut down cell division, but this effect could be overcome by mutations in either the ftsW or ftsI genes, despite the fact that DidA bound to neither protein directly. Instead, the authors propose that, in the absence of DidA, the three proteins form an active complex that promotes constriction, and when DidA binds FtsN, it converts the complex into an inactive state. Finally, the authors showed that expression of didA was driven by the transcriptional regulator DriD. Treatment with zeocin, a didA-specific DNA toxin, caused DriD to bind to didA's promoter and increase transcription. Zeocin did not, however, up-regulate the driD gene itself, indicating that damage-induced posttranslational modification of preexisting DriD protein is a key step in the regulatory pathway. Together, these results identify a novel mechanism of cell division control in Caulobacter. While details will probably differ in other types of bacteria, the identification of a second control system is likely to lead to the search for similar systems elsewhere. In addition, since even disabling both SOS and non-SOS systems did not entirely prevent normal regulation of cell division in Caulobacter, the authors note, yet more control systems may remain to be discovered. Modell JW, Kambara TK, Perchuk BS, Laub MT (2014) A DNA Damage-Induced, SOS-Independent Checkpoint Regulates Cell Division in Caulobacter crescentus . doi:10.1371/journal.pbio.1001977

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery.

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC(50) values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.

The genomics of poplar-rust interactions to improve tree resistance against fungal disease

With their long life cycle, trees must have accurate mechanisms to perceive microbial invasion and elaborate signalling networks in order to activate the appropriate defense response through transcriptional reprogramming. Transcriptional activators and repressors participate in the tight regulation of the stress response, which is key to minimise the fitness costs associated with an activated response. With the availability of its whole genome sequence, ease of growth and clonal propagation, and routine transformation, poplar (Populus spp.) is considered a model tree species for genomics research and also a good system in forest pathology [1]. Moreover, genomes of a cortege of associated microorganisms are being sequenced including a tree pathogen, Melampsora poplar rust. We pursued various approaches to identify poplar genes involved in the interaction with the biotrophic Melampsora rust pathogen. Recent transcriptome analyses from our lab have shown that the expression of genes encoding several transcription factors are up-regulated during infection by Melampsora rust. Similarly we have also shown that mitogen activated protein kinases (MAPKs) are associated with poplar disease resistance against Melampsora rust [2]. New data obtained from various experimental approaches have directed our focus on two important families of transcription factors; the jasmonate ZIM-domain (JAZ) and Cys2/His2 zinc-finger protein (ZFP) families. We have identified a novel MAPK-interacting partner, PtiZFP1, which belongs to the C2H2 ZFP family of transcriptional EAR repressors.The JAZ family of transcriptional repressors were recently identified as key negative regulators of jasmonate (JA) responses.Transcript analyses show that some ZFP and JAZ members exhibit hormone-related expression profiles and up-regulation by rust infection. This up-regulation of JAZ and ZFP transcripts after rust infection strongly suggests that a hormonal response including JA is a key component of the poplar defence response against Melampsora. Late and sustained kinetics of PtiZFP1 and specificJAZ up-regulation suggest that the corresponding proteins may be required for late regulation of defense mechanisms. Several recent data have shownthat proteolytic cleavage of transcriptional repressors is a general mechanism used by plants to activate gene induction. This mechanism is now well documented for jasmonate signaling, which depends on proteasome-mediated degradation of the JAZ repressors [3,4]. In the present work, we also obtain clear evidence that MAPKs promote degradation of PtiZFP1 through the 26S proteasome.Our work suggests that PtiZFP1 and JAZs are part of Melampsora specific hormone-related responses, and by correlation as putative transcriptional repressors, participate in the regulation of the transcriptional responses downstream of these stress hormones.We hypothesize that the observed gene induction play a negative feedback loop needed to replace PtiZFP1 and JAZ proteins that were degraded following through the 26S proteasome. Newly synthesized repressor proteins could contribute to defense attenuation and therefore prevent a runaway response. Based on our data, we propose a model (Figure ​(Figure1)1) where two important families of transcription factors play a predominant role in poplar defense response to the biotrophic fungus Melampsora. The activation of poplar MAPKs would lead toPtiZFP1 degradation and as a result to transcriptional activation of defense-related genes. In parallel we also observed that several genes involved in the JA response (including JAZ) are induced following Melampsora infection. This model highlights the importance of the 26S proteasome in regulating protein pools of transcriptional repressor. For future studies we intend to uncover which cis elements and target genes are recognized by PtiZFP1 and perform functional approaches to uncover JAZ interacting proteins in poplar. Figure 1 Proposed model of PtiZFP1 function in the plant defense response. During normal conditions, direct transcriptional repressors such as PtiZFP1 inhibit the expression of stress-related genes. Following stress perception, a MAPK signaling cascade is activated leading to phosphorylation of PtiZFP1. Phosphorylation targets PtiZFP1 for degradation via the 26S proteasome (P26S), thus relieving repression of defense genes. Defense signaling in turn activates PtiZFP1 gene transcription in order to replenish normal PtiZFP1 protein levels and complete a regulatory cycle necessary to attenuate the defense response. A similar regulatory cycle exists for jasmonic acid (JA) signaling. JA biosynthesis is induced by stress which then promotes proteasome-mediated degradation of JAZ proteins. These indirect transcriptional repressors sequester the bHLH MYC2, thus inhibiting expression of defense genes under non-stressed conditions. Upon release, MYC2 ensures both positive and negative feedback loops by activating LOX3 and JAZ genes respectively. Confirmed and hypothetical pathways are presented in bold and dashed lines respectively.

Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors.

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.

Two Modes of Binding of DinI to RecA Filament Provide a New Insight into the Regulation of SOS Response by DinI Protein

RecA protein plays a principal role in bacterial SOS response to DNA damage. The induction of the SOS response is well understood and involves the cleavage of the LexA repressor catalyzed by the RecA nucleoprotein filament. In contrast, our understanding of the regulation and termination of the SOS response is much more limited. RecX and DinI are two major regulators of RecA's ability to promote LexA cleavage and strand exchange reaction, and are believed to modulate its activity in ongoing SOS events. DinI's function in the SOS response remains controversial, since its interaction with the RecA filament is concentration dependent and may result in either stabilization or depolymerization of the filament. The 17 C-terminal residues of RecA modulate the interaction between DinI and RecA. We demonstrate that DinI binds to the active RecA filament in two distinct structural modes. In the first mode, DinI binds to the C-terminus of a RecA protomer. In the second mode, DinI resides deeply in the groove of the RecA filament, with its negatively charged C-terminal helix proximal to the L2 loop of RecA. The deletion of the 17 C-terminal residues of RecA favors the second mode of binding. We suggest that the negatively charged C-terminus of RecA prevents DinI from entering the groove and protects the RecA filament from depolymerization. Polymorphic binding of DinI to RecA filaments implies an even more complex role of DinI in the bacterial SOS response.

RecA-Mediated Cleavage of λ cI Repressor Accepts Repressor Dimers: Probable Role of Prolyl Cis-Trans Isomerization and Catalytic Involvement of H163, K177, and K232 of RecA

The λ cI repressor is found to be cleaved in the presence of activated RecA in its DNA- bound dimeric form at a rate similar to that in the absence of operator DNA in contrast to previous studies inferring repressor monomer as a preferred substrate. Though activated RecA does not possess any measurable isomerase activity against a standard peptide substrate, prolyl isomerase inhibitors cyclosporin A and rapamycin do inhibit RecA-mediated cleavage. Histidine and lysine to a smaller extent, are shown to cleave cI repressor in a non- enzymatic fashion whereas arginine and glutamate do not. When activated RecA filament is covalently modified by using an excess of diethyl pyrocarbonate or maleic anhydride, RecA- mediated cleavage of cI repressor is inhibited. Combining our chemical modification data with model building and earlier mutagenesis data, it is argued that H163, K177, and K232 in RecA are crucial residues involved in cI repressor cleavage by combining with the catalytic Ser149 and K192 in the repressor. It is suggested by model building that subunits n, n+4, and n+5 in the RecA filament contribute one loop each for holding the C-terminal domain of the repressor during cleavage within the RecA helical groove, explaining why its ADP-form is inactive and its ATP-form is active regarding repressor cleavage.

Serendipity

Serendipity

Cleavage of Bacteriophage λ cI Repressor Involves the RecA C-Terminal Domain

The SOS response to DNA damage in Escherichia coli involves at least 43 genes, all under the control of the LexA repressor. Activation of these genes occurs when the LexA repressor cleaves itself, a reaction catalyzed by an active, extended RecA filament formed on DNA. It has been shown that the LexA repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. Bacteriophages, such as λ, have repressors (cI) that are structural homologs of LexA and also undergo self-cleavage when SOS is induced. It has been puzzling that some mutations in RecA that affect the cleavage of repressors are in the C-terminal domain (CTD) far from the groove where cleavage is thought to occur. In addition, it has been shown that the rate of cleavage of cI by RecA is dependent upon both the substrate on which RecA is polymerized and the ATP analog used. Electron microscopy and three-dimensional reconstructions show that the conformation and dynamics of RecA's CTD are also modulated by the polynucleotide substrate and ATP analog. Under conditions where the repressor cleavage rates are the highest, cI is coordinated within the groove by contacts with RecA's CTD. These observations provide a framework for understanding previous genetic and biochemical observations.

Crystal structure of RecX: A potent regulatory protein of RecA fromXanthomonas campestris

Crystal structure of RecX: A potent regulatory protein of RecA from<i>Xanthomonas campestris</i>

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

The bacterial LexA transcriptional repressor.

Bacteria respond to DNA damage by mounting a coordinated cellular response, governed by the RecA and LexA proteins. In Escherichia coli, RecA stimulates cleavage of the LexA repressor, inducing more than 40 genes that comprise the SOS global regulatory network. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation. In some well-characterised pathogens, induction of the SOS response modulates the evolution and dissemination of drug resistance, as well as synthesis, secretion and dissemination of the virulence. In this review, we discuss the structure of LexA protein, particularly with respect to distinct conformations that enable repression of SOS genes via specific DNA binding or repressor cleavage during the response to DNA damage. These may provide new starting points in the battle against the emergence of bacterial pathogens and the spread of drug resistance among them.

RecA-Dependent Cleavage of LexA Dimers

A critical step in the SOS response of Escherichia coli is the specific proteolytic cleavage of the LexA repressor. This reaction is catalyzed by an activated form of RecA, acting as a co-protease to stimulate the self-cleavage activity of LexA. This process has been reexamined in light of evidence that LexA is dimeric at physiological concentrations. We found that RecA-dependent cleavage was robust under conditions in which LexA is largely dimeric and conclude that LexA dimers are cleavable. We also found that LexA dimers dissociate slowly. Furthermore, our evidence suggests that interactions between the two subunits of a LexA dimer can influence the rate of cleavage. Finally, our evidence suggests that RecA stimulates the transition of LexA from its noncleavable to its cleavable conformation and therefore operates, at least in part, by an allosteric mechanism.

Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach.

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.