Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.     

Repression of the E coli recA gene requires at least two LexA protein monomers

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.     

Binding of the Bacillus subtilis LexA protein to the SOS operator

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5′-CGAACN₄GTTCG-3′. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5′ end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5′-CGAACATATGTTCG-3′. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.     

The protein HU can displace the LexA repressor from its DNA-binding sites

The major bacterial histone-like protein HU is a small, basic, dimeric protein composed of two closely related subunits. HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division. It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites. In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites. We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes. The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive. Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding. Like HU, this drug disrupted LexA–operator complexes. These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Identification of a lexA Gene in, and Construction of a lexA Mutant of, Xanthomonas campestris pv. citri

The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri. Received: 1 September 1999 / Accepted: 25 October 1999     

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Both the amino-terminal and the carboxy-terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA-catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non-specific binding to DNA, the purified amino-terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3-fold if the DNA lattice is fully saturated with protein. Further, the amino-terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino-terminal domain harbours a substantial amount of residues in alpha-helical conformation, a prerequisite for DNA recognition via a helix--turn--helix structural motif as proposed for many other regulatory proteins.     

Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain

Summary One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis. Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA. In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor. Previous works have shown that activated RecA plays a second role, i.e. it is required for the establishment of base pair substitution mutations promoted by UV irradiation. Using a forward mutatonal assay and recA441 lexA(Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF). In the recA441 lexA(Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively. The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42° C. We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42° C. On the other hand, we show that N-AcO-AAF can promote mutations even at 30° C; the number of mutations being increased when the bacteria were grown at 42° C. Analysis of the mutants obtained at 30° C indicate that they belong to both type of mutations, UmuC-dependent or UmuC-independent. The much higher ability of N-AcO-AAF to induce RecA as compared to N-OH-AF strongly suggests that the former mutagen is able to induce at least partially the activated form of RexA441 even at 30°C in a strain which overproduces RecA, [lexA(Def)]. Furthermore, we show that the UmuC-independent type of mutagenesis induced by N-AcO-AAF depends on gene(s) that are part of the lexA regulon.     

Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA.

Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA. These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation. This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface. The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers. Each half-operator contains seven interfering phosphates. These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target. On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis. We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein. A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain. We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.     

Analysis of the expression of the Rhodobacter sphaeroides lexA gene

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN₇GAACN₇GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation. Received: 31 January 2000 / Accepted: 3 April 2000     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12.

The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein. In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions. DNA sequence analysis identified 20 different mutations. In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA. In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction.     

大肠杆菌（Escherichia coli）LexA蛋白的结构与功能

LexA蛋白首先在大肠杆菌（Escherichia coli）中作为SOS反应的重要调节因子之一被发现. LexA蛋白含有202个氨基酸,由N端DNA结合结构域和C端催化核心结构域构成. 细胞中LexA蛋白大都以二聚体形式存在,并且有可切割和不可切割两种构象. 在正常生理条件下,LexA特异性结合16 bp的保守序列5′-CTGTN8ACAG-3′,即SOS盒,抑制约50个基因的表达. 当发生DNA损伤时,活化的RecA蛋白通过稳定LexA蛋白可切割构象,促进LexA蛋白Ala84-Gly85间肽键的切割,产生的C端LexA85 202和N端LexA1 84被蛋白酶ClpXP和Lon快速降解. LexA蛋白切割后,SOS基因以一定的顺序开始表达,并且完成DNA损伤修复. 本文回顾和总结了LexA蛋白分子结构,自我切割分子机制和影响因素,以及在SOS反应中的作用等方面的研究进展. 同时,也讨论了LexA蛋白在原核细胞中的进化保守性.     

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12

Summary Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA ⁺ gene product. The requirement of SDR for recA ⁺ can be suppressed by rin mutations (for recA⁺-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA ⁺-dependent SDR seen in rnh ^- rin⁺ lexA⁺ strains, recA ⁺-independent in rnh ^- rin^- lexA⁺, and recA ⁺-independent in rnh ^- rin⁺ lexA(Def). The expression of SDR in rin ^- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA ⁺ requirement by rin mutations was shown to depend on some new function of the recF ⁺ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF ⁺. The lexA3 mutation inhibited recA ⁺-dependent SDR via reducing the amount of recA ⁺ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh ^- rin^- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA ⁺-regulated gene product in the recA ⁺-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA ⁺, rin⁺ and recF ⁺ genes.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.