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PubMed1997

Characterization of DinR, the Bacillus subtilis SOS repressor.

In Bacillus subtilis, exposure to DNA damage and the development of natural competence lead to the induction of the SOS regulon. It has been hypothesized that the DinR protein is the cellular repressor of the B. subtilis SOS system due to its homology to the Escherichia coli LexA transcriptional repressor. Indeed, comparison of DinR and its homologs from gram-negative and -positive bacteria revealed conserved structural motifs within the carboxyl-terminal domain that are believed to be important for autocatalysis of the protein. In contrast, regions within the DNA binding domain were conserved only within gram-negative or -positive genera, which possibly explains the differences in the sequence specificities between gram-negative and gram-positive SOS boxes. The hypothesis that DinR is the repressor of the SOS regulon in B. subtilis has been tested through overexpression, purification, and characterization of the DinR protein. Like E. coli LexA, B. subtilis DinR undergoes an autocatalytic reaction at alkaline pH at a siscile Ala91-Gly92 bond. The cleavage reaction can also be mediated in vitro under more physiological conditions by the E. coli RecA protein. By using electrophoretic mobility shift assays, we demonstrated that DinR interacts with the previously characterized SOS box of the B. subtilis recA gene, but not with sequences containing single base pair mutations within the SOS box. Together, these observations strongly suggest that DinR is the repressor of the SOS regulon in B. subtilis.

Amino Acid Sequence↗
PubMed1987

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques.

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.

Bacterial Proteins↗
PubMed1992

Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor.

A new genetic screening method has been developed to isolate Escherichia coli promoters which are components of the SOS regulon. Plasmids containing the regulatory regions of polB (dinA) and two new loci, dinG and dinH, were characterized. Galactokinase gene fusion experiments indicated that transcription of these genes is inducible by treatment with mitomycin and conforms to a classical model of SOS regulation involving simple LexA repression. Mapping studies using the E. coli DNA library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) revealed that dinG and dinH are located at 17.8 and 19.8 min on the chromosome, respectively. The nucleotide sequence of the dinH regulatory region contains a segment which is very similar to previously characterized binding sites for LexA protein. An asymmetric, noncanonical 20-bp LexA operator in the cloned dinG promoter region was identified. Additional experiments have revealed that the nucleotide sequence of the gene immediately downstream of the DNA damage-inducible polB locus encodes a polypeptide which has extensive sequence homology to several known and putative DNA and RNA helicase proteins. This gene, which is not regulated by the LexA repressor, has been designated hepA. The predicted amino acid sequence of the product of hepA contains several highly conserved sequence motifs that are also found in enzymes such as the RecQ and UvrB proteins of E. coli and the Rad3 protein of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1981

A general approach for purifying proteins encoded by cloned genes without using a functional assay: isolation of the uvrA gene product from radiolabeled maxicells.

The uvrA protein (UVRA) of E. coli has been extensively purified from a strain in which UVRA is overproduced and specifically labeled with 35S-methionine. This approximately 100-fold overproduction relative to normal strains is a result of having the uvrA gene present on a multicopy plasmid in a spr recA cell that makes defective lexA protein, the normal repressor of the uvrA gene, while the specific labeling of UVRA is done with maxicells. This approach facilitates the preparation of the protein since enzyme assays do not have to be carried out during the intermediate stages of purification. The purified UVRA binds to DNA and has ATPase activity but does not have intrinsic endonuclease activity. When added to extracts of uvrA- cells, the purified UVRA does promote the specific cutting of UV-irradiated DNA. Since this approach for working out rapid purification procedures by specifically labeling the proteins encoded by cloned genes does not require the use of a functional assay, it is a general one that can be applied to a wide variety of other gene products in addition to UVRA.

Adenosine Triphosphatases↗
PubMed1995

Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities.

The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro67-->Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of lambda cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)6 and (dT)6, as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro67-->Lys) and homologous recombination (Pro67-->Lys and Pro67-->Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Pro67 is located in a functionally important position in the RecA structure.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of the RecA protein from Streptococcus pneumoniae.

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.

Adenosine Triphosphate↗
PubMed1995

A model for the LexA repressor DNA complex.

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein-DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two beta-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed.

Amino Acid Sequence↗
PubMed1995

Cloning, sequence and regulation of expression of the lexA gene of Aeromonas hydrophila.

The lexA gene of Aeromonas hydrophila (Ah) has been isolated by using a specific one-step cloning system. The Ah LexA repressor is able to block Escherichia coli (Ec) SOS gene expression and is likely to be cleaved by the activated RecA protein of this bacterial species after DNA damage. Ah lexA would encode a protein of 207 amino acids (aa), which is 75% identical to the LexA repressor of Ec. Two Ec-like SOS boxes have been located upstream from Ah lexA, the distance between them being 4 bp, whereas this same distance in Ec lexA is 5 bp. The structure and sequence of the DNA-binding domain of the LexA repressor of Ec, as well as the region at which its hydrolysis occurs, are highly conserved in Ah LexA. Moreover, a residue of the region implicated in the specific cleavage reaction, and which is present in all known RecA-cleavable repressors, is changed in the Ah LexA. Expression of Ah lexA is DNA-damage inducible in both the Ah and Ec genetic backgrounds to the same extent. In contrast, Ec lexA is poorly induced in DNA-injured Ah cells.

Aeromonas hydrophila↗
PubMedDate not supplied

Binding of Escherichia coli LexA repressor to the RecA operator.

Equilibrium binding of Escherichia coli LexA repressor to the recA operator was studied by the polyacrylamide gel mobility shift assay as a function of solution conditions. In the presence of NaCl at 20 degrees C, there was a significant salt dependence in binding to the recA operator, typical for protein-nucleic acid interactions with some electrostatic contribution to the binding free energy. In preliminary experiments in which the anion of the Na+ salt was changed from chloride to fluoride, little change was found with anion identity. This indicates that the salt effect on the binding interaction arises solely from the polyelectrolyte effect, not from anion binding or release by the protein upon complex formation. Increasing the temperature to 37 degrees C changed the binding affinity for complex formation at any given salt concentration and resulted in a change in the sensitivity of complex formation to NaCl concentration. Quantitative analysis of the data to obtain equilibrium binding constants is discussed.

Anions↗
PubMed2006

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.

Anilino Naphthalenesulfonates↗
PubMed1988

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.

Alkylation↗
PubMed1998

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Bacterial Proteins↗
PubMed1992

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.

Bacterial Proteins↗
PubMed1997

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis.

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli. The recombinant protein bound to the promoter regions of the M. leprae lexA, M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis. Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis. Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti-M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.

Bacterial Proteins↗
PubMed1988

The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor.

LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).

Bacterial Proteins↗
PubMed1996

SOS response as an adaptive response to DNA damage in prokaryotes.

Escherichia coli possesses an elaborate adaptive mechanism called the "SOS response" to cope with various types of DNA damage. More than 20 SOS genes, most of which are known to be involved in the functions that promote the survival of DNA-damaged cells, are induced by treatments that damage DNA or inhibit DNA synthesis. All the SOS genes share similar sequences in the regulatory regions called the "SOS box", to which LexA repressor binds to repress the transcription in the absence of DNA damage. The SOS signal appears to be the single-stranded DNA produced in vicinity of DNA damage, to which RecA protein binds to be activated as a coprotease. The activated RecA promotes autocleavage of LexA protein by allosteric interaction, which activates the latent serine protease activity of LexA. The induced products of the SOS genes repair DNA lesions by various mechanisms, including recombination, excision repair and error-prone repair, and as the consequence, the SOS signal in the cell decreases and the repression of the SOS genes is restored.

Bacterial Proteins↗
PubMed1985

Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli.

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.

Bacterial Proteins↗
PubMed1993

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli.

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind-) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations oc in the umuDC and umuD'C operons and also used the oc98-recA mutation. The oc1-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutagenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.

Bacterial Proteins↗