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PubMed1983

Characterisation of the promoter for the LexA regulated sulA gene of Escherichia coli.

The sulA gene of Escherichia coli, which encodes an inducible inhibitor of cell division, plays a role in the SOS response. Its expression, like that of other SOS genes, is repressed by the LexA protein. This paper reports the identification and characterisation of the promoter for the cloned sulA gene. The promoter bears good resemblance to the consensus promoter sequence and directs the synthesis of two major RNA species both in vitro and in vivo. Comparison of the sequence around the sulA promoter with the operator sequences of other SOS genes revealed the presence of an SOS box, the LexA protein binding site. This overlaps the -10 region of the promoter and covers the transcriptional initiation sites. LexA protein bound to this SOS box, would, therefore, effectively block transcription.

Bacterial Proteins↗
PubMed1992

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.

Adenosine Triphosphate↗
PubMed2004

Rapid spontaneous accessibility of nucleosomal DNA.

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.

Bacterial Proteins↗
PubMed1997

Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box.

The gene coding for the Mycobacterium tuberculosis homologue of LexA has been cloned and sequenced. Amino acids required for autocatalytic cleavage are conserved, whereas those important for specific DNA binding are not, when compared with Escherichia coli LexA. The transcriptional start site was mapped and a DNA sequence motif was identified which resembled the consensus Cheo box sequence involved in the regulation of DNA-damage-inducible genes in Bacillus subtilis. The M. tuberculosis-LexA protein was overexpressed in E. coli and purified by means of a His tag. The purified LexA was shown to bind to the Cheo box sequence found upstream of its own gene.

Amino Acid Sequence↗
PubMed1977

Cell division in Escherichia coli BS-12 is hypersensitive to deoxyribonucleic acid damage by ultraviolet light.

Escherichia coli BS-12 uvrA lon is hypersensitive to ultraviolet light. On minimal agar plates at densities in excess of about 10(7) bacteria per plate, as few as one or two photoreversible pyrimidine dimers in the entire genome are sufficient to cause inhibition of cell division. Most of the resulting filaments are unable to divide or form a viable colony. Inhibition of cell division appears to be a rapid consequence of replication of deoxyribonucleic acid containing a pyrimidine dimer. Photoreversibility of the inhibition of cell division persists indefinitely, indicating that the continued presence of the pyrimidine dimers (or the continued generation of daughter strand gaps) is necessary to maintain the division-inhibited state. In view of the kinetics for the production of filamentation by ultraviolet light and the extremely low average inducing fluence (0.03 J/m2), it is concluded that the initiating signal is not the same as that causing other inducible phenomena such as prophage induction or Weigle reactivation.

Bacterial Proteins↗
PubMed1987

An inducible pathway is required for mutagenesis in Salmonella typhimurium LT2.

UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA+ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate (K. Overbye and P. Margolin, J. Bacteriol. 146:170-178, 1981; K. Overbye, S. M. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Mitomycin C induction of the phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead us to deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.

Bacterial Proteins↗
PubMed1994

Analysis of the SOS inducing signal in Bacillus subtilis using Escherichia coli LexA as a probe.

We analyzed the Bacillus subtilis SOS response using Escherichia coli LexA protein as a probe to measure the kinetics of SOS activation and DNA repair in wild-type and DNA repair-deficient strains. By examining the effects of DNA-damaging agents that produce the SOS inducing signal in E. coli by three distinct pathways, we obtained evidence that the nature of the SOS inducing signal has been conserved in B. subtilis. In particular, we used the B. subtilis DNA polymerase III inhibitor, 6-(p-hydroxyphenylazo)-uracil, to show that DNA replication is required to generate the SOS inducing signal following UV irradiation. We also present evidence that single-stranded gaps, generated by excision repair, serve as part of the UV inducing signal. By assaying the SOS response in B. subtilis dinA, dinB, and dinC mutants, we identified distinct deficiencies in SOS activation and DNA repair that suggest roles for the corresponding gene products in the SOS response.

Amino Acid Sequence↗
PubMed2005

The Leptospira interrogans lexA gene is not autoregulated.

Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated.

Bacterial Proteins↗
PubMed1994

The 90-kDa heat shock protein is essential for Ah receptor signaling in a yeast expression system.

In an effort to provide a more powerful system to study the Ah receptor (AHR) signaling pathway, we expressed the AHR, its dimerization partner ARNT, and a beta-galactosidase (lacZ) reporter gene, driven by two dioxin-responsive enhancers, in the yeast Saccharomyces cerevisiae. In this system, the agonists beta-naphthoflavone and alpha-naphthoflavone induced transcription of the lacZ gene, with EC50 values of 7.9 x 10(-8) and 3.0 x 10(-7) M, respectively, while the nonagonist dexamethasone was without effect. As a first application of this system, we examined the relationship between the 90-kDa heat shock protein (hsp90) and AHR function. To accomplish this in a manner that was independent of the ARNT protein, we constructed a chimeric receptor in which the DNA binding and primary dimerization domains of the AHR were swapped with analogous domains from the LexA protein. Coexpression of this AHR-LexA chimera and a lacZ reporter gene driven by eight LexA operator sites in a yeast strain with regulatable levels of hsp90, yielded pharmacology that closely mirrored that of the AHR/ARNT/dioxin-responsive enhancer system described above, but only when hsp90 levels were held near their wild type levels. When hsp90 levels were reduced to approximately 5% of normal, AHR signaling in response to agonist was completely blocked despite normal cell growth. These results provide the first genetic evidence for the role of hsp90 in AHR signaling and provide the basis for a powerful new system in which to study this pathway.

Bacterial Proteins↗
PubMed2001

Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli.

The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

Bacterial Proteins↗
PubMed2003

DNA array analysis of gene expression in response to UV irradiation in Escherichia coli.

The capacity of DNA macroarrays that contain all 4290 predicted open reading frames of the E. coli K12 genome was evaluated by measuring changes in gene expression in response to irradiation by ultraviolet light (UV). UV and other DNA damaging agents are known to trigger the induction of the SOS response. This is a coordinated increase in the level of expression of a set of approximately 30 unlinked genes, the SOS genes, negatively regulated by the LexA repressor. The analysis was performed on a set of isogenic strains with mutations that affect expression of genes of the SOS system: (i) the lexA+ strain, in which the SOS system can be induced after DNA damage, (ii) lexAind- mutants in which the SOS system cannot be induced, and (iii) lexAdef mutants in which the SOS system is induced constitutively. We found that a large set of genes appeared to be either upregulated or downregulated following UV irradiation. Among the genes which appeared to be upregulated in a LexA-dependent manner, we correctly identified 9 out of 27 SOS genes printed on the arrays and one gene containing a LexA binding site. One gene, dnaN, encoding the beta subunit of DNA polymerase III holoenzyme, was identified as an upregulated gene in a LexA-independent manner. Our results were compared to those of similar studies previously published. Although the SOS response as a whole could not be illustrated by using DNA arrays, the data suggest that regulation of some SOS genes might be more complex than previously thought.

Bacterial Proteins↗
PubMed1983

Transcription of the sulA gene and repression by LexA.

The Escherichia coli sulA gene product is a highly unstable protein, whose synthesis in response to DNA damage is associated with an inhibition of septation. Genetic evidence as well as sequence information suggests that the sulA gene is part of the E. coli SOS system and is induced after DNA damage. We have constructed a plasmid carrying only the sulA gene; this plasmid is stable only when it contains an amber mutation in the sulA structural gene. Using fragments of this plasmid, we have carried out in vitro transcription experiments and demonstrated one major start site for RNA transcription. We have mapped this initiation point to an adenylate residue 30 nucleotides before the protein start. Purified LexA protein completely abolishes this transcription, in agreement with the prediction made from the genetic and sequence information previously available.

Bacterial Proteins↗
PubMed1988

Mutagenic DNA repair in Escherichia coli. XVI. Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains.

Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal. Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele. No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present. It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect. Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.

Alleles↗
PubMed1982

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12.

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.

Bacterial Proteins↗
PubMed1997

The Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide.

The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H2O2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H2O2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H2O2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways.

Bacterial Proteins↗
PubMed1996

Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

Adaptor Proteins, Signal Transducing↗
PubMed1981

Regulation of SOS functions: purification of E. coli LexA protein and determination of its specific site cleaved by the RecA protein.

The LexA protein of Escherichia coli was purified to more than 96% purity from cells harboring a recombinant plasmid carrying the lexA gene with the lacZ promoter sequence. The amino acid composition of the LexA protein and its amino-terminal sequence were analyzed. The results are in agreement with the prediction from the nucleotide sequence of the lexA gene. The LexA protein is cleaved into two polypeptides by E. coli RecA protein in the presence of ATP and single-stranded DNA. The site of the specific cleavage was determined by analyzing amino acid sequences of the cleaved products at the amino and carboxyl termini. The cleavage of the LexA protein by the RecA protein was found to occur at a single site between Ala84 and Gly85.

Bacterial Proteins↗
PubMed1983

Nucleotide sequence of the regulatory region of the uvrD gene of Escherichia coli.

We have sequenced the control region of the Escherichia coli uvrD gene and demonstrated the presence of a nucleotide sequence which is a perfect match for the consensus LexA protein binding site [Little and Mount, Cell 29 (1982) 11-22]. Upstream of this presumed LexA binding site is a promoter sequence, uvrD P1 which would be under LexA control while farther downstream is another possible promoter, uvrD P2, which would be independent of LexA control. Downstream of the LexA binding site is a potential transcription terminator in the form of a stem-loop structure followed by a series of T residues. On the basis of this sequence analysis, expression of the uvrD gene would be expected to increase after DNA damage or replication inhibition as part of the SOS response, as is reported in the preceding paper [Arthur and Eastlake, Gene 25 (1983) 309-316].

Bacterial Proteins↗