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At least 469 records · Page 26Linked to original sources
PubMed1991

Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria.

A broad host range plasmid containing an operon fusion between the recA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria-30 species belonging to 20 different genera - to study the expression of the recA gene after DNA damage. These included species of the families Enterobacteriaceae, Pseudomonadaceae. Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae and Azotobacteraceae. Results obtained show that all bacteria tested, except Xanthomonas campestris and those of the genus Rhodobacter, are able to repress and induce the recA gene of E. coli in the absence and in the presence of DNA damage, respectively. All these data indicate that the SOS system is present in bacterial species of several families and that the LexA-binding site must be very conserved in them.

DNA Damage↗
PubMed1991

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage.

Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.

Bacterial Proteins↗
PubMed1992

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage.

The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.

Bacterial Proteins↗
PubMed1990

Proteolytic processing of MucA protein in SOS mutagenesis: both processed and unprocessed MucA may be active in the mutagenesis.

The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ'-'MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA+ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.

Bacterial Proteins↗
PubMed1991

Heat-inducible reactivation of UV-damaged bacteriophage lambda.

Induction of the SOS response in UV-irradiated bacteria leads to an increase in the survival of an infecting irradiated bacteriophage lambda (Weigle 1953). We report that a similar reactivation of irradiated phage lambda was induced by shifting the culture of recipient bacteria from 30 degrees to 47 degrees C. However, this repair process was nonmutagenic. The amplitude of the phenomenon was increased with the quantity of UV lesions in the phage DNA. It was present despite mutations affecting the SOS response or the heat shock response in the infected strains (recA, lexA, umuC or rpoH mutations respectively). In contrast, the heat-inducible repair process was abolished in uvrA derivatives. Also, pretreatment with chloramphenicol largely enhanced phage reactivation after heat shock. Therefore, it appears that the excision repair mechanism of UV lesions was stimulated both by temperature shift-up and blockage of protein synthesis.

Bacteriophage lambda↗
PubMed1993

SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light.

In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mp18 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvr A fpg-1 double mutant than that seen in uvr A, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GC-->TA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly G-->T transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.

Bacteriophage M13↗
PubMed1994

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides.

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA- mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium meliloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 bp upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactivated in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.

Amino Acid Sequence↗
PubMed1994

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli.

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective epsilon subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122::Tn5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC+ plasmid. Interestingly, while the frequency of UV-induced GC-->AT transitions is greatly reduced, the frequency of AT-->TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC+; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.

Bacterial Proteins↗
PubMed1991

Specificity of recA441-mediated (tif-1) mutational events.

To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI- mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G:C----C:G, G:C----T:A and A:T----T:A transversion events were specifically enhanced after SOS induction. A preferential 5'-Y-Purine-3' neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5'-C/GTGG-3' sequences is also observed. Fifty events were recovered at the lacI "frameshift hotspot site" and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA+ background and is a consequence of the fourfold induction of the (-)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI- mutations.

Amino Acids↗
PubMed1993

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo.

Evidence is presented that the pR bat gene is essential for plasmid replication and for spontaneous induction of the SOS response in Escherichia coli. Mutations preventing single-stranded DNA production, needed for pR plasmid replication, also prevent the induction of the SOS system. The following experimental design was used. Firstly, we identified the minima rep region, defined as the minimal DNA sequence necessary for pR plasmid replication and, secondly, analyzed the nucleotide sequence of this region. This identified structures and functions (ori-plus, ori-minus and Rep protein) homologous to those found in phages and plasmids replicating by the rolling-circle mechanism. Finally, mutations were introduced either in the replication protein catalytic site or in the nick site consensus sequence, which caused the pR plasmid to lose its ability to induce the SOS system. We conclude that, in this system, the in vivo SOS-inducing signal appears to be the single-stranded DNA produced during pR replication.

Amino Acid Sequence↗
PubMed1995

Altered SOS induction associated with mutations in recF, recO and recR.

The SOS system of Escherichia coli aids survival following damage to DNA by promoting DNA repair while cell division is delayed. Induction of the SOS response is dependent on RecA and also on the product of recF. We show that normal induction also requires the products of recO and recR. SOS induction was monitored using a sfiA-lacZ fusion strain. Induction was delayed to a similar degree by mutation in recF, recO or recR. A similar effect was observed following overexpression of RecR from a recombinant recR+ plasmid. We show that the overexpression of RecR also reduces the UV resistance of a recBC sbcBC strain and of a sfiA strain, but not of a rec+sfiA+ strain. The implications of these data for the kinetics of DNA repair are discussed.

Bacterial Proteins↗
PubMed1987

Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively.

We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh). The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions. In E. coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed. The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription. Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription.

DNA Polymerase III↗
PubMed1987

Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli.

To measure the degree of phr gene induction by DNA-damaging agents, the promoter region was fused to the coding region of the lacZ gene in plasmid pMC1403. The new plasmids were introduced into Escherichia coli cells having different repair capabilities. More efficient induction of phr gene expression was detected in a uvrA- strain as compared with the wild-type strain. In addition, obvious induction was detected in uvrA- cells treated by 4-nitroquinoline 1-oxide and mitomycin C. Nalidixic acid, an inhibitor of DNA gyrase, also induced phr gene expression. In contrast, little induced gene expression was noted in UV-irradiated lexA- and recA- strains. It is suggested from these results that induction of the phr gene is one of the SOS responses. Possible nucleotide sequences which could be considered to constitute an SOS box were found at the regulator region of the phr gene.

Bacterial Proteins↗
PubMed1987

A new mutator strain of Bacillus subtilis.

Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).

Bacillus subtilis↗
PubMed1988

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli.

Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ+. dnaQ49 is a recessive allele that confers temperature-sensitive and salt-suppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.

Alleles↗
PubMed1988

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells.

The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.

Alleles↗