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PubMed1999

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation.

The recA gene of Escherichia coli is the prototype of the recA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination. In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants. Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system. All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage. The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover. Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA-independent, lethal filamentation of cells.

Adenosine Triphosphate↗
PubMed1999

On the in vivo function of the RecA ATPase.

The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.

Adenosine Triphosphatases↗
PubMed2000

Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments.

The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination. In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies. These data revealed preferential binding at filament ends (most likely, at one end). End-specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis.

Binding Sites↗
PubMed1995

Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains.

The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E. coli cultures when the drug was added simultaneously to the thermal induction. A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E. coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells. This feature was not detected in other E. coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.

DNA, Bacterial↗
PubMed1995

Promoters of the phr gene in Escherichia coli K-12.

We have identified two promoters of the Escherichia coli phr gene by DNA deletion mapping, S1 mapping of transcripts and sequence homology. The weaker promoter, P2, located approximately 530 bp upstream from the start codon, extends beyond the previously known nucleotide sequence. The stronger, P1, lies 90 bp from the gene and is distinct from three previously described promoter-like sequences nearby. beta-Galactosidase production from a plasmid-borne gene, promoted by a synthetic copy of P1, increases after DNA damage, but the increase does not depend on the SOS-box-like sequences normally present in the vicinity of P1. Induction still requires intact recA and lexA genes, and also intact sulA.

Base Sequence↗
PubMed1988

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound.

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.

Benzaldehydes↗
PubMed1990

The role of dam methyltransferase in the control of DNA replication in E. coli.

The timing and control of initiation of DNA replication in E. coli was studied under conditions where the cellular level of dam methyltransferase was controlled by a temperature-inducible promoter. Flow cytometry was used to demonstrate that the synchrony of initiation at the several origins within each cell was critically dependent on the level of dam methyltransferase. Initiations were shown to be synchronous only in a narrow temperature range. The data are explained by a model where a newly replicated and therefore hemimethylated oriC is inert for reinitiation. Such a model may be applicable to eukaryotic cells, where classes of origins are initiated in synchrony and only once per cell cycle.

DNA Replication↗
PubMed1990

Participation of rec genes of Escherichia coli K 12 in W-reactivation of UV-irradiated phage lambda.

The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA]. W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation.

Bacterial Proteins↗
PubMed1991

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli.

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.

Antioxidants↗
PubMed1991

Activation of RecA protein in recombination-deficient strains of Escherichia coli following DNA-damaging treatments.

Activation of the RecA protein following UV-irradiation or bleomycin (BM) treatment was measured in rec mutants of E. coli by monitoring beta-galactosidase activity. We provide evidence here that the defect in the recN mutant results in high constitutive and induced levels of activated RecA protein. In all rec mutants studied, with the exception of the recN mutant, induction of enzyme activity, following DNA-damaging treatments, was reduced relative to the wild type. The kinetics of induced sfiA expression indicates that the DNA-unwinding activity of the RecBCD enzyme plays a major role in SOS-signal formation. The RecF protein is not needed for BM induction in strains with a functional RecBCD pathway of recombination. However, a functional product of recF gene is implied in the formation of an efficient inducing signal after UV-irradiation, as well as in the additional processing of BM-induced lesions after exposure to the drug. A fully expressed RecF pathway of recombination does not provide a high level of activated RecA protein following DNA-damaging treatments.

Bleomycin↗
PubMed1996

Genetic barriers among bacteria.

Barriers to chromosomal gene transfer between bacterial species control their genetic isolation. These barriers, such as different microhabitats, the host ranges of genetic exchange vectors and restriction-modification systems, limit gene exchange, but the major limitation is genomic sequence divergence. The mismatch-repair system inhibits interspecies recombination, the inducible SOS system stimulates interspecies recombination, while natural selection determines the effective recombination frequencies.

Bacteria↗
PubMed2005

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more).

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.

Bacterial Proteins↗
PubMed2005

The SOS-LUX-TOXICITY-Test on the International Space Station.

For the safety of astronauts and to ensure the stability and integrity of the genome of microorganisms and plants used in bioregenerative life support systems, it is important to improve our knowledge of the combined action of (space) radiation and microgravity. The SOS-LUX-TOXICITY test, as part of the TRIPLE-LUX project (accepted for flight at Biolab in Columbus on the International Space Station, (ISS)), will provide an estimation of the health risk resulting from exposure of astronauts to the radiation environment of space in microgravity. The project will: (i) increase our knowledge of biological/health threatening action of space radiation and enzymatic DNA repair; (ii) uncover cellular mechanisms of synergistic interaction of microgravity and space radiation; (iii) provide specified biosensors for spacecraft milieu examination; and (iv) provide experimental data on stability and integrity of bacterial DNA in spacecrafts. In the bacterial biosensor "SOS-LUX-Test" developed at DLR (patent), bacteria are transformed with the pBR322-derived plasmid pPLS-1 or the similar, advanced plasmid SWITCH, both carrying the promoterless lux operon of Photobacterium leiognathi as the reporter element controlled by a DNA damage-dependent SOS promoter as sensor element. A short description of the space experiment is given, and the current status of adaptation of the SOS-LUX-Test to the ISS, i.e. first results of sterilization, biocompatibility and functional tests performed with the already available hardware and bread board model of the automated space hardware under development, is described here.

Bacteriological Techniques↗
PubMed2004

Rewiring the cell: synthetic biology moves towards higher functional complexity.

A steady stream of research has fueled excitement in the field of synthetic biology. Logic gates, oscillators, and memory elements constructed using genetic and biochemical components have all been demonstrated. However, the nagging question remains as to how higher levels of complexity can be designed into these synthetic systems. A recent paper from Collins' group provides some answers to this question.

Biomimetics↗
PubMed1999

A phylogenomic study of DNA repair genes, proteins, and processes.

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.

Adenosine Triphosphatases↗
PubMedDate not supplied

Bacillus subtilis homologous recombination: genes and products.

Homologous recombination plays a critical role in maintaining gene diversification and genome stability. Fourteen Bacillus subtilis recombination gene products have been genetically characterised and classified into five different epistatic groups. At least seven other recombination genes could be predicted. Recombination gene products which define activities that help RecA to process DNA repair and recombination have been studied, but those that processed recombination intermediates into products (post-synaptic stage) await elucidation.

Bacillus subtilis↗
PubMed1997

Analysis of ssb mutations in vivo implicates SSB protein in two distinct pathways of SOS induction and in recombinational DNA repair.

Site-directed mutations in the Escherichia coli ssb gene were tested for the ability to complement a chromosomal ssb deletion for viability, and only the ssb W54-->G mutation failed to do so at the pSC101 copy level. Non-aromatic amino acid substitutions for SSB Trp-54 (ssb W54-->L and ssb W54-->S) produced the greatest effects on in vivo protein function including altered marker linkage subsequent to generalized transduction, extreme UV sensitivity, and a lack of ability to support SOS induction. Additionally, the ssb-113 (ssb P176-->S) mutation demonstrated the existence of both uvrA-dependent and uvrA-independent components of SOS induction. Although nucleotide excision repair appeared unaffected by alterations in the SSB protein, the mutational analysis suggests a direct role for SSB in recombinational repair.

Bacteriophage P1↗