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At least 73 records · Page 4Linked to original sources
PubMed1998

Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae.

Transcriptional activation of the recA gene of Streptococcus pneumoniae was previously shown to occur at competence. A 5.7 kb recA-specific transcript that contained at least two additional genes, cinA and dinF, was identified. We now report the complete characterization of the recA operon and investigation of the role of the competence-specific induction of recA. The 5.7 kb competence-specific recA transcript is shown to include lytA, which encodes the pneumococcal autolysin, a protein previously shown to contribute to virulence of S. pneumoniae. Uncoupling (denoted Ind-) of recA and/or the downstream genes was achieved through the placement of transcription terminators within the operon, either upstream or downstream of recA. Prevention of the competence-specific induction of recA severely affected spontaneous transformation. Transformation efficiencies of recA+ (Ind-) and of wild-type cells were compared under various conditions and with different donor DNA. Chromosomal transformation was reduced 17-(chromosomal donor) to 45-fold (recombinant plasmid donor), depending on the donor DNA, and plasmid establishment was reduced 129-fold. Measurement of uptake of radioactively labelled donor DNA in transformed cells in parallel with scoring for transformants (chromosomal donor) revealed normal uptake, but a 21-fold reduction in recombination in a recA+ (Ind-) strain, indicating that the transformation defect was primarily in recombination. Strikingly enough, a much larger (460-fold) reduction in recombination was observed for the shortest homologous donor fragment used (878 nucleotides long). Possible interpretations of the observation that basal RecA appears unable to promote efficient recombination whatever the number and the length of donor fragments taken up are proposed. The role of recA induction is discussed in view of the potential contribution of transformation to genome plasticity in this pathogen.

Amidohydrolases↗
PubMed1998

DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence?

The genome sequence of Mycobacterium tuberculosis was analysed by searching for homologues of genes known to be involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes necessary to perform nucleotide excision repair (NER), base excision repair (BER), recombination, and SOS repair and mutagenesis were identified. In particular, all of the genes known to be directly involved in the repair of oxidative and alkylative damage are present in M. tuberculosis. In contrast, we failed to identify homologues of genes involved in mismatch repair. This finding has potentially significant implications with respect to genome stability, strain variability at repeat loci and the emergence of chromosomally encoded drug resistance mutations.

DNA Damage↗
PubMed2001

Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2::kan mutant.

Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution. Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli. PriA mutants are Rec(-) and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression. PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4',6-diamidino-2-phenylindole-stained log phase cells. Two populations of cells were seen. Eighty four percent appeared like wild type, and 16% of the cells were filamented and had poorly partitioned chromosomes (Par(-)). To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant. Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning. Mutating either recA or recB virtually eliminated the Par(-) phenotype. Filamentation in the recB mutant decreased to 3%, but increased to 28% in the recA mutant. The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal. Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK. Mutation of dif showed no change in phenotype whereas ftsK1cat was lethal with priA2kan. A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.

Adenosine Triphosphatases↗
PubMed2001

Historical overview: searching for replication help in all of the rec places.

For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism. Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination. In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks. Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions. The new results feature multiple pathways for repair and the involvement of many enzymatic systems. The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents. In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse. Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination. This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research.

Animals↗
PubMed2002

Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics.

A basic challenge in systems biology is to understand the dynamical behavior of gene regulation networks. Current approaches aim at determining the network structure based on genomic-scale data. However, the network connectivity alone is not sufficient to define its dynamics; one needs to also specify the kinetic parameters for the regulation reactions. Here, we ask whether effective kinetic parameters can be assigned to a transcriptional network based on expression data. We present a combined experimental and theoretical approach based on accurate high temporal-resolution measurement of promoter activities from living cells by using green fluorescent protein (GFP) reporter plasmids. We present algorithms that use these data to assign effective kinetic parameters within a mathematical model of the network. To demonstrate this, we employ a well defined network, the SOS DNA repair system of Escherichia coli. We find a strikingly detailed temporal program of expression that correlates with the functional role of the SOS genes and is driven by a hierarchy of effective kinetic parameter strengths for the various promoters. The calculated parameters can be used to determine the kinetics of all SOS genes given the expression profile of just one representative, allowing a significant reduction in complexity. The concentration profile of the master SOS transcriptional repressor can be calculated, demonstrating that relative protein levels may be determined from purely transcriptional data. This finding opens the possibility of assigning kinetic parameters to transcriptional networks on a genomic scale.

Algorithms↗
PubMed1992

Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.

Adenosine Triphosphate↗
PubMed1997

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA.

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated lambda phage infecting UV-preirradiated bacterial cells (termed lambdaUTM for lambda untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for lambdaUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F'lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.

Bacterial Proteins↗
PubMed2000

Histone-like protein HU is required for recA gene-dependent DNA repair and SOS induction pathways in UV-irradiated Escherichia coli.

PURPOSE: Escherichia coli HU protein exists as a heterodimer composed of two highly homologous subunits, HU-1 and HU-2, encoded by the hupB and hupA genes, respectively. It introduces negative supercoils into a relaxed circular DNA. Various roles of HU have been suggested in cellular processes such as DNA replication and transcription. The present experiments were designed to understand the role of HU in DNA repair processes in E. coli. MATERIALS AND METHODS: The sensitivity of hupA/hupB mutants of E. coli to the lethal and mutagenic effects of UV was compared with that of a wild-type strain. The effect of the hupAhupB mutations in SOS induction was also examined. RESULTS: The hupAhupB mutations increased the UV sensitivity of E. coli. Nucleotide excision repair was unaffected by the deficiency of HU. On the other hand, E. coli hupAhupB mutants were sensitive to UV in the recA+recB+recF background but not in the recArecB+recF+ or recA+recBrecF+ background. The frequency of UV-induced mutation to rifampicin resistance was significantly reduced in the hupAhupB mutants, and the induction of the recA::lacZ and umuC::lacZ fusion genes was also suppressed in the mutants. CONCLUSIONS: HU protein plays a critical role in the recA, recB-dependent recombinational DNA repair and SOS induction pathways in UV-irradiated E. coli.

Bacterial Proteins↗
PubMed2003

The directed mutation controversy in an evolutionary context.

Neo-Darwinists have long held that random mutations produce genetic differences among individuals, and selection increases the frequency of advantageous alleles. In 1988, Cairns et al. claimed that an environmental pressure can cause advantageous mutations to occur in specific genes to alleviate that particular pressure. Directed mutation, as proposed by Cairns, has been all but eradicated from evolutionary thinking. However, more than a decade of research spurred by the Cairns et al. paper has cast doubt on three neo-Darwinian principles: (1) mutations occur independently of the environment, (2) mutations are due to replication errors, and (3) mutation rates are constant. This mini-review explores the history of the controversy and the decade of research that followed so as to place it in an evolutionary context. Several of the cellular mechanisms and models that explain the increased genetic diversity in populations experiencing adverse environmental pressure are described. In most cases it is clear that the increased genetic diversity is due to breakdowns of cellular machinery or alleles evolved for a purpose other than increasing genetic diversity, rather than to cellular systems that have been evolutionarily selected to increase the genetic diversity in times of stress.

Bacteria↗
PubMed1999

FtsK-dependent and -independent pathways of Xer site-specific recombination.

Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division. Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers. Two recombinases, XerC and XerD, act at the E. coli chromosomal recombination site, dif, and at related sites in plasmids. We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors. We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif. The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers. Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates.

Bacterial Proteins↗
PubMed1991

Control of large chromosomal duplications in Escherichia coli by the mismatch repair system.

Excessive recombination between repeated, interspersed, and diverged DNA sequences is a potential source of genomic instability. We have investigated the possibility that a mechanism exists to suppress genetic exchange between these quasi-homologous (homeologous) sequences. We examined the role of the general mismatch repair system of Escherichia coli because previous work has shown that the mismatch repair pathway functions as a barrier to interspecies recombination between E. coli and Salmonella typhimurium. The formation of large duplications by homeologous recombination in E. coli was increased some tenfold by mutations in the mutL and mutS genes that encode the mismatch recognition proteins. These findings indicate that the mismatch recognition proteins act to prevent excessive intrachromosomal exchanges. We conclude that mismatch repair proteins serve as general controllers of the fidelity of genetic inheritance, acting to suppress chromosomal rearrangements as well as point mutations.

Chromosomes, Bacterial↗
PubMed1993

Tn7 transposition creates a hotspot for homologous recombination at the transposon donor site.

Homologous recombination at the bacterial transposon Tn7 donor site is stimulated 10-fold when Tn7 is activated to transpose at high frequency in RecD- Escherichia coli, where recombination is focused near the ends of double-chain breaks. This is observed as an increase in recombination between two lacZ heteroalleles when one copy of lacZ carries within it a Tn7 that is transposing at high frequency. This stimulation of recombination is dependent upon the presence of homology with the donor site, is independent of SOS induction, and is not due to a global stimulation of recombination. When stimulated by Tn7 transposition, the conversion events giving rise to Lac+ recombinants occur preferentially at the site of Tn7, suggesting that transposition is stimulating gene conversion at the donor site. These results support the model that Tn7 transposition occurs by a "cut and paste" mechanism, leaving a double-chain break at the donor site that is repaired by the host homologous recombination machinery; normally, repair would use homology in a sister chromosome to regenerate a copy of the transposon. This proposed series of events allows transposition that is nonreplicative, per se, to be effectively replicative.

Chromosomes, Bacterial↗
PubMed1995

Molecular analysis of lambda bio transducing phage produced by oxolinic acid-induced illegitimate recombination in vivo.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of lambda Spi- phage during prophage induction. The frequency of Spi- phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalAr bacteria with the drug. RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes. Analyses of att region and recombination junctions of Spi- phages revealed that they have essentially the same structures as lambda bio transducing phages but are classified into two groups with respect to recombination sites. In the majority class of the transducing phages, there were not more than 3-bp homologies between the parental E. coli bio and lambda recombination sites. In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites. These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism.

Bacteriophage lambda↗
PubMed1988

Quantification of the genotoxic effects of alkylating agents in bacterial assays.

The genotoxic effects of 11 monofunctional alkylating carcinogens in the Escherichia coli Multitest, SOS Chromotest and Salmonella assay were quantified using the 'yield versus lethal hit plot' method. In this method, the genotoxic yield-potency is defined as the integral under the yield versus lethal hit curve, whereas the genotoxic yield-efficiency is defined as the maximum yield obtained. With the series of substances tested, these two genotoxicity indices were found to be equivalent in the E. coli Multitest. The three bacterial short-term tests were compared and the following observations made: (i) the mutagenic efficiencies in the Salmonella assay and in the Multitest are correlated; (ii) the SOS-inducing efficiency in the SOS Chromotest correlates with both the SOS-inducing efficiency and the recombinogenic efficiency in the Multitest; (iii) mutagenesis and SOS induction measured by the same protocol in the Multitest are clearly distinct phenomena; (iv) when quantified with the 'yield versus lethal hit plot' method, the data from the SOS Chromotest do not correlate any more with the data from the Salmonella assay. Therefore the SOS Chromotest should be considered as a complementary rather than an alternative test to the Salmonella assay. Consequently, the Multitest appears as a relevant candidate for the basis of a battery of tests since it permits the measurement of at least two independent genotoxic effects in a single bacterial system. The method of quantification of genotoxic potencies using the 'yield versus lethal hit plot' method demonstrates the quantitative relationships existing between the data obtained using different systems measuring the same genotoxic effect and differentiates different genotoxic effects from each other.

Alkylating Agents↗
PubMed2002

The role of multiple SOS boxes upstream of the Mycobacterium tuberculosis lexA gene--identification of a novel DNA-damage-inducible gene.

Four potential binding sites for LexA were identified upstream of the Mycobacterium tuberculosis lexA gene. A mutational analysis of these sites in a lexA-lacZ reporter construct revealed that only one of these SOS boxes was required for DNA-damage-mediated regulation of lexA expression. A novel DNA-damage-inducible gene, Rv2719c, was identified that was divergently transcribed relative to lexA; the other three SOS boxes were found to be involved in regulating expression of this novel mycobacterial-specific gene. The SOS boxes lay in the respective promoter regions of the genes that they regulated.

Amino Acid Sequence↗
PubMed1994

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions.

Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.

Bacillus subtilis↗
PubMed1993

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis.

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.

Bacterial Proteins↗