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PubMed1988

Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.

Bacteriophage lambda↗

PubMed1997

A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats.

Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes). This recombinational rearrangement event is efficient and independent of RecA and RecBCD. In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats. In this model, the inverted repeats promote the formation of a DNA cruciform which is processed by an endonuclease into a linear DNA with two hairpin loops at its ends. Upon DNA replication, this linear dumbbell-like DNA is then converted to the inverted dimer. In support of this model, linear dumbbell DNA molecules with unidirectional origin of DNA replication (ColE1 ori ) have been constructed and shown to transform E.coli efficiently resulting in the formation of the inverted dimer. The ability of linear dumbbell DNA to transform E.coli suggests that the terminal loops may be important in bypassing the requirement of DNA supercoiling for initiation of replication of the ColE1 ori.

DNA↗

PubMed1999

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.

DNA Transposable Elements↗

PubMed2006

DNA mechanics as a tool to probe helicase and translocase activity.

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

DNA↗

PubMed2006

Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.

All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

Bacterial Proteins↗

PubMed2003

A real-time DNase assay (ReDA) based on PicoGreen fluorescence.

DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed.

Binding, Competitive↗

PubMed1993

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli.

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

ATP-Dependent Proteases↗

PubMed1999

Characterization of the recD gene of Neisseria gonorrhoeae MS11 and the effect of recD inactivation on pilin variation and DNA transformation.

Pilin antigenic variation in Neisseria gonorrhoeae may result following intrachromosomal recombination between homologous pil genes. Despite extensive study, recA is the only previously characterized gene known to be involved in this process. In this study, the gonococcal recD gene, encoding one subunit of the putative RecBCD holoenzyme, was characterized and its role in pilin variation assessed. The complete recD gene of N. gonorrhoeae MS11 was cloned and its nucleotide sequence determined. The gonococcal recD gene complemented a defined Escherichia coli recD mutant, based on plaque formation of bacteriophage lambda and the restoration of ATP-dependent nuclease activity. Inactivation of the gonococcal recD gene had no measurable effect on cell viability or survival following UV exposure, but did decrease the frequency of DNA transformation approximately threefold. The frequency at which non-parental pilin phenotypes were spawned was 12-fold greater in MS11 recD mutants compared with the parental MS11 rec+ strain. Similar results were obtained using recD mutants that were not competent for DNA transformation. Complementation of the MS11 recD mutant with a wild-type recD gene copy restored the frequency of pilin phenotypic variation to approximately wild-type levels. The nucleotide changes at pilE in the recD mutants were confined to the variable regions of the gene and were similar to changes previously attributed to gene conversion.

Amino Acid Sequence↗

PubMed1999

Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.

It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.

Aerobiosis↗

PubMed2005

Coupling of two motor proteins: a new motor can move faster.

We study the effect of a coupling between two motor domains in highly processive motor protein complexes. A simple stochastic discrete model, in which the two parts of the protein molecule interact through some energy potential, is presented. The exact analytical solutions for the dynamic properties of the combined motor species, such as the velocity and dispersion, are derived in terms of the properties of free individual motor domains and the interaction potential. It is shown that the coupling between the motor domains can create a more efficient motor protein that can move faster than individual particles. The results are applied to analyze the motion of RecBCD helicase molecules.

DNA↗

PubMed1992

Escherichia coli DNA helicases: mechanisms of DNA unwinding.

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.

Binding Sites↗

PubMed1995

Collapse and repair of replication forks in Escherichia coli.

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.

Chromosomes, Bacterial↗

PubMed2006

The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites.

Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species.

Bacterial Proteins↗

PubMed2001

What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function.

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.

Bacteriophage lambda↗

PubMed1999

Mechanisms of genome-wide hypermutation in stationary phase.

Stationary-phase mutation (a subset of which was previously called adaptive mutation) occurs in apparently nondividing, stationary-phase cells exposed to a nonlethal genetic selection. In one experimental system, stationary-phase reversion of an Escherichia coli F'-borne lac frameshift mutation occurs by a novel molecular mechanism that requires homologous recombination functions of the RecBCD system. Chromosomal mutations at multiple loci are detected more frequently in Lac+ stationary-phase revertants than in cells that were also exposed to selection but did not become Lac+. Thus, mutating cells represent a subpopulation that experiences hypermutation throughout the genome. This paper summarizes current knowledge regarding stationary-phase mutation in the lac system. Hypotheses for the mechanism of chromosomal hypermutation are discussed, and data are presented that exclude one hypothetical mechanism in which chromosomal mutations result from Hfr formation.

DNA Repair↗

PubMed1994

Recombination in adaptive mutation.

The genetic requirements for adaptive mutation in Escherichia coli parallel those for homologous recombination in the RecBCD pathway. Recombination-deficient recA and recB null mutant strains are deficient in adaptive reversion. A hyper-recombinagenic recD strain is hypermutable, and its hypermutation depends on functional recA and recB genes. Genes of subsidiary recombination systems are not required. These results indicate that the molecular mechanism by which adaptive mutation occurs includes recombination. No such association is seen for spontaneous mutation in growing cells.

DNA Damage↗

PubMed2002

Phenotypes of a naturally defective recB allele in Neisseria meningitidis clinical isolates.

Neisseria meningitidis strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. The phenotype is consequent to the presence of a nonfunctional recB(ET-37) allele carrying multiple missense mutations. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recB(ET-37) allele. Congenic recB(ET-37) meningococci, in addition to being sensitive to UV, were defective both in repair of DNA lesions induced by UV treatment and, partially, in recombination-mediated transformation. Consistently, the wild-type, but not the recB(ET-37), allele was able to complement the Escherichia coli recB21 mutation to UV resistance and proficiency in recombination. recB(ET-37) meningococci did not exhibit higher frequencies of spontaneous mutation to rifampin resistance than recB-proficient strains. However, mutation rates were enhanced following UV treatment, a phenomenon not observed in the recB-proficient counterpart. Interestingly, the results of PCR-based assays demonstrated that the presence of the recB(ET-37) allele considerably increased the frequency of recombination at the pilin loci. The main conclusion that can be drawn is that the presence of the defective recB(ET-37) allele in N. meningitidis isolates causes an increase in genetic diversity, due to an ineffective RecBCD-dependent DNA repair and recombination pathway, and an increase in pilin antigenic variation.

Alleles↗

PubMed2006

Analysis of involvement of the RecF pathway in p44 recombination in Anaplasma phagocytophilum and in Escherichia coli by using a plasmid carrying the p44 expression and p44 donor loci.

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single p44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the A. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved in p44 gene conversion. The recombination intermediate structure between a donor p44 and the p44 expression locus of A. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor p44 locus connected to the 3' conserved region of the recipient p44 in the p44 expression locus in direct orientation. To determine whether the p44 recombination intermediate structure can be generated in a RecF-active Escherichia coli strain, we constructed a double-origin plasmid carrying the p44 expression locus and a donor p44 locus and introduced the plasmid into various E. coli strains. The recombination intermediate was recovered in an E. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the p44 gene conversion in A. phagocytophilum occurs through the RecF pathway.

Anaplasma phagocytophilum↗