RuvAB Acts at Arrested Replication Forks

AbstractReplication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage

Extending the cereus group genomics to putative food-borne pathogens of different toxicity

The cereus group represents sporulating soil bacteriacontaining pathogenic strains which may cause diarrheic or emetic foodpoisoning outbreaks. Multiple locus sequence typing revealed a presencein natural samples of these bacteria of about thirty clonal complexes.Application of genomic methods to this group was however biased due tothe major interest for representatives closely related to B. anthracis.Albeit the most important food-borne pathogens were not yet defined,existing dataindicate that they are scattered all over the phylogenetictree. The preliminary analysis of the sequences of three genomesdiscussed in this paper narrows down the gaps in our knowledge of thecereus group. The strain NVH391-98 is a rare but particularly severefood-borne pathogen. Sequencing revealed that the strain must be arepresentative of a novel bacterial species, for which the name Bacilluscytotoxis is proposed. This strain has a reduced genome size compared toother cereus group strains. Genome analysis revealed absence of sigma Bfactor and the presence of genes encoding diarrheic Nhe toxin, notdetected earlier. The strain B. cereus F837/76 represents a clonalcomplex close to that of B. anthracis. Including F837/76, three such B.cereus strains had been sequenced. Alignment of genomes suggests that B.anthracis is their common ancestor. Since such strains often emerge fromclinical cases, they merit a special attention. The third strain, KBAB4,is a typical psychrotrophe characteristic to unbiased soil communities.Phylogenic studies show that in nature it is the most active group interms of gene exchange. Genomic sequence revealed high presence ofextra-chromosomal genetic material (about 530 kb) that may account forthis phenomenon. Genes coding Nhe-like toxin were found on a big plasmidin this strain. This may indicate a potential mechanism of toxicityspread from the psychrotrophic strain community. The results of thisgenomic work and ecological compartments of different strains incite toconsider a necessity of creating prophylactic vaccines against bacteriaclosely related to NVH391-98 and F837/76. Presumably developing of suchvaccines can be based on the properties of non-pathogenic strains such asKBAB4 or ATCC14579 reported here or earlier. By comparing the proteincoding genes of strains being sequenced in this project to others weestimate the shared proteome in the cereus group to be 3,000?b200 genesand the total proteome to be 20-25,000 genes

Replication slippage involves DNA polymerase pausing and dissociation

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process

Replication fork collapse at replication terminator sequences

Replication fork arrest is a source of genome re arrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the first blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein

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Impairment of lagging strand synthesis triggers the formation of a RuvABC substrate at replication forks

The holD gene codes for the ψ subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the γ complex clamp loader. A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions. In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA. They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant. The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature. DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD. We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype

A Two-Protein Strategy for the Functional Loading of a Cellular Replicative DNA Helicase

International audienceThe delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB

Extending the cereus group genomics to putative food-borne pathogens of different toxicity

The cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about thirty clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to B. anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain must be a representative of a novel bacterial species, for which the name Bacillus cytotoxis is proposed. This strain has a reduced genome size compared to other cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical psychrotrophe characteristic to unbiased soil communities. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530 kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrotrophic strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome in the cereus group to be 3,000?b200 genes and the total proteome to be 20-25,000 genes