Yoshino Kubota

Genome sequence of Vibrio parahaemolyticus : a pathogenic mechanism distinct from that of V. cholerae

BACKGROUND Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. METHODS The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. FINDINGS The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. INTERPRETATION The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157: H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

RAD6-RAD18-RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light

In nature, organisms are exposed to chronic low-dose ultraviolet light (CLUV) as opposed to the acute high doses common to laboratory experiments. Analysis of the cellular response to acute high-dose exposure has delineated the importance of direct DNA repair by the nucleotide excision repair pathway and for checkpoint-induced cell cycle arrest in promoting cell survival. Here we examine the response of yeast cells to CLUV and identify a key role for the RAD6–RAD18–RAD5 error-free postreplication repair (RAD6 error-free PRR) pathway in promoting cell growth and survival. We show that loss of the RAD6 error-free PRR pathway results in DNA-damage-checkpoint-induced G2 arrest in CLUV-exposed cells, whereas wild-type and nucleotide-excision-repair-deficient cells are largely unaffected. Cell cycle arrest in the absence of the RAD6 error-free PRR pathway was not caused by a repair defect or by the accumulation of ultraviolet-induced photoproducts. Notably, we observed increased replication protein A (RPA)– and Rad52–yellow fluorescent protein foci in the CLUV-exposed rad18Δ cells and demonstrated that Rad52-mediated homologous recombination is required for the viability of the rad18Δ cells after release from CLUV-induced G2 arrest. These and other data presented suggest that, in response to environmental levels of ultraviolet exposure, the RAD6 error-free PRR pathway promotes replication of damaged templates without the generation of extensive single-stranded DNA regions. Thus, the error-free PRR pathway is specifically important during chronic low-dose ultraviolet exposure to prevent counter-productive DNA checkpoint activation and allow cells to proliferate normally.

Functional and Physical Interaction of Yeast Mgs1 with PCNA: Impact on RAD6-Dependent DNA Damage Tolerance

ABSTRACT Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

Degradation of Escherichia coli RecN Aggregates by ClpXP Protease and Its Implications for DNA Damage Tolerance

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in ΔclpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance.

Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication forks in Escherichia coli

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA‐dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV‐induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature‐dependent growth defect of dnaE486 (Pol III α‐catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of ΔmgsA ΔrecA cells or the lethality of polA12 ΔrecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.

Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement

The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.

RecA protein recruits structural maintenance of chromosomes (SMC)-like RecN protein to DNA double-strand breaks.

Background: RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand breaks (DSBs) repair. Results: We identified a RecA mutant that is deficient in interacting with RecN. Conclusion: A functional interaction between RecN and RecA is required for assembly of RecN at the sites of DSBs. Significance: RecN is critical for protecting the structural integrity of chromosomes during DSBs repair. Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recAQ300R, is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recAQ300R fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecNK35A binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

ABSTRACT Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G2 arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G2 arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G2 arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

Epigenetically induced paucity of histone H2A.Z stabilizes fission-yeast ectopic centromeres

In most eukaryotes, centromeres are epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A–loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at noncentromeric regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe that show reduced association with the CENP-A chaperone Scm3, owing to persistence of the histone H2A variant H2A.Z. After the acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, thus leading to subsequent stabilization of the neocentromeres. These findings provide new insights into histone variant–mediated epigenetic control of neocentromere establishment.