Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR)

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation

Neutral genomic microevolution of a recently emerged pathogen, salmonella enterica serovar agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks

X-irradiation of cells on glass slides has a dose doubling impact

Immunofluorescence detection of γH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks (DSBs) induced by ionising radiation. We observed that X-irradiation of mammalian cells exposed on glass slides induced twofold higher foci numbers compared to irradiation with γ-rays. Here, we show that the excess γH2AX foci after X-irradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and 137Cs γ-rays induce ∼20 γH2AX foci per Gy in cells growing on thin (∼2 μm) plastic foils immersed in water. The same yield is obtained following γ-irradiation of cells growing on glass slides. However, 120 kV X-rays produce ∼40 γH2AX foci per Gy in cells growing on glass, twofold greater than obtained using cells irradiated on plastic surfaces. The same increase in γH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess γH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X- or γ-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner

Characteristics of vancomycin-resistant enterococci (VRE) isolated from medical fields in Japan

学位記番号：医博甲169

Chromosome breakage after G2 checkpoint release

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain

Chromosomal integrity after UV irradiation requires FANCD2-mediated repair of double strand breaks

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.Fil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vallerga, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Radl, Daniela Betiana. Autoridad Regulatoria Nuclear; ArgentinaFil: Paviolo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Di Giorgio, Marina. Autoridad Regulatoria Nuclear; ArgentinaFil: Soria, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Characterization of an emergent clone of enteroinvasive Escherichia coli circulating in Europe

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness indistinguishable from that caused by Shigella, mainly in developing countries. Recently an upsurge of cases of EIEC infections has been observed in Europe, with two large outbreaks occurring in Italy and in the United Kingdom. We have characterized phenotypically and genotypically the strains responsible for these epidemics together with an additional isolate from a sporadic case isolated in Spain. The three isolates belonged to the same rare serotype O96:H19 and were of sequence type ST-99, never reported before in EIEC or Shigella. The EIEC strains investigated possessed all the virulence genes harboured on the large plasmid conferring the invasive phenotype to EIEC and Shigella while showing only some of the known chromosomal virulence genes and none of the described pathoadaptative mutations. At the same time, they displayed motility abilities and biochemical requirements resembling more closely those of the non-pathogenic E. coli rather than the EIEC and Shigella strains used as reference. Our observations suggested that the O96:H19 strains belong to an emerging EIEC clone, which could be the result of a recent event of acquisition of the invasion plasmid by commensal E. coli

Insertion/deletion-related polymorphisms in the human T cell receptor beta gene complex.

Insertion/deletion related polymorphisms (IDRP) involving stretches of 15-30 kb within the human TCR-beta gene complex were revealed by pulse-field gel electrophoresis. Two independent IDRP systems were detected by analysis of Sfi I- and Sal I-digested human DNA samples using probes for TCR C and V region gene segments. The allelic nature of these systems was verified in family studies, and mapping data allowed localization of one area of insertion/deletion among the V gene segments and the other near the C region genes. All but one of 50 individuals tested could be typed for the two allelic systems, and gene frequencies for the two allelic forms were 0.37/0.61 and 0.46/0.54, indicating that these polymorphisms are widespread