DNA double strand break repair in Escherichia coli perturbs cell division and chromosome dynamics

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum

SbcCD regulation and localization in Escherichia coli

The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. In Escherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of the sbcDC operon and the cellular localization of the SbcC and SbcD proteins. Transcription of the sbcDC operon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm

Repair on the Go:E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division

RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site

Antagonistic Roles of SEPALLATA3, FT and FLC Genes as Targets of the Polycomb Group Gene CURLY LEAF

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering

Symmetries and asymmetries associated with non-random segregation of sister DNA strands in Escherichia coli

AbstractThe successful inheritance of genetic information across generations is a complex process requiring replication of the genome and its faithful segregation into two daughter cells. At each replication cycle there is a risk that new DNA strands incorporate genetic changes caused by miscopying of parental information. By contrast the parental strands retain the original information. This raises the intriguing possibility that specific cell lineages might inherit “immortal” parental DNA strands via non-random segregation. If so, this requires an understanding of the mechanisms of non-random segregation. Here, we review several aspects of asymmetry in the very symmetrical cell, Escherichia coli, in the interest of exploring the potential basis for non-random segregation of leading- and lagging-strand replicated chromosome arms. These considerations lead us to propose a model for DNA replication that integrates chromosome segregation and genomic localisation with non-random strand segregation

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene ($\beta$-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.Transformation génétique du pin maritime (Pinus pinaster Ait.). En appliquant nos protocoles d'embryogenèse somatique développés pour Pinus pinaster, des tissus et plantes transgéniques ont été obtenus après bombardement avec des microparticules (biolistique) ou coculture de masses embryonnaires (ESM) avec Agrobacterium tumefaciens. Les expériences de transformation ont été conduites à l'aide du gène de sélection hpt (résistance à l'hygromycine B) et du gène rapporteur gus (activité $\beta$-glucuronidase). L'hygromycine a permis de sélectionner efficacement les cellules transformées par ces deux méthodes en 4 à 19 semaines. Le nombre moyen de lignées résistantes à l'hygromycine exprimant le gène gus obtenu par gramme d'ESM varie de 7,0 à 8,5 (biolistique) ou de 0 à 67,3 (Agrobacterium). Les embryons matures obtenus à partir de certaines de ces lignées ont pu être convertis en plantules élevées en serre. Seulement 12 mois sont nécessaires de la transformation des ESM jusqu'à l'acclimatation des plantes. La nature transgénique des ESM, embryons somatiques et plantes, a été confirmée à l'aide de tests histochimiques “ GUS " et de méthodes moléculaires

Effects of suppressor mutants upon flowering time.

<p>Flowering time was recorded as the number of rosette leaves at bolting, thus late flowering plants have more rosette leaves. Plants were grown in long days unless otherwise stated. Error bars show standard error of mean calculated from at least 10 plants. (<b>A</b>) The <i>clf-50 fpa-10</i> mutant shows a strong vernalization reponse. (<b>B</b>) The <i>clf-50 ft-12</i> mutant does not respond to vernalization treatment. (<b>C</b>) The <i>clf-50 sep3-7</i> mutant flowers at similar time to wild type, thus <i>SEP3</i>⁺ activity is needed for early flowering in the <i>clf</i> background. (<b>D</b>) The <i>flc-3</i> mutation enhances the early flowering of <i>clf-28</i> mutants, revealing that <i>FLC</i> activity delays flowering in the <i>clf</i> background. Plants grown in short days, where the effects of <i>clf</i> on flowering time are most obvious (<b>E</b>) <i>clf-28 ft-10</i> mutants flower later than <i>ft-10</i> mutants due to <i>FLC</i>⁺ activity.</p

Gene expression in suppressor mutants.

<p>(<b>A</b>) Western blot analysis of FPA protein levels. Three independent <i>clf-50 fpa-10</i> samples were processed. Note that no protein is detected in the null <i>fpa-7</i> control, whereas in extracts from a <i>35S::FPA-YPA</i> transgenic line a larger product corresponding to the <i>FPA-YFP</i> fusion protein is detected, confirming the specificity of the antibody for FPA. No FPA protein is detectable in <i>fpa-10</i> extracts, indicating that <i>fpa-10</i> is likely a null allele. (<b>B</b>) Real time PCR analysis of <i>FLC</i> expression. (<b>C</b>) Real time PCR analysis of <i>AG</i> expression, showing high <i>AG</i> expression in suppressor mutants. (<b>D</b>) Western blot analysis of AG protein expression. The AG antibody detects two proteins of about 29 kDa that are specific for AG, the smaller band possibly representing a truncated product or spurious translation initiation event (Riechmann et al., 1999). AG protein is strongly detected in wild type flowers but not in leaves. Weak expression is found in <i>clf-50</i> and <i>clf-50 fpa-10</i> leaves. (<b>E</b>) Real time PCR analysis of <i>SEP3</i> expression. (<b>F</b>) Histochemical staining of GUS reporter gene activity. <i>SEP3::GUS</i> is not expressed in wild type leaves but shows weak expression in vasculature of <i>clf-81</i> leaves (enlarged in inset). (<b>G</b>) Real time PCR analysis of <i>SEP2</i> expression. (<b>H</b>) Real time PCR analysis of <i>FT</i> expression (<b>I</b>) Real time PCR analysis of <i>SEP3</i> expression. Error bars in real time PCR experiments represent standard error of mean of three independent samples (biological replicates). Expression was normalised relative to the <i>EiF4A</i> gene, and is expressed relative to expression in wild type. In <b>B</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>H</b> whole seedlings less roots of 20 day old short day grown seedlings were used. In <b>I</b> rosette leaves of long day plants at 21 days were used.</p