Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. DNA replication is tightly monitored to ensure that the genome is replicated precisely once per cell cycle and that DNA replication is complete before mitosis begins. Conditions for DNA synthesis are rarely ideal, and a number of obstacles must often be dealt with, such as a damaged DNA template and shortage of deoxynucleoside triphosphates (dNTPs), to allow replication fork progression. Stalling replication forks pose serious threats to genome integrity because they can collapse through disassembly of the replication complex and break (6,11,24). Such damaged forks may subsequently undergo incorrect repair, leading to genetic changes like chromosomal rearrangements (6,24). Recent data have also revealed that activated oncogenes can induce DNA replication stress, defined here as replication-associated DNA damage (2, 3, 10). Oncogene-induced replication stress can lead to additional tumor-promoting genetic changes, but it may also serve as a tumor barrier by activation of cell cycle arrest, apoptosis, and/or senescence during early tumor development (32).WEE1 and CHK1 kinases have major roles in suppressing DNA replication stress (4,23,27,42), and attenuation of their function can contribute to carcinogenesis and cause cell death (40). The massive amount of DNA breakage is likely mediated by DNA endonuclease activity, and recent studies suggest that this is mediated by the endonuclease MUS81 (12,14,15). Notably, the mechanisms by which oncogenes or inhibition of checkpoint kinases can lead to endonuclease-mediated DNA breakage are poorly understood. It is also not fully understood if these breaks also play a role in inducing fork stalling or if they are temporally delayed events secondary to the fork stalling.As both oncogenes and checkpoint kinases are regulators of cyclin-dependent kinase (CDK) activity, we previously proposed that most of the DNA replication str...

SummaryThe phyllosphere mycobiome in cereals is an important determinant of crop health. However, an understanding of the factors shaping this community is lacking.Fungal diversity in leaves from a range of cultivars of winter wheat (Triticum aestivum), winter and spring barley (Hordeum vulgare) and a smaller number of samples from oat (Avena sativa), rye (Secale cereale) and triticale (Triticum 9 Secale) was studied using next-generation sequencing. The effects of host genotype, fungicide treatment and location on fungal communities were explored.In total, 635 251 fungal internal transcribed spacer (ITS) reads were obtained from 210 leaf samples. Visual disease assessments and relative read abundance of Zymoseptoria tritici and Ramularia collo-cygni were strongly positively related. Crop genotype at the species level explained 43% of the variance in the total dataset, followed by fungicide treatment (13%) and location (4%). Indicator species, including plant pathogens, responding to factors such as crop species, location and treatment were identified.Host genotype at both the species and cultivar level is important in shaping phyllosphere fungal communities, whereas fungicide treatment and location have minor effects. We found many host-specific fungal pathogens, but also a large diversity of fungi that were relatively insensitive to host genetic background, indicating that host-specific pathogens live in a 'sea' of nonspecific fungi.

When environmental conditions are unsuitable to support nematode reproduction, Caenorhabditis elegans arrests development before the onset of sexual maturity and specialised 'dauer' larvae, adapted for dispersal, and extended diapause are formed. Dauer larvae do not feed and their metabolism is dependent on internal food reserves. Adult worms which express defects in the insulin/insulin-like growth factor receptor DAF-2 also display enhanced longevity. Whole genome mRNA expression profiling has demonstrated that C. elegans dauer larvae and daf-2 adults have similar transcription profiles for a cohort of longevity genes. Important components of this enhanced longevity system are the a-crystallin family of small heat shock proteins, anti-ROS defence systems, increased activity of cellular detoxification processes and possibly also increased chromatin stability and decreased protein turnover. Anaerobic fermentation pathways are upregulated in dauer larvae, while long-lived daf-2 adults appear to have normal oxidative metabolism. Anabolic pathways are down regulated in dauer larvae (and possibly in daf-2 adults as well), and energy consumption appears to be diverted to enhanced cellular maintenance and detoxification processes in both systems. q

Aims: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. Methods and Results: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were puri®ed to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the puri®ed bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each puri®ed bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. Conclusions: All bacteriocins puri®ed in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining puri®ed bacteriocins have been isolated from environments other than malted barley. Signi®cance and Impact of the Study: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the ®rst time, the variety of previously identi®ed and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.

Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.