Phosphorylation events surrounding the DNA damage response in "Saccharomyces cerevisiae"

Protein phosphorylation mediated by checkpoint kinases is crucial for the cellular response to DNA damage. The sensor kinases Mec1 and Tel1 initiate the checkpoint signaling cascade by directly activating the checkpoint effector kinase Rad53. This checkpoint pathway, however, is responsive to normal endogenous replication as well. As a result an S-phase specific threshold for Rad53 activation exists, which allows cells to tolerate endogenous damage-like structures. Here we show that Rad53 itself is phosphorylated in a cell-cycle dependent manner independent of DNA damage signaling (Chapter 2). We propose that this is part of the cell-cycle regulated sensitivity of Rad53 to activation. This phosphorylation occurs in G2/M, persists until S phase onset and depends on both the polo-like kinase, Cdc5, and the cyclin-dependent kinase, Cdc28. These cell-cycle dependent phosphorylation events are located in the C-terminal part of Rad53. Serines 774 and 789 were shown to be phosphorylated by mass spectrometry. Mutation of these sites eliminated the cell-cycle dependent phosphorylation of Rad53 and partially impaired the activation of Rad53 in response to minor amounts of DNA damage in G2/M. This led to more rapid checkpoint adaptation in response to irreparable DNA damage. Thus, cell-cycle dependent phosphorylation in the C-terminal part of Rad53 enhances Rad53 activation in response to DNA double strand breaks (DSBs). Mec1 and Tel1 initiate a response to DNA damage independently of Rad53. The phosphorylation of histone H2A at serine 129 (γH2A) at DSBs by Mec1 and Tel1 has an important role in mediating DNA repair. This study shows that the occurrence of γH2A is not limited to DSBs, but also occurs at stalled replication forks (Chapter 3). Using chromatin immunoprecipitation high γH2A levels were monitored at hydroxyurea-stalled replication forks and depended nearly exclusively upon Mec1 kinase activity. Furthermore our study showed that γH2A not only occurs at damaged chromatin but in regions of normally replicating chromatin and near telomeres (Chapter 4). High levels of γH2A could be monitored both in the rDNA of normally growing yeast cells and at telomeres. Here γH2A depended mainly on Tel1 and γH2A levels increased during S phase and during the elongation of critically short telomeres. We also provide evidence that γH2A contributes to telomeric anchoring in S phase yeast cells in addition to the yKu and Sir4 anchoring pathways

SalmoNet, an integrated network of ten Salmonella enterica strains reveals common and distinct pathways to host adaptation

Salmonella enterica is a prominent bacterial pathogen with implications on human and animal health. Salmonella serovars could be classified as gastro-intestinal or extra-intestinal. Genome-wide comparisons revealed that extra-intestinal strains are closer relatives of gastro-intestinal strains than to each other indicating a parallel evolution of this trait. Given the complexity of the differences, a systems-level comparison could reveal key mechanisms enabling extra-intestinal serovars to cause systemic infections. Accordingly, in this work, we introduce a unique resource, SalmoNet, which combines manual curation, high-throughput data and computational predictions to provide an integrated network for Salmonella at the metabolic, transcriptional regulatory and protein-protein interaction levels. SalmoNet provides the networks separately for five gastro-intestinal and five extra-intestinal strains. As a multi-layered, multi-strain database containing experimental data, SalmoNet is the first dedicated network resource for Salmonella. It comprehensively contains interactions between proteins encoded in Salmonella pathogenicity islands, as well as regulatory mechanisms of metabolic processes with the option to zoom-in and analyze the interactions at specific loci in more detail. Application of SalmoNet is not limited to strain comparisons as it also provides a Salmonella resource for biochemical network modeling, host-pathogen interaction studies, drug discovery, experimental validation of novel interactions, uncovering new pathological mechanisms from emergent properties and epidemiological studies. SalmoNet is available at http://salmonet.org

Hydropower and fish – Report and messages from workshop on research and innovation in the context of the European policy framework

Hydropower is the world's largest renewable electricity source and will have an important role in the future energy system with increased requirements to integrate environmental and socioeconomic aspects of sustainability. One important field of interaction is between hydropower and fish. The aim of optimizing hydropower production as well as fish production via Research and Innovation in the context of the European policy framework was the topic of the workshop “Hydropower and Fish – Research and Innovation in the context of the European Policy Framework” organized in May 2017 in Brussels. This paper reports the main messages from the workshop sessions including future research needs, collaboration strategies and knowledge exchange. In particular, the workshop emphasized the need for standardized monitoring and mitigation approaches and of following a balanced approach in addressing challenges between renewable energy production and river and fish ecology. Future research in the area is needed. As perspective and primer for future discussions, the interrelations of hydropower and fish to the different spheres of the total environment are presented and discussed.acceptedVersio

Posttranslational modifications of repair factors and histones in the cellular response to stalled replication forks.

DNA damage during replication requires an integration of checkpoint response with replication itself and distinct repair pathways, such as replication pausing, recombination and translesion synthesis. Here we focus on recent advances in our understanding of how protein posttranslational modifications contribute to the maintenance of fork integrity. In particular, we examine the role of histone modifications and chromatin remodeling complexes in this process

Fundamental Aspects of Parahydrogen Enhanced Low-Field Nuclear Magnetic Resonance

We report new phenomena in low-field 1H nuclear magnetic resonance (NMR) spectroscopy using parahydrogen induced polarization (PHIP), enabling determination of chemical shift differences, δν, and the scalar coupling constant J. NMR experiments performed with thermal polarization in millitesla magnetic fields do not allow the determination of scalar coupling constants for homonuclear coupled spins in the inverse weak coupling regime (δν<J). We show here that low-field PHIP experiments in the inverse weak coupling regime enable the precise determination of δν and J. Furthermore we experimentally prove that observed splittings are related to δν in a nonlinear way. Naturally abundant 13C and 29Si isotopes lead to heteronuclear J-coupled 1H-multiplet lines with amplitudes significantly enhanced compared to the amplitudes for thermally prepolarized spins. PHIP-enhanced NMR in the millitesla regime allows us to measure characteristic NMR parameters in a single scan using samples containing rare spins in natural abundance

Cohesin and the nucleolus constrain the mobility of spontaneous repair foci

The regulation of chromatin mobility in response to DNA damage is important for homologous recombination in yeast. Anchorage reduces rates of recombination, whereas increased chromatin mobility correlates with more efficient homology search. Here we tracked the mobility and localization of spontaneous S‐phase lesions bound by Rad52, and find that these foci have reduced movement, unlike enzymatically induced double‐strand breaks. Moreover, spontaneous repair foci are positioned in the nuclear core, abutting the nucleolus. We show that cohesin and nucleolar integrity constrain the mobility of these foci, consistent with the notion that spontaneous, S‐phase damage is preferentially repaired from the sister chromatid