Investigations on phosphospecific interactions in the DNA damage response

Double strand breaks (DSBs) are one of the most dangerous forms of DNA damage. A globular cellular response is activated upon induction of DSBs. Mediator of DNA damage checkpoint 1 (MDC1) is one of those proteins involved in the DNA damage response (DDR) that contains an N-terminal forkhead associated (FHA) domain and a tandem BRCA1 C-terminal (BRCT) domain. MDC1 is recruited to sites of DNA damage upon phosphorylation of variant histone 2A (H2AX) by the kinase ataxia telangiectasia mutated (ATM). In order to identify the amino acids that are targeted by ATM, we took a candidate approach and mutated a Threonine residue at the N-terminus of MDC1 to Alanin. We then performed an ATM kinase assay using radioactive ATP in order to check whether the T4A mutant fragment was still phosphorylated by ATM. Results of this study clearly revealed that ATM phosphorylates Thr 4 in MDC1 in vitro. We suggest that Thr4 is the major ATM phosphorylation site within the N-terminus of MDC1. In a second part of this work, we sought to develop a proteomics approach to identify novel components of the DDR. We set up a tandem affinity purification (TAP) system using the BRCA1-BRCT domains fused to enhanced yellow fluorescent protein (EYFP) and triple-tagged at the C-terminus by a Streptavidin-binding peptide (Strep-tag II) a Flag-tag and an S-tag. As the Strep-tag II did not efficiently bind to Streptavidin, the performance of a TAP was not successful. DNS Doppelstrangbrüche (DSB) gehören zu den gefährlichsten DNS Schäden und führen zu einer weitgreifenden zellulären Antwort. Mediator of DNA damage checkpoint 1 (MDC1) ist ein Protein dieser DNA damage response (DDR) und besitzt eine N-terminale FHA als auch eine C-terminale tandem BRCT Domäne. MDC1 wird als Folge der Phosphorylierung von variant Histone 2A (H2AX) durch die Kinase Ataxia Telangiectasia mutated (ATM) zum Ort eines DNS Schadens rekrutiert. Um die von ATM phosphorylierte Aminosäure zu identifizieren, wurde Thr4 als potentieller Kandidat zu Ala ummutiert. Mit radioaktivem ATP wurde ein ATM- Kinase Assay durchgeführt um herauszufinden ob das T4A mutierte Fragment immer noch phosphoryliert wird. Die Ergebnisse zeigten, dass T4A von ATM in vitro phosphoryliert wird. Folglich ist T4 die einzige ATM Phosphorylierungsstelle am N- Terminus von MDC1. In einem zweiten Teil entschieden wir uns ein Proteomicsverfahren zu etablieren um neue Komponenten der DDR zu identifizieren. Wir entwickelten eine Tandem Affinity Purification (TAP), bestehend aus der BRCA1 BRCT Domäne verbunden mit dem enhanced yellow fluorescent protein (EYFP) und einem Trippel-tag am C-Terminus gebildet von einem Streptavidin-bindenden Peptid (Strep-tag II), einem Flag-tag und einem S-tag. Da der Strep-tag II nicht effizient von Streptavidin gebunden wurde, blieb die Durchführung der TAP erfolglos

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damag

Salmonella enterica serotype Virchow associated with human infections in Switzerland: 2004-2009

BACKGROUND: Salmonellosis is one of the most important foodborne diseases and a major threat to public health. Salmonella serotype Virchow ranks among the top five serovars in Europe. METHOD: A total of 153 strains isolated from different patients from 2004 through 2009 in Switzerland were further characterized by (i) assessing phenotypic antibiotic resistance profiles using the disk diffusion method and (ii) by genotyping using pulsed-field gel electrophoresis (PFGE) after macrorestriction with XbaI in order to evaluate strain relationship. RESULTS: The relative frequency of S. Virchow among other Salmonella serovars varied between 4th to 8th rank. The annual incidence ranged from 0.45/100'000 in 2004 to 0.40/100'000 in 2009. A total of 48 strains (32%) were resistant to one to 3 antimicrobials, 54 strains (36%) displayed resistance patterns to more than three antibiotics. No trend was identifiable over the years 2004 to 2009. We found a high prevalence (62%) of nalidixic acid resistant strains, suggesting an equally high rate of decreased fluoroqionolone susceptibility, whereas intermediate resistance to ciprofloxacin was negligible. Two strains were extended spectrum β-lactamase (ESBL) producers. Analysis of PFGE patterns uncovered a predominant cluster (similarity coefficient above 80%) consisting of 104 of the 153 strains. CONCLUSION: The worldwide increase of antibiotic resistances in Salmonella is an emerging public health problem. For Switzerland, no clear trend is identifiable over the years 2004 to 2009 for S. Virchow. Antimicrobial susceptibility and resistance profiles varied considerably within this period. Nevertheless, the situation in Switzerland coincided with findings in other European countries. Genotyping results of this strain collection revealed no evidence for an undetected outbreak within this time period

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage

<it>Salmonella enterica </it>serotype Virchow associated with human infections in Switzerland: 2004-2009

Abstract Background Salmonellosis is one of the most important foodborne diseases and a major threat to public health. Salmonella serotype Virchow ranks among the top five serovars in Europe. Method A total of 153 strains isolated from different patients from 2004 through 2009 in Switzerland were further characterized by (i) assessing phenotypic antibiotic resistance profiles using the disk diffusion method and (ii) by genotyping using pulsed-field gel electrophoresis (PFGE) after macrorestriction with XbaI in order to evaluate strain relationship. Results The relative frequency of S. Virchow among other Salmonella serovars varied between 4th to 8th rank. The annual incidence ranged from 0.45/100'000 in 2004 to 0.40/100'000 in 2009. A total of 48 strains (32%) were resistant to one to 3 antimicrobials, 54 strains (36%) displayed resistance patterns to more than three antibiotics. No trend was identifiable over the years 2004 to 2009. We found a high prevalence (62%) of nalidixic acid resistant strains, suggesting an equally high rate of decreased fluoroqionolone susceptibility, whereas intermediate resistance to ciprofloxacin was negligible. Two strains were extended spectrum β-lactamase (ESBL) producers. Analysis of PFGE patterns uncovered a predominant cluster (similarity coefficient above 80%) consisting of 104 of the 153 strains. Conclusion The worldwide increase of antibiotic resistances in Salmonella is an emerging public health problem. For Switzerland, no clear trend is identifiable over the years 2004 to 2009 for S. Virchow. Antimicrobial susceptibility and resistance profiles varied considerably within this period. Nevertheless, the situation in Switzerland coincided with findings in other European countries. Genotyping results of this strain collection revealed no evidence for an undetected outbreak within this time period.</p

The low affinity neurotrophin receptor CD271 regulates phenotype switching in melanoma

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity. According to the "phenotype switching" model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state. Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma. CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness. Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression. Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival