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München, Germany

Byrne R.T.,Ludwig Maximilians University of Munich | Schuller J.M.,Max Planck Institute of Biochemistry | Unverdorben P.,Max Planck Institute of Biochemistry | Forster F.,Max Planck Institute of Biochemistry | And 3 more authors.
FEBS Letters | Year: 2014

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Herbach N.,Institute of Veterinary Pathology | Bergmayr M.,Institute of Veterinary Pathology | Goke B.,Ludwig Maximilians University of Munich | Wolf E.,Gene Center | Wanke R.,Institute of Veterinary Pathology
PLoS ONE | Year: 2011

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR dn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR dn transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR dn transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR dn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR dn transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR dn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis. © 2011 Herbach et al.

During the process of cell division, chromosomes must be distributed equally between the two emerging daughter cells. One copy of each chromosome is created and remains glued to the original until threads, called microtubules, pull the chromosome pairs apart and distribute them to the two new cells. Researchers from the Max Planck Institute of Molecular Physiology in Dortmund and the Gene Center of the University of Munich (LMU) have now analyzed and modelled the structure of the point of attachment of the chromosomes to the threads, called the kinetochore. In the process, they have discovered how the different kinetochore proteins work together to bind the chromosomes securely to the microtubules. Cell division is vital for the continuation of life. If something goes wrong in, say, the distribution of chromosomes, abnormalities or serious diseases such as cancer may result. This is why scientists are keen to get to grips with the details of this fundamentally important process. "What I cannot create, I do not understand." This quote from physicist Richard Feynman is a guiding principle for Andrea Musacchio, Director at the Max Planck Institute and head of the study. He uses it to make a virtue of necessity, as the interplay of the individual components of the kinetochore during cell division in real cells does easily not lend itself to examination. "Only by taking the system apart and simplifying it do we have a chance of understanding how the kinetochore works - so we modelled it in the lab", explains Musacchio. The nuclear complex of a kinetochore contains about 30 proteins, making synthesis in the laboratory very difficult – like a construction kit with Lego blocks that all have different shapes and functions. But it gets worse: "Unlike Lego, these protein building blocks in the kinetochore interact with each other – but we didn't know how. Besides, you can't just walk into a store and pick the blocks you need off the shelf", reports John Weir, lead author of the study. The scientists began to synthesize the various building blocks of the kinetochore individually and eventually managed to construct an artificial kinetochore with 21 parts, which can connect chromosomes to microtubules. The whole system is far more complex in the natural world, as even more proteins have roles to play in real cells. Using the model, the scientists were able to examine the details of kinetochore function and structure. They found that the seven subunits of the protein complex CHIKMLN interact with each other. "This increases their binding strength with certain partners", explains Alex Faesen, who participated in the study. CHIKMLN is connected to the chromosome by a protein and binds to a ten-unit assembly (the KMN network), which is responsible for microtubule contact. "The whole structure consists of 21 subunits that form a bridge between the chromosome and the microtubules", says Kerstin Klare, another member of the research team, in summary. By modelling the kinetochore, the team has laid the foundation for further studies into the complex architecture and functionality of this vital structure. Their goal: to create an artificial model of cell division as a whole. "Because only when we can recreate these processes and cell components will we be in a position to truly understand how they work", says Musacchio. Explore further: Two unsuspected proteins may hold the key to creating artificial chromosomes More information: John R. Weir et al. Insights from biochemical reconstitution into the architecture of human kinetochores, Nature (2016). DOI: 10.1038/nature19333

Viruses essentially consist of a protein coat that encapsulates the viral genetic material – usually one or more molecules of RNA. During an infection, the only viral component that actually gets into the host cell is the RNA (the coat protein is stripped off). But vertebrates like ourselves have an innate immune system that can detect viral intruders and initiate appropriate countermeasures. An immune sensor called RIG-I (the protein encoded by retinoic acid-inducible gene I) recognizes the foreign RNA and activates an immune reaction against the virus. But since host cells are themselves full of RNAs that are essential for their survival the identification of exotic viral RNA is no easy task. "We had already shown that the process is based on the recognition of two specific structural features of viral RNAs, as Professor Karl-Peter Hopfner (LMU Gene Center) explains. "But this finding alone did not fully explain exactly how RIG-I discriminates viral from cellular RNA," he adds. Now Hopfner and his research team have shown that RIG-I must be actively removed from cellular RNA in order to prevent it from triggering false alarms. The new findings appear in the online journal eLife. RIG-I distinguishes viral RNAs from cellular RNAs on the basis of their structural peculiarities. Once viral RNA is detected in the cell, a signal cascade is triggered which ultimately leads to the synthesis of antiviral proteins. "Interestingly, RIG-I is an protein that can hydrolyze ATP, the currency unit of metabolic energy in the cell, thus releasing the chemical energy stored in the compound," says Hopfner. "We have previously shown that RIG-I makes use of this energy to propel itself along the double-stranded RNA like a train on a railway track. But how this activity is linked to the recognition of viral RNA remained a mystery." The breakthrough came with the recent discovery of a mutation in RIG-I, which deprives the protein of its ability to hydrolyze bound ATP. This mutation turns out to be the underlying cause of Singleton-Merten Syndrome, a rare form of autoimmune disease characterized by tooth loss, bone demineralization and calcification of the vasculature. Hopfner's team has now shown that the mutant form of RIG-I is also unable to tell the difference between friend and foe, such that the RIG-I-dependent signal relay is activated by cellular, as well as viral, RNA molecules. "So we took a closer look at the set of cellular RNAs that interact with the mutated form of RIG-I," Hopfner continues. And to everyone's surprise, we found that the mutant RIG-I localizes to the ribosomes, which serve as the cell's protein factories. Ribosomes are made up of proteins and RNAs, and the mutant RIG-I binds predominantly to a double-stranded stretch of RNA that extends from the ribosomal surface. With the help of Hopfner's colleague Prof. Roland Beckmann and his team, RIG-I could be located on a Ribosomal RNA element by using cryo electron microscopy. "We concluded from this finding that RIG-I must hydrolyze ATP in order to detach itself from cellular RNA. If this mechanism is defective, RIG-I binds to double-stranded regions in cellular RNAs – in particular to those associated with the ribosomes – and the continuing activation of the signal cascade precipitates an autoimmune reaction. This discovery could contribute to the development of new therapeutic possibilities in the future," Hopfner says. Explore further: Team learns how cellular protein detects viruses and sparks immune response More information: Charlotte Lässig et al. ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA, eLife (2015). DOI: 10.7554/eLife.10859

Scientists at the Helmholtz Zentrum München, working with colleagues from the Ludwig-Maximilians-Universität München, have developed a method for the thorough analysis of protein modifications. They mapped the phosphorylation sites of the RNA polymerase II enzyme, which is responsible for expressing our genes. The results have now been published in the Molecular Cell scientific journal. The contents in our genetic information are actually silent (meaning inactive) and first have to be made to "speak". Like the read head in a tape recorder, RNA polymerase II, Pol II for short, runs over the DNA (tape) and transcribes the genetic and epigenetic information into RNA. In order to keep the enzyme from working randomly, however, it is dynamically modified at many different points in order to control its activity depending on the situation. "Phosphorylation makes it possible to influence the activity of the enzyme at 240 different sites," explains Prof. Dirk Eick, the study's last author and head of the Research Unit Molecular Epigenetics at Helmholtz Zentrum München. Together with colleagues from the Biomedical Center and Gene Center of the Ludwig-Maximilians-Universität München and the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, he and his team have developed a method for simultaneously examining all 240 sites in Pol II. "The trick is a combination of genetic and mass spectrometric methods," reveals first author Dr. Roland Schüller. "By producing genetically modified variants of the regions in question, we can examine each individual phosphorylation site with a mass spectrometer." This allows the researchers to determine exactly how and precisely where certain enzymes that influence phosphorylation act. The scientists also successfully compared the Pol II modification patterns in humans and in yeast. "The regulation of the transcription of genes by Pol II is an elementary process in life and gene regulation deviations are the basis for many human disorders," study leader Eick explains the work's background. "Research into the phosphorylation pattern at certain times during the transcription cycle is therefore necessary in order to be able to gain an understanding of the underlying mechanisms of gene regulation at the transcription level sometime in the future." Explore further: Lab identifies new role for factor critical to transcription More information: Schüller, R. et al. (2015). Heptad-specific Phosphorylation of RNA Polymerase II CTD, Molecular Cell, DOI: 10.1016/j.molcel.2015.12.003

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