Stephan Diekmann

Publications of Stephan Diekmann

• CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin.

  Authors: Silvia Dambacher, Wen Deng, Matthias Hahn, Dennis Sadic, Jonathan Fröhlich, Alexander Nuber, Christian Hoischen, Stephan Diekmann, Heinrich Leonhardt, Gunnar Schotta

  Nucleus (Austin, Tex.). 01/2012; 3(1).

  Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes,Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric proteins. The Mis18 complex, and, in particular, its member M18BP1 was shown to be essential for both incorporation and maintenance of CENP-A. Here we show that M18BP1 displays a cell cycle-regulated association with centromeric chromatin in mouse embryonic stem cells. M18BP1 is highly enriched at centromeric regions from late anaphase through to G1 phase. An interaction screen against 16 core centromeric proteins revealed a novel interaction of M18BP1 with CENP-C. We mapped the interaction domain in M18BP1 to a central region containing a conserved SANT domain and in CENP-C to the C-terminus. Knock-down of CENP-C leads to reduced M18BP1 association and lower CENP-A levels at centromeres, suggesting that CENP-C works as an important factor for centromeric M18BP1 recruitment and thus for maintaining centromeric CENP-A.

• Dynamics of CENP-N kinetochore binding during the cell cycle.

  Authors: Daniela Hellwig, Stephan Emmerth, Tobias Ulbricht, Volker Döring, Christian Hoischen, Ronny Martin, Catarina P Samora, Andrew D McAinsh, Christopher W Carroll, Aaron F Straight, Patrick Meraldi, Stephan Diekmann

  Journal of cell science. 11/2011; 124(Pt 22):3871-83.

  Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding ofAccurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

• Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state.

  Authors: Lisa Prendergast, Chelly van Vuuren, Agnieszka Kaczmarczyk, Volker Doering, Daniela Hellwig, Nadine Quinn, Christian Hoischen, Stephan Diekmann, Kevin F Sullivan

  PLoS biology. 06/2011; 9(6):e1001082.

  Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They areCentromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

• Segrosome assembly at the pliable parH centromere.

  Authors: Meiyi Wu, Massimiliano Zampini, Malte Bussiek, Christian Hoischen, Stephan Diekmann, Finbarr Hayes

  Nucleic acids research. 03/2011; 39(12):5082-97.

  The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere.The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere. Here we demonstrate that the distinctive parH site (∼100-bp) consists of an array of degenerate tetramer boxes interspersed by AT-rich spacers. Although numerous consecutive AT-steps are suggestive of inherent curvature, parH lacks an intrinsic bend. Sequential deletion of parH tetramers progressively reduced centromere function. Nevertheless, the variant subsites could be rearranged in different geometries that accommodated centromere activity effectively revealing that the site is highly elastic in vivo. ParG cooperatively coated parH: proper centromere binding necessitated the protein's N-terminal flexible tails which modulate the centromere binding affinity of ParG. Interaction of the ParG ribbon-helix-helix domain with major groove bases in the tetramer boxes likely provides direct readout of the centromere. In contrast, the AT-rich spacers may be implicated in indirect readout that mediates cooperativity between ParG dimers assembled on adjacent boxes. ParF alone does not bind parH but instead loads into the segrosome interactively with ParG, thereby subtly altering centromere conformation. Assembly of ParF into the complex requires the N-terminal flexible tails in ParG that are contacted by ParF.

• Dynamic as well as stable protein interactions contribute to genome function and maintenance.

  Authors: Peter Hemmerich, Lars Schmiedeberg, Stephan Diekmann

  Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 11/2010; 19(1):131-51.

  The cell nucleus is responsible for the storage, expression, propagation, and maintenance of the genetic material it contains. Highly organized macromolecular complexes are required for theseThe cell nucleus is responsible for the storage, expression, propagation, and maintenance of the genetic material it contains. Highly organized macromolecular complexes are required for these processes to occur faithfully in an extremely crowded nuclear environment. In addition to chromosome territories, the nucleus is characterized by the presence of nuclear substructures, such as the nuclear envelope, the nucleolus, and other nuclear bodies. Other smaller structural entities assemble on chromatin in response to required functions including RNA transcription, DNA replication, and DNA repair. Experiments in living cells over the last decade have revealed that many DNA binding proteins have very short residence times on chromatin. These observations have led to a model in which the assembly of nuclear macromolecular complexes is based on the transient binding of their components. While indeed most nuclear proteins are highly dynamic, we found after an extensive survey of the FRAP literature that an important subset of nuclear proteins shows either very slow turnover or complete immobility. These examples provide compelling evidence for the establishment of stable protein complexes in the nucleus over significant fractions of the cell cycle. Stable interactions in the nucleus may, therefore, contribute to the maintenance of genome integrity. Based on our compilation of FRAP data, we propose an extension of the existing model for nuclear organization which now incorporates stable interactions. Our new "induced stability" model suggests that self-organization, self-assembly, and assisted assembly contribute to nuclear architecture and function.

• Theoretical study of lipid biosynthesis in wild-type Escherichia coli and in a protoplast-type L-form using elementary flux mode analysis.

  Authors: Dimitar Kenanov, Christoph Kaleta, Andreas Petzold, Christian Hoischen, Stephan Diekmann, Roman A Siddiqui, Stefan Schuster

  The FEBS journal. 02/2010; 277(4):1023-34.

  In the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producingIn the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producing unsaturated and saturated fatty acids, phospholipids, lipid A, and cardiolipin. The present analysis was carried out both for the wild-type and for several in silico knockout mutants, using the concept of elementary flux modes. Our results confirm that, in the wild type, there are four main products: L1-phosphatidylethanolamine, lipid A, lipid A (cold-adapted), and cardiolipin. We found that each of these compounds is produced on several different routes, indicating a high redundancy of the system under study. By analysis of the elementary flux modes remaining after the knockout of genes of lipid biosynthesis, and comparison with publicly available data on single-gene knockouts in vivo, we were able to determine the metabolites essential for the survival of the cell. Furthermore, we analyzed a set of mutations that occur in a cell wall-free mutant of Escherichia coli W1655F+. We postulate that the mutant is not capable of producing both forms of lipid A, when the combination of mutations is considered to make a nonfunctional pathway. This is in contrast to gene essentiality data showing that lipid A synthesis is indispensable for the survival of the cell. The loss of the outer membrane in the cell wall-free mutant, however, shows that lipid A is dispensable as the main component of the outer surface structure in this particular E. coli strain.

• The role of localization in the operation of the mitotic spindle assembly checkpoint.

  Authors: Maiko Lohel, Bashar Ibrahim, Stephan Diekmann, Peter Dittrich

  Cell cycle (Georgetown, Tex.). 09/2009; 8(16).

  The mitotic spindle assembly checkpoint ((M)SAC) is an important regulatory mechanism of the cell cycle, ensuring proper chromosome segregation in mitosis. It delays the transition to anaphase untilThe mitotic spindle assembly checkpoint ((M)SAC) is an important regulatory mechanism of the cell cycle, ensuring proper chromosome segregation in mitosis. It delays the transition to anaphase until all chromosomes are properly attached to the mitotic spindle by emitting a diffusible "wait anaphase"-signal from unattached kinetochores. Current models of the checkpoint disregard important spatial properties like localization, diffusion and realistic numbers of kinetochores. To allow for in silico studies of the dynamics of these models in a more realistic environment, we introduce a mathematical framework for quasi-spatial simulation of localized biochemical processes that are typically observed during checkpoint activation and maintenance. The "emitted inhibition" model of the (M)SAC by Doncic et al. (Proc Natl Acad Sci USA 2005; 102:6332-7) assumes instantaneous activation of the diffusible "wait anaphase"-signal upon kinetochore encounter. We modify this model to account for binding kinetics with finite rates and use the developed framework to determine the feasible range of the binding parameters. We find that for proper activation, the binding rate constant has to be fast and above a critical value. Furthermore, this critical value depends significantly on the amount of local binding sites at each kinetochore. The critical values lie in a physiological realistic regime (10(4)-10(6) M(-1)s(-1)). We also determine the feasible parameter range for fast checkpoint activation of the "Mad2 template" model, for which the kinetic parameters have recently been studied in vitro by Simonetta et al. (PLoS Biology 2009; 7:1000010). We find critical values for binding and catalysis rate constants, both significantly higher than the measured values. Our results suggest that yet unknown mechanisms at the kinetochores facilitate binding and catalysis in vivo. We conclude that quantitative models of the (M)SAC have to account for the limited availability of binding sites at kinetochores.

• Acceptor-photobleaching FRET analysis of core kinetochore and NAC proteins in living human cells.

  Authors: D Hellwig, C Hoischen, T Ulbricht, Stephan Diekmann

  European biophysics journal : EBJ. 08/2009; 38(6):781-91.

  Faithful chromatin segregation is mediated and controlled by the kinetochore protein network which assembles at centromeres. In this study, the neighbourhood relations of inner kinetochore andFaithful chromatin segregation is mediated and controlled by the kinetochore protein network which assembles at centromeres. In this study, the neighbourhood relations of inner kinetochore and nucleosome-associated complex (NAC) proteins were analysed in living human interphase cells by acceptor photobleaching FRET. The data indicate that CENP-U is in close vicinity to CENP-I as well as to CENP-B and that CENP-M is close to CENP-T.

• Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation.

  Authors: Malte Bussiek, Christian Hoischen, Stephan Diekmann, Martin L Bennink

  Journal of structural biology. 04/2009;

  Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cellSatellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35 nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (-18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM-AS chromatin.

• In silico study of kinetochore control, amplification, and inhibition effects in MCC assembly.

  Authors: Bashar Ibrahim, Peter Dittrich, Eberhard Schmitt, Stephan Diekmann

  Bio Systems. 08/2008;

  Eukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes areEukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. In different organisms, a mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1/Mad3, and Cdc20 inhibits the anaphase promoting complex (APC/C) to initiate promotion into anaphase. The mechanism of MCC formation and its regulation by the kinetochore are unclear. Here, we constructed dynamical models of MCC formation involving different kinetochore control mechanisms including amplification as well as inhibition effects, and analysed their quantitative properties. In particular, in this system, fast and stable metaphase to anaphase transition can only be triggered when the kinetochore controls the Bub3:BubR1-related reactions; signal amplification and inhibition play a subordinate role. Furthermore, when introducing experimentally determined parameter values into the models analysed here, we found that effective MCC formation is not combined with complete Cdc20 sequestering. Instead, the MCC might bind and completely block the APC/C. The SACM might function by an MCC:APC/C complex rearrangement.

• Mad2 binding is not sufficient for complete Cdc20 sequestering in mitotic transition control (an in silico study).

  Authors: Bashar Ibrahim, Peter Dittrich, Stephan Diekmann, Eberhard Schmitt

  Biophysical chemistry. 05/2008; 134(1-2):93-100.

  For successful mitosis, metaphase has to be arrested until all centromeres are properly attached. The onset of anaphase, which is initiated by activating the APC, is controlled by the spindleFor successful mitosis, metaphase has to be arrested until all centromeres are properly attached. The onset of anaphase, which is initiated by activating the APC, is controlled by the spindle assembly checkpoint (M)SAC. Mad2, which is a constitutive member of the (M)SAC, is supposed to inhibit the activity of the APC by sequestering away its co-activator Cdc20. Mad1 recruits Mad2 to unattached kinetochores and is compulsory for the establishment of the Mad2 and Cdc20 complexes. Recently, based on results from in vivo and in vitro studies, two biochemical models were proposed: the Template and the Exchange model. Here, we derive a mathematical description to compare the dynamical behaviour of the two models. Our simulation analysis supports the Template model. Using experimentally determined values for the model parameters, the Cdc20 concentration is reduced down to only about half. Thus, although the Template model displays good metaphase-to-anaphase switching behaviour, it is not able to completely describe (M)SAC regulation. This situation is neither improved by amplification nor by p31(comet) inhibition. We speculate that either additional reaction partners are required for total inhibition of Cdc20 or an extended mechanism has to be introduced for (M)SAC regulation.

• Dynamics of inner kinetochore assembly and maintenance in living cells.

  Authors: Peter Hemmerich, Stefanie Weidtkamp-Peters, Christian Hoischen, Lars Schmiedeberg, Indri Erliandri, Stephan Diekmann

  The Journal of cell biology. 04/2008; 180(6):1101-14.

  To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells.To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells. CENP-A and CENP-I are stable centromere components that are incorporated into centromeres via a "loading-only" mechanism in G1 and S phase, respectively. A subfraction of CENP-H also stays stably bound to centromeres. In contrast, CENP-B, CENP-C, and some CENP-H and hMis12 exhibit distinct and cell cycle-specific centromere binding stabilities, with residence times ranging from seconds to hours. CENP-C and CENP-H are immobilized at centromeres specifically during replication. In mitosis, all inner CENPs become completely immobilized. CENPs are highly mobile throughout bulk chromatin, which is consistent with a binding-diffusion behavior as the mechanism to scan for vacant high-affinity binding sites at centromeres. Our data reveal a wide range of cell cycle-specific assembly plasticity of the centromere that provides both stability through sustained binding of some components and flexibility through dynamic exchange of other components.

• Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

  Authors: Andrew Derome, Christian Hoischen, Malte Bussiek, Ruth Grady, Malgorzata Adamczyk, Barbara Kedzierska, Stephan Diekmann, Daniela Barillà, Finbarr Hayes

  Proceedings of the National Academy of Sciences of the United States of America. 03/2008; 105(6):2151-6.

  Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is aMultidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.

• In-silico modeling of the mitotic spindle assembly checkpoint.

  Authors: Bashar Ibrahim, Stephan Diekmann, Eberhard Schmitt, Peter Dittrich

  PLoS ONE. 02/2008; 3(2):e1555.

  BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression fromBACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (M)SAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (M)SAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.

• Assembly of the inner kinetochore proteins CENP-A and CENP-B in living human cells.

  Authors: Sandra Orthaus, Christoph Biskup, Birgit Hoffmann, Christian Hoischen, Sabine Ohndorf, Klaus Benndorf, Stephan Diekmann

  Chembiochem : a European journal of chemical biology. 02/2008; 9(1):77-92.

  DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex resultsDNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the "inner kinetochore", is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP-A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP-A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP-A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP-A and CENP-B by fluorescence intensity and lifetime-based FRET measurements in living human HEp-2 cells. We observed Förster resonance energy transfer (FRET) between CENP-A and CENP-B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein-protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP-A and histone H4 as well as between CENP-A molecules themselves. On the other hand, no FRET was detected between CENP-A and H2A.1 or H3.1. Our data support the view that two CENP-A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.

• Organisation of nucleosomal arrays reconstituted with repetitive African green monkey alpha-satellite DNA as analysed by atomic force microscopy.

  Authors: Malte Bussiek, Gabriele Müller, Waldemar Waldeck, Stephan Diekmann, Jörg Langowski

  European biophysics journal : EBJ. 01/2008; 37(1):81-93.

  Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement andAlpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45-50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.

• RNAi knockdown of human kinetochore protein CENP-H.

  Authors: Sandra Orthaus, Sabine Ohndorf, Stephan Diekmann

  Biochemical and biophysical research communications. 10/2006; 348(1):36-46.

  The inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of theThe inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of the inner kinetochore proteins which is conserved amongst many eukaryotes. By specific RNAi knockdown, we reduced the CENP-H protein level in human HEp-2 cells down to less than 5% of its normal value. In these CENP-H knocked-down cells, we observed severe mitotic phenotypes like misaligned chromosomes and multipolar spindles, however, no mitotic arrest. Strong reduction of CENP-H resulted in a slightly reduced CENP-C level at the kinetochores and normal localisation of hBubR1, indicating a functional mitotic checkpoint at the hBubR1 protein level. In CENP-H knocked-down human cells, the misaligned chromosomes contained only reduced levels of CENP-E. Our data clearly indicate that CENP-H has an important impact on the architecture and function of the human kinetochore complex.

• The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

  Authors: Roman A Siddiqui, Christian Hoischen, Otto Holst, Ivonne Heinze, Bernhard Schlott, Johannes Gumpert, Stephan Diekmann, Frank Grosse, Matthias Platzer

  FEMS microbiology letters. 06/2006; 258(2):305-11.

  Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divideCell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

• Distance determination in protein-DNA complexes using fluorescence resonance energy transfer.

  Authors: Mike Lorenz, Stephan Diekmann

  Methods in molecular biology (Clifton, N.J.). 02/2006; 335:243-55.

  Fluorescence resonance energy transfer (FRET) provides distance information between a donor and an acceptor dye in the range of 10-100 A. Knowledge of the exact positions of some dyes (e.g.,Fluorescence resonance energy transfer (FRET) provides distance information between a donor and an acceptor dye in the range of 10-100 A. Knowledge of the exact positions of some dyes (e.g., fluorescein, rhodamine, or Cy3) with respect to nucleic acids and DNA design enables us to translate these data into precise structural information using molecular modeling. Here we describe this in vitro approach from the design and synthesis of the DNA FRET samples to the fluorescence spectroscopy methods and analysis. Advances in the preparation of dye-labeled nucleic acid molecules and modern techniques like the measurement of FRET in vivo lead to an increased importance of FRET studies in structural and molecular biology.

• Molecular basis of the interaction specificity between the human glucocorticoid receptor and its endogenous steroid ligand cortisol.

  Authors: Johannes von Langen, Karl-Heinrich Fritzemeier, Stephan Diekmann, Alexander Hillisch

  Chembiochem : a European journal of chemical biology. 07/2005; 6(6):1110-8.

  We analyzed the binding of five steroids to the human glucocorticoid receptor (hGR) experimentally as well as theoretically. In vitro, we measured the binding affinity of aldosterone, cortisol,We analyzed the binding of five steroids to the human glucocorticoid receptor (hGR) experimentally as well as theoretically. In vitro, we measured the binding affinity of aldosterone, cortisol, estradiol, progesterone, and testosterone to hGR in competition with the ligand dexamethasone. The binding affinity relative to the endogenous ligand cortisol (100%) is reduced for progesterone (22%) and aldosterone (20%) and is very weak for testosterone (1.5%) and estradiol (0.2%). In parallel, we constructed a homology model of the hGR ligand-binding domain (LBD) based on the crystal structure of the human progesterone receptor (hPR). After docking the five steroids into the hGR model ligand-binding pocket, we performed five separate 4-ns molecular dynamics (MD) simulations with these complexes in order to study the complex structures. We calculated the binding affinities with two different approaches (MM/PBSA, FlexX) and compared them with the values of the experimentally determined relative binding affinities. Both theoretical methods allowed discrimination between strongly and weakly binding ligands and recognition of cortisol as the endogenous ligand of the hGR in silico. Cortisol binds most strongly due to a nearly perfect steric and electrostatic complementarity with the hGR binding pocket. Chemically similar ligands such as estradiol, testosterone, and progesterone also fit into the hGR binding pocket, but they are unable to form all those contacts with the amino acids of the protein that are necessary to yield a stable, transcriptionally active receptor conformation. Our analysis thus explains the selectivity of the human glucocorticoid receptor for its endogenous ligand cortisol at a molecular level.