Chromatin at the Nanolevel - Development of a single molecule FRET experiment and analysis of the structure and stability of individual nucleosomes

The structure and stability of individual nucleosome complexes is analysed on the single molecule level. Both aspects are important for the organisation of chromatin inside the nucleus, e.g. by controlling the accessibility of DNA to transcription factors. On the level of individual nucleosomes in vitro experiments provide valuable information on the processes responsible for dynamic changes in the nucleosome structure. An experimental setup is presented which monitors the conformation of freely diffusing complexes. Nucleosomal DNA is labeled with small fluorophores and Fluorescence Resonance Energy Transfer (FRET) is used to monitor changes in nucleosome structure with nm accuracy. Experiments are presented in which various remodelling factors induce detectable changes in the nucleosome conformation. A major focus is laid on the stability of nucleosomes under the influence of various factors such as ionic strength, total nucleosome concentration, histone tail acetylation and the use of different DNA sequences. Nucleosomes dissociate spontaneously at low sample concentrations and sequence-specific changes in nucleosome structure occur on the ms time scale. Histone tail acetylation also results in a destabilisation of the nucleosome complex. The dissociation at larger ionic strength correlates with an opening of the overall nucleosome structure which predominantly affects the linker DNA region

Nucleosome accessibility governed by the dimer/tetramer interface

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A–H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3–H4)2 tetramer/(H2A–H2B) dimer interface, followed by H2A–H2B dimer release from the DNA and, lastly, (H3–H4)2 tetramer removal. We estimate that the open intermediate state is populated at 0.2–3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery

Superconducting nanowire photon number resolving detector at telecom wavelength

The optical-to-electrical conversion, which is the basis of optical detectors, can be linear or nonlinear. When high sensitivities are needed single-photon detectors (SPDs) are used, which operate in a strongly nonlinear mode, their response being independent of the photon number. Nevertheless, photon-number resolving (PNR) detectors are needed, particularly in quantum optics, where n-photon states are routinely produced. In quantum communication, the PNR functionality is key to many protocols for establishing, swapping and measuring entanglement, and can be used to detect photon-number-splitting attacks. A linear detector with single-photon sensitivity can also be used for measuring a temporal waveform at extremely low light levels, e.g. in long-distance optical communications, fluorescence spectroscopy, optical time-domain reflectometry. We demonstrate here a PNR detector based on parallel superconducting nanowires and capable of counting up to 4 photons at telecommunication wavelengths, with ultralow dark count rate and high counting frequency

Design principles for an enterprise systems chartering method

Our research follows a design science approach to develop a method that supports the initialization of ES implementation projects – the chartering phase. This project phase is highly relevant for implementation success, but is understudied in IS research. In this paper, we derive design principles for a chartering method based on a systematic review of ES implementation literature and semi-structured expert interviews. Our analysis identifies differences in the importance of certain success factors depending on the system type. The proposed design principles are built on these factors and are linked to chartering key activities. We specifically consider system-type-specific chartering aspects for process-centric Business Intelligence & Analytics (BI&A) systems, which are an emerging class of systems at the intersection of BI&A and business process management. In summary, this paper proposes design principles for a chartering method – considering specifics of process-centric BI&A

How histone modifications change nucleosome stability : FRET studies on single molecules and in bulk

Abstract is not available in fulltext.Published versio