Characterizing the nucleosome site selectivity, adduct structures and impact on chromatin for a variety of metal-based anticancer compounds and structural studies of nucleosome-linker histone interactions

• Main Author: Adhireksan, Zenita
• Other Authors: Curtis Alexander Davey
• Format: Theses and Dissertations
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences::Biochemistry; DRNTU::Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/66028
• Institution: Nanyang Technological University

The enormous length of DNA molecules is managed in the nucleus of eukaryotic cells by wrapping the double helix through histone protein association to form chromatin. The basic repeating unit of chromatin is the nucleosome. Due to this packaging, nucleosomal DNA and histones have different conformations and accessibility compared to that of naked DNA or chaperone-bound histones. The structure and dynamics of chromatin are important for many cellular functions. Different nuclear factors bind to nucleosomes to regulate chromatin dynamics and signal downstream cellular processes, like DNA replication and transcription. As a consequence, nucleosomes and their binding partners can be specifically targeted to modulate these functions. This is especially relevant to cancer cell function, whereby numerous epigenetic differences, such as alterations in nucleosome positioning, between transformed and healthy cells result from extensive changes in gene expression and the frequency of DNA replication (cell division). Distinguishing epigenetic features of cancer cells could be potentially exploited through the design of site-specific chromatin-targeting anticancer agents. Because of the severe side effects and resistance problems associated with platinum anticancer drugs, many different compounds based on alternative heavy metals have been explored as potential therapeutic agents and many more are presently under investigation. However, the cellular targets and mechanisms of action for these newer agents are largely unknown. Based on studies with cisplatin and other platinum drugs, DNA is generally assumed to be the cellular target for reactive metal-based compounds. However, this thesis work and other recent evidence show that proteins are the main targets for at least some of these compounds. Since the nucleosome contains both DNA and protein components and is an important therapeutic target, it is useful for investigating the DNA- versus protein-binding preferences of different metal-based agents. We investigated the site selectivity, adduct structures and impact on chromatin for many different metal-based compounds, including ruthenium, osmium, gold and rhodium agents. This involved a broad spectrum of biochemical, X-ray crystallographic and electron microscopic (EM) techniques in conjunction with collaborations on cellular, analytical chemistry and computational approaches. The striking discovery is that, with the exception of one class of ruthenium agent, the compounds investigated show a preference for binding to histone protein sites as opposed to the DNA of the nucleosome. Moreover, by comparing two structurally similar ruthenium compounds, one a cytotoxic anti-primary tumor agent and the other an effectively non-cytotoxic antimetastasis agent, we could show the basis for their differential DNA versus histone protein targeting and consequently linkages to their distinct cellular impact. For the compounds that preferentially form adducts at histone protein sites, we also discovered that there are two distinct classes of binding site, which are distinguishable by virtue of electrostatic components, affinity for histidine, steric access and hydrophobic interactions. Beyond this, the results shed light on the basis for cleavage of ‘carrier’ ligands, the influence of different metal centers and the basis for a novel nucleosomal drug-drug synergy. Finally, we show that adducts formed by certain metalloagents can have a direct and dramatic influence on the structure and dynamics of the chromatin fiber. Together with the other findings, this suggests strong therapeutic potential surrounding many different features of chromatin, yielding new leads for the rational design of improved metal-based anticancer agents. By compacting chromatin in a site specific manner, linker histone association with the nucleosome changes the structure and accessibility of defined genomic sites. However, the details of linker histone binding location on the nucleosome are still not clear, and in particular a high-resolution atomic structure for a nucleosome-linker histone assembly has not been solved. In addition to the biological significance, structural characterization of linker histone-nucleosome interactions could hold importance for drug development initiatives, for instance new targets for metal-based compounds. In this thesis, we investigated binding and crystallization of complexes of nucleosome with linker histones, focusing largely on the minimal sized assembly, the ‘chromatosome’. We found different affinities for nucleosome binding to the full length versus globular domain-only linker histone and also some degree of DNA length and sequence dependence in the interaction. Different crystals of the chromatosome were obtained, however the best so far diffract X-rays to only ~5.5 Å resolution. Nonetheless, the initial results of the study provide a platform for further optimization of chromatosome crystallizations and possible structural characterization.