Crystallographic studies of chromatin-targeting metal-based compounds and nucleosome core particles assembled with engineered cohesive end DNA fragments

• Main Author: Louis, DeFalco Jr.
• Other Authors: Curtis Alexander Davey
• Format: Theses and Dissertations
• Language: English
• Published: 2019
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/89610; http://hdl.handle.net/10220/47720
• Institution: Nanyang Technological University

In eukaryotic cells, related functions like the storage, protection, and precise regulation of genomic DNA are achieved by its compaction into protein–nucleic acid complex structures known as chromosomes. During the initial steps of compaction, naked DNA is wound around an octamer of histone proteins in a process that forms the nucleosome core. This nucleoprotein complex creates increasingly higher-order structures that, with the addition of linker histone proteins, ultimately generate densely packed chromatin. It is established that tumor cells present distinct dissimilarities in chromatin dynamics, nucleosome positioning, and epigenetic modifications like DNA methylation and post-translational modifications relative to healthy cells, and this distinction is significant in the context of genomic regulation during the eukaryotic cell cycle. With this knowledge, chromatin reactivity toward exogenous molecules is the primary principle concerning rational design of therapeutic compounds that separately, or simultaneously, target protein and DNA motifs of the nucleosome core. Beyond the nucleosome core, a comprehensive understanding of genomic structure and nucleosome–nucleosome arrangement requires the availability of minimal and reproducible systems for investigation. Understanding gained from new nucleosome structures will illuminate heretofore unknown details regarding the interplay between nucleosome dynamics and genetic regulation and their collective genome-wide impact. Earlier studies from our collaborators have shown a synergistic effect between the antirheumatic compound auranofin (AUF), possessing a gold(I) reactive center, and the ruthenium(II)-based anticancer drug RAPTA-T. Remarkably, the synergism observed as a decrease in tumor cell viability coincides with an allosteric mechanism by which binding of RAPTA-T to its characteristic target sites on the H2A:H2B acidic patch (sites RU1 and RU2) facilitates the binding of AUF to a distinct histidine residue (H113) found on core histone H3 near the nucleosome dyad. Here in a follow-up study, Compound III (C3), a novel hetero-dinuclear compound containing distantly-linked ruthenium(II) and gold(I) metal centers, was found to form adducts at canonical nucleosome ruthenium and gold binding sites by exploiting the allosteric crosstalk between distant regions of the histone octamer surface to cross-link tetramer with dimer. Mononuclear C3 precursors, a trinuclear C3 derivative, and two viral peptide–metalloreagent conjugates possessing AUF-like gold(I) reactive centers were additionally employed in an attempt to capitalize on the dimer–tetramer allosteric pathway towards therapeutic gain, with structural data for each presenting varying degrees of effectiveness. Adapting established cloning techniques to produce palindromic nucleosome DNA sequences, we were able to construct nucleosomes containing linker regions that maintain the capacity to form base pairs at their termini. Controlling this process allowed for the creation of well diffracting crystals of nucleosome-linker histone assemblies. Building on this concept, several new nucleosome DNA construct designs are presented here, all of which are based on the 601 Widom high affinity sequence. This has allowed improvement of technical issues involved in nucleosome core particle (NCP) DNA production as well as the elucidation of NCP crystallization kinetic principles, which offer a degree of control over NCP crystal lattice formation. As such, this approach holds potential for facilitating acquisition of NCP structures with diverse, genetically significant DNA sequences, histone proteins and associated chromatin factors. Additionally, a high-resolution crystal structure of a cohesive-end NCP reveals a novel multimeric arrangement that may approximate specific instances of chromatin fiber packing.