Repository logo

Browsing by Author "Hansen, Jeffrey C., committee member"

Now showing 1 - 6 of 6
  • Thumbnail Image
    ItemOpen Access
    Biophysical, structural, and functional studies of histone binding proteins
    (Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
    Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
  • Thumbnail Image
    ItemEmbargo
    Mapping the metabolic protein interactome that supports energy conservation at the limits of life
    (Colorado State University. Libraries, 2024) Williams, Seré Anne, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Pilon, Marinus, committee member; Anderson, G. Brooke, committee member; Snow, Christopher, committee member
    Distinct metabolic strategies yield energetic gains from a wide variety of substrates, yet only three overarching methods of energy conservation have been defined: substrate level phosphorylation, the generation of a charged membrane, and electron bifurcation. The dominant theme of known energy conservation mechanisms suggests that energy is conserved through the selective movement and management of electrons, thus essentially all life relies on redox (reduction and oxidation) reactions. Small molecule redox cofactors (such as NAD(P)+) and proteinaceous electron carriers (such as ferredoxins) are employed as electron carriers throughout the biosphere. Proteinaceous electron carriers offer the potential for selective protein-protein interactions to bridge reductive flow from catabolic reactions to the membrane, providing a "proteinaceous electron highway" for efficient electron shuttling. Specific redox protein partnerships have been shown to adapt to changing physiological conditions, suggesting that proteinaceous electron flux is tunable and provides a level of selectivity not possible with small molecule electron transport. While electron flux through a tunable and regulated system of protein interactions can offer exceptional energy conservation strategies, large gaps remain in our knowledge of how electron flux is regulated in vivo. Identification of bona fide in vivo protein assemblies – and how such assemblies dictate the totality of electron flow and thus cellular metabolism – is an important milestone to understand the regulation imposed on metabolism, energy-production, and energy conservation. Resolving the dynamic nature of nanoscale interactions in living systems is arguably the current frontier of molecular biology, and combinatorial methods – which layer multiple in vitro and in vivo techniques with large data analysis – have come to the forefront. This dissertation addresses energy conservation strategies of in vivo protein associations in a model, genetically accessible, hyperthermophilic archaeon (Thermococcus kodakarensis) by mapping the metabolic protein interactome using affinity purification mass spectrometry (AP-MS) and generating engineered strains where fusion proteins selectively redirect electron flux in vivo. Twenty-five proteins involved in distinct metabolic functions were tagged to reveal that each tagged-protein interacts with ~ thirty proteins on average. These interactions connected disparate functions suggesting catabolic and anabolic activities may occur in concert -- in temporal and spatial proximity in vivo. The AP-MS method also refined our understanding of previously determined stable complexes suggesting that protein complexes in vivo likely adapt to redox conditions. Engineered strains linking a proteinaceous electron donor to a proposed electron acceptor were viable and impacted electron flux in vivo. Fusion strains linking a ferredoxin to the hydrogen-generating respiratory system increased hydrogen gas output ~8% on average with one strain showing a ~45% increase over wild type. Fusion strains impacting lipid saturation were shown to inhibit saturation, and future studies aim to determine if electrons can be redirected from the vast reductant sink of lipids to the generation of hydrogen gas, a valuable biofuel.
  • Thumbnail Image
    ItemOpen Access
    Post-initiation activities of the archaeal RNA polymerase in a chromatin landscape
    (Colorado State University. Libraries, 2020) Sanders, Travis James, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Peersen, Olve B., committee member; Ben-Hur, Asa, committee member
    The machineries that control transcription initiation and elongation in Archaea and Eukarya are highly homologous. These similarities support the prevailing evolutionary theory of Archaea being the progenitor of Eukarya. Due to the retention of a core transcription apparatus, while lacking complexities of the eukaryotic counterpart, archaeal systems offer the unique potential to study and characterize the basic protein components necessary for transcription. Transcription termination was less well understood in both Archaea and Eukarya. Shared homology of the initiation and elongation phases argued for a homologous method of termination in Archaea and Eukarya. Additionally, both the archaeal and eukaryotic transcription apparatuses are frequently impeded by histone proteins bound to DNA. Like the transcription complex, archaeal histones are a simplified mirror to eukaryotic histones, permitting evaluation of all steps in the transcription cycle in the context of a chromatin landscape. This thesis summarizes the core molecular machineries involved in the regulation of archaeal transcription during elongation and termination in the greater context of archaeal histone-based chromatin. Thus, the discoveries made have contributed to both the transcription and chromatin fields by providing mechanistic details of the core, conserved transcription apparatus in the framework of evolution.
  • Thumbnail Image
    ItemOpen Access
    Post-initiation regulatory mechanisms of transcription in the Archaea
    (Colorado State University. Libraries, 2023) Wenck, Breanna Renée, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Osborne Nishimura, Erin, committee member; Wilusz, Carol, committee member
    Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. Archaeal systems contain simplified, basal regulatory transcription components and mechanisms homologous to their eukaryotic counterparts, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. Many archaeal genomes are organized with histone proteins that resemble the core eukaryotic histone fold, which permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that impacts transcription regulation and gene expression. Despite such semblance between the eukaryotic and archaeal core histone folds, archaeal genomes lack the canonical N and C terminal extensions that are abundantly modified to regulate transcription in eukaryotic genomes. Much of what is known regarding factor-mediated transcription regulation in the Archaea is limited; however combined and continued efforts across the field provide tidbits of information, but many pieces are still missing. This thesis aims to i) delineate the role key residues within the histone-DNA complex and archaeal histone-based architecture and key residues within the histone-DNA complex have on the progression of the transcription apparatus, characterize factor-mediated transcription termination, and explore chromatin- and TFS-mediated regulatory effects on transcription via global RNA polymerase (RNAP) positions.
  • Thumbnail Image
    ItemEmbargo
    Spn1, Spt4, Spt5, and Spt6 preserve chromatin structure over promoters and open reading frames
    (Colorado State University. Libraries, 2024) Tonsager, Andrew Jordan, author; Stargell, Laurie A., advisor; Hansen, Jeffrey C., committee member; Santangelo, Thomas, committee member; Argueso, Juan Lucas, committee member
    The eukaryotic chromatin landscape presents formidable nucleosomal barriers for processes that require access to DNA, such as transcription. These barriers are overcome through the action of many factors, including histone chaperones Spn1, Spt5, Spt6, and FACT and transcription elongation factor Spt4. However, it is poorly understood how each contributes to this process. To ascertain the role that these factors play on preserving chromatin structure over the genome, this thesis has utilized micrococcal nuclease digestion followed by sequencing (MNase-seq) to analyze chromatin protections in the yeast genome in cells expressing numerous mutant alleles of these factors. Extensive characterization of MNase-protected fragments in a wide range of sizes established that the essential histone chaperone Spn1 preserves both nucleosomal and subnucleosomal structures over both promoters and open reading frames across the genome. Additional analyses from existing MNase-seq datasets demonstrated the extent to which Spn1 and other RNAPII-associated factors maintain nucleosome features over genes of varied characteristics. The study of factors described in this thesis is performed in living cells, which have been genetically modified to express mutant alleles of chromatin factors. This thesis also describes a course-based undergraduate research experience (CURE) developed to introduce upper-level biochemistry students to techniques in yeast genome engineering in an authentic research setting.
  • Thumbnail Image
    ItemOpen Access
    The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates
    (Colorado State University. Libraries, 2013) Clark, Nicholas James, author; Luger, Karolin, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey C., committee member; Woody, Robert, committee member
    Eukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.