Browsing by Author "Williams, Gareth J."

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    Integrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joining
    (2020-06-04) Hepburn, Morgan Rose; Schriemer, David C.; Lees-Miller, Susan P.; Ng, Kenneth Kai Sing; Williams, Gareth J.; Huang, Lan
    Non-homologous end joining (NHEJ) performs untemplated repair of DNA double strand breaks (DSBs). Despite lack of a template, intricate repair, coordinated by the core NHEJ factors, can repair breaks with minimal to no alterations. Initiating repair, Ku70/80 binds to the free DNA ends, and interacts with the large protein kinase, DNA dependent protein kinase catalytic subunit (DNA-PKcs), forming the holoenzyme DNA-PK. Holoenzymes can synapse across the break to tether the DNA ends. Assembly of the initial synaptic complex and its role in NHEJ is poorly understood, as final ligation requires a structural rearrangement of this initial complex. To better understand DNA-PKcs’ role in NHEJ, an integrative structural model of DNA-PKcs in the initial stages of NHEJ was developed using mass spectrometry (MS) techniques. Due to technical challenges working with DNA-PKcs, each of the MS techniques were optimized for the system. Hydrogen deuterium exchange (HX) methods were optimized on a nano-spray HX system, allowing for differential HX analysis of bead bound DNA-PKcs complexes with high sequence coverage, and 5X improvement in protein consumption. Reversible crosslinking and peptide fingerprinting (RCAP) was optimized to allow for direct detection of DNA binding peptides, using a single sample. Finally, given the benefits of DNA-PKcs complex assembly on beads to limit heterogeneity, an on-bead crosslinking method was developed. Mass Spec Studio was used to accurately identify many crosslinks, which can be utilized for a label free quantitation comparison of states. Using HX-MS to explore DNA-PKcs conformational changes from binding to activation of the kinase, an allosteric pathway was identified in DNA-PKcs connecting DNA-binding with the kinase domain. Nucleotide loading of the kinase domain revealed that DNA-PK occupies a tensed state when active. From integrative structural modelling, with the XL-MS restraints, a model with a precision of 13.5Å was reported, revealing a symmetric DNA-PK dimer, with head-to-head interactions. In our synaptic model, the DNA ends are positioned with a large offset, protected by a previously uncharacterized plug domain of DNA-PKcs. We propose the initial formation of the synaptic complex allows for a hierarchical processing of DNA ends and assembly of a core NHEJ scaffold.
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    The Role of RAD51 Paralogs and Their Interactions in the RAD51- Mediated Homologous Recombination DNA Repair
    (University of Calgary, 2018-09-24) Patel, Deepak; Pepper, Jordan; Williams, Gareth J.
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    Structure-function studies of viral RNA-dependent RNA polymerases
    (2018-09-18) Samanta, Soumya; Ng, Kenneth Kai Sing; Zimmerly, Steven; Noskov, Sergei Yu; Williams, Gareth J.; Glover, Mark
    Human norovirus (NV) and hepatitis C virus (HCV) are two major and related human pathogens belonging to the positive-strand RNA virus superfamily. NV belongs in the Caliciviridae family and is a major cause of gastroenteritis outbreaks throughout the world. Although there is substantial interest in the development of vaccines and antiviral therapeutics for norovirus infections, there are currently no effective antiviral treatments. Using a fluorescence-based primer extension assay, I have identified a series of structurally-related compounds that inhibit norovirus polymerase at micromolar concentrations. Previous work in our group has helped to structurally characterize human norovirus polymerase complexes with RNA, nucleoside triphosphates, and divalent metal ions. These structures have revealed the enzyme trapped indifferent conformations that are likely important for the catalytic cycle. Several enzyme-inhibitor complexes with alendronic acid and medronic acid inhibitors have been crystallized, and the structure determination of NV Pol complexes has been undertaken. This thesis presents new results correlating enzyme inhibition kinetics with crystallographic structure determination for several novel inhibitor complexes. In the second part of this thesis, work is described on characterizing interactions between RNA and HCV non-structural protein NS5B, the RNA-dependent RNA Polymerase (RDRP) central to HCV replication. HCV NS5B is highly-conserved and forms the core of the catalytic site of the replicase complex. I have determined the structure of a novel polymerase-RNA complex that shows the binding of a short RNA oligonucleotide to a region of the enzyme near a commonly-targeted site for the binding of allosteric inhibitors.