Browsing by Author "Hepburn, Morgan Rose"

Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemOpen Access
    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.