Integrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joining

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dc.contributor.advisorSchriemer, David C.
dc.contributor.authorHepburn, Morgan Rose
dc.contributor.committeememberLees-Miller, Susan P.
dc.contributor.committeememberNg, Kenneth Kai Sing
dc.contributor.committeememberWilliams, Gareth J.
dc.contributor.committeememberHuang, Lan
dc.date2020-11
dc.date.accessioned2020-06-09T18:37:44Z
dc.date.available2020-06-09T18:37:44Z
dc.date.issued2020-06-04
dc.description.abstractNon-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.en_US
dc.identifier.citationHepburn, M. R. (2020). Integrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joining (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/37902
dc.identifier.urihttp://hdl.handle.net/1880/112161
dc.language.isoengen_US
dc.publisher.facultyCumming School of Medicineen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en_US
dc.subjectMass Spectrometryen_US
dc.subjectNon-Homologous End Joiningen_US
dc.subjectHydrogen Deuterium Exchangeen_US
dc.subjectCrosslinkingen_US
dc.subjectDNA dependent protein kinase catalytic subunit (DNA-PKcs)en_US
dc.subject.classificationBiochemistryen_US
dc.titleIntegrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joiningen_US
dc.typedoctoral thesisen_US
thesis.degree.disciplineMedicine – Biochemistry and Molecular Biologyen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameDoctor of Philosophy (PhD)en_US
ucalgary.item.requestcopytrueen_US
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