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Role of human polynucleotide kinase/phosphatase (PNKP) in nuclear and mitochondrial DNA repair

  • Author / Creator
    Subedi, Sudip
  • DNA is continuously under stress from the insults inflicted by both endogenous and exogenous agents. These agents include endogenous reactive oxygen species (ROS), ionizing radiation (IR) and many common chemotherapeutic drugs, which can inflict a plethora of DNA lesions including abasic sites, DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). Such damages can be mutagenic or lethal and are often linked to cancer and degenerative diseases. Cells are equipped with sophisticated repair machineries to prevent the mutational and cytotoxic consequences of DNA damage, which involve the orchestration of various repair proteins. Frequently, IR, ROS and topoisomerase 1 inhibitor-induced strand breaks bear termini with enzymatically incompatible ends such as 5’-hydroxyl and 3’-phosphate, which need to be processed to generate ligatable ends, i.e. 5’-phosphate and 3’-hydroxyl termini. The dual functioning enzyme polynucleotide kinase/phosphatase (PNKP) contains both a kinase domain to phosphorylate 5’-OH termini and a phosphatase domain to replace 3’-phosphate with 3’-OH termini. PNKP’s role in nuclear repair pathways like base excision/single strand-break repair (BER/SSBR) and non-homologous end joining (NHEJ) for DSB repair has been well established. Mitochondria of human cells contain a 16.5 kbp circular DNA molecule (mtDNA), which is even more under threat of endogenous ROS owing to its proximity to the electron transport chain and lack of chromatin-associated protection unlike nuclear DNA. Natural mutation frequencies in some regions of mtDNA are 20-100 fold higher than the nuclear DNA. The oxidative damages and breaks of the mitochondrial genome have been implicated in various human degenerative diseases, aging and cancer. Several DNA repair pathways have been shown to be involved in the maintenance of mtDNA. Here we show that functionally active full-length PNKP localizes to human mitochondria. Based on these findings, we hypothesized that mitochondrial PNKP (mtPNKP) must be critical for cell survival. In order to begin to examine the importance of mtPNKP in cell survival, we set out to generate cell lines that express PNKP exclusively either in the nucleus or mitochondria. To achieve that, we mapped the nuclear localization signal (NLS) and mitochondrial targeting sequence (MTS) of PNKP and used site-directed mutagenesis to mutate them in an attempt to impair PNKP trafficking into the organelle of choice, e.g. mutating the NLS to impair PNKP import into the nucleus. As an alternative approach, we used the pShooter organelle directing vector system to guide PNKP to the organelle of choice. Once the cell lines are generated, we will challenge them with various genotoxic and chemotherapeutic agents. As part of the targeting approach we generated human A549 PNKP knockout cells using CRISPR technology.

  • Subjects / Keywords
  • Graduation date
    2015-11
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3JM23W6K
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Department of Oncology
  • Specialization
    • Experimental Oncology
  • Supervisor / co-supervisor and their department(s)
    • Michael Weinfeld
  • Examining committee members and their departments
    • Gordon Chan (Oncology)
    • Lynne-Marie Postovit (Oncology)
    • Mark Glover (Biochemistry)