Item – Theses Canada

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OCLC number
46575990
Link(s) to full text
LAC copy
LAC copy
Author
Ting, Nicholas S. Y.,1970-
Title
Identification and characterization of proteins that interact with the human DNA-dependent protein kinase (DNA-PK).
Degree
Ph. D. -- University of Calgary, 1999
Publisher
Ottawa : National Library of Canada = Bibliothèque nationale du Canada, [1999]
Description
2 microfiches.
Notes
Includes bibliographical references.
Abstract
One of the key proteins required during cellular responses to DNA damage is the DNA dependent protein kinase (DNA-PK). DNA-PK is a serine/threonine protein kinase composed of a 465 kDa catalytic subunit called DNA-PKcs and a heterodimeric DNA-targeting component of 70 and 80 kDa, known as Ku. DNA-PK is activated by ends of double stranded DNA, 'in vitro', to phosphorylate substrates such as the tumor suppressor protein, p53, transcription factors, SpI and Serum Response Factor (SRF), the carboxy terminal domain (CTD) of RNA Polymerase II, and a protein required for DNA repair, XRCC4. Biochemical and genetic studies with various radiosensitive cell lines and mouse knockout models for Ku80, Ku70 and DNA-PKcs have shown that DNA-PK is required for the process of non-homologous end-joining during the repair of DNA double-strand breaks and V(D)J recombination. During the initial purification of DNA-PK from Hela cells, several polypeptides that were phosphorylated in a DNA dependent manner were present in a partially purified active kinase fraction. Three of these polypeptides were identified as DNA-PKcs, Ku8O and Ku70. Similar polypeptides were observed to be present in a partially purified DNA-PK fraction from human placenta. Here, two of these polypeptides have been identified as the translation initiation factor, eIF-2 and a heterodimeric transcription factor, NF90/45. eEF-2[beta] and NF90/45 are shown to be 'in vitro' substrates of DNA-PK. Electromobility shift assays have demonstrated that a pool of polypeptides which contained eIF-2 and NF90/45 or recombinant NF90 alone was able to support the formation of a stabilized protein-DNA complex between purified DNA-PKcs and Ku on linear double stranded DNA. Moreover, antibodies to recombinant NF90 and NF45 immunoprecipitated DNA-PKcs, NF90 and NF45 from a partially purified protein fraction from human placenta and from crude cell extracts. However, Ku was not detected in these immune complexes. Based on these results, we propose that the interaction between NF90/45 and DNA-PKcs is required for the formation of a stable trimeric DNA-PKcs/Ku80/70 complex on linear double-stranded DNA, which may be important for the function of DNA-PK during non-homologous end-joining 'in vivo '.
Subject
Protein kinases.
Protéines kinases.
ISBN
0612385116
9780612385115