Wechsler, Thomas (2005): Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs. Dissertation, LMU München: Faculty of Biology |
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Abstract
Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. To identify new proteins, which contribute to double-strand break repair by nonhomologous end joining, we previously performed a yeast two-hybrid screen with fragments of human DNA-PKcs as bait. From the identified putative interaction partners of DNA-PKcs we chose two for further characterization: Protein phosphatase 5 (PP5) and Ku70 binding protein 3 (Kub3). With PP5 we have identified the first protein phosphatase with a function in doublestrand break repair. We show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites of it. Cells with either hypo or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity. For the characterization of Kub3 we describe its correct reading frame and a putative metalloprotease domain. Using a rabbit polyclonal antibody against human Kub3 we demonstrate that Kub3 is a nuclear protein which co-precipitates with DNA-PKcs. When Kub3 is overexpressed in HeLa cells, DNA-PKcs phosphorylation at T2609 is increased after ionizing radiation. However, when Kub3 is knocked down in drosophila cells by RNAi, cell survival after ionizing radiation is not affected. In summary, with PP5 and Kub3 we have characterized two new interaction partners of DNA-PKcs. While PP5 clearly functions in double-strand break repair, further experiments are necessary to confirm such a role for Kub3.
Item Type: | Theses (Dissertation, LMU Munich) |
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Keywords: | DNA damage, DNA double-strand break repair, nonhomologous end joining, DNA-PKcs, Protein phosphatase 5, Kub3 |
Subjects: | 500 Natural sciences and mathematics 500 Natural sciences and mathematics > 570 Life sciences |
Faculties: | Faculty of Biology |
Language: | English |
Date of oral examination: | 18. August 2005 |
1. Referee: | Weiß, Elisabeth |
MD5 Checksum of the PDF-file: | 6987ea2245879359c4bd92a466a2b8d3 |
Signature of the printed copy: | 0001/UMC 14933 |
ID Code: | 4246 |
Deposited On: | 02. Dec 2005 |
Last Modified: | 24. Oct 2020 10:10 |