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Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs
Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs
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.
DNA damage, DNA double-strand break repair, nonhomologous end joining, DNA-PKcs, Protein phosphatase 5, Kub3
Wechsler, Thomas
2005
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Wechsler, Thomas (2005): Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs. Dissertation, LMU München: Fakultät für Biologie
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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.