Untersuchung des Mechanismus, der das ringförmige Heterodimer Ku70/80 nach erfolgreicher Doppelstrangbruch-Reparatur von der DNA entfernt.
Final Report Abstract
Unrepaired DNA double-strand breaks (DSBs) can lead to apoptosis or tumorigenesis. In mammals, double-strand breaks are repaired mainly by non-homologous end-joining (NHEJ). The core proteins of this pathway include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the heterodimer Ku70/80, the two tightly associated proteins XRCC4 and Ligase IV and the XRCC4-like factor (XLF/Cernunnos). Although the early steps of DSB repair by NHEJ are well understood, little is known about how the participating proteins are removed from the DNA after repair has been completed. The fate of the Ku70/80 heterodimer, which encloses the dsDNA like a ring, is particularly interesting. For this DFG-funded project we had proposed the following specific aims to address this question: Specific aim 1: Detection of the Ku heterodimer on a NHEJ substrate. We will attempt to detect the Ku70/80 heterodimer on a NHEJ substrate after completion of DNA end-joining. Specific aim 2: Characterization of the Ku removal activity. If we are not able to detect Ku, we will identify by biochemical methods the factors that facilitate Ku removal. If we are able to detect Ku on DNA after NHEJ, we will test if Ku can be removed by chromatin remodeling factors. Over the course of this project we have successfully developed a Ku removal assay using cellfree extracts carrying out NHEJ. In these extracts we were able to study Ku removal by generating circular NHEJ products that trap this heterodimer. We further used the in vitro NHEJ assay to study the ability of cells from RIDDLE patients to repair DSBs. In summary, the results of this project showed that Ku is swiftly removed from DNA after successful NHEJ but that the newly established assay cannot be further developed to address other questions or to characterize the Ku removal activity in more detail. We further found that cell-free extracts from RIDDLE patients carrying mutations in the ubiquitin ligase RNF168 are fully competent in NHEJ, but seem to have increased DNA end-processing activity.
Publications
- RIDDLE immunodeficiency syndrome is linked to defects in 53BP1- mediated DNA damage signaling. Proc Natl Acad Sci U S A 104, 16910-16915, (2007)
Stewart, G. S. et al.
