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Analysis of the functions of the AAA-ATPase p97 in DNA double-strand break repair ; Analyse der Funktionen der AAA-ATPase p97 in DNA-Doppelstrangbruchreparatur
Ionizing radiation (IR) can cause DNA double-strand breaks (DSBs) in the cellular genome, which result in genomic instability, if left unrepaired. The two major repair pathways to cope with DSBs are non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). DSB repair by NHEJ is initiated by the Ku heterodimer that binds the open DNA ends and recruits all factors needed for ligation to the break. Each subunit of the Ku molecule fully encircles the DNA and is therefore trapped by ligation of the DSB. To restore the DNA integrity, Ku needs to be actively removed after successful repair by NHEJ. In addition, it was claimed that Ku blocks end resection and therefore needs to be removed from open DNA ends to enable HRR. If a DSB is repaired by HRR, the 5’ ends are extensively resected and the single-stranded DNA regions are covered by RPA. The DNA-bound RPA can be phosphorylated by DNA-PK, before it is replaced by Rad51 filaments. The Rad51 filaments mediate the search for the homologous sister chromatid for template-based repair. In this study, clear evidence was found for p97 removing sterically trapped Ku80 from the DNA after successful DSB repair by NHEJ in human cells. Inhibition or depletion of the AAA+-ATPase p97 significantly delays the removal of Ku80 from the DNA after IR. Together with data from pulsed-field gel-electrophoresis and in vitro studies, this data demonstrates that p97 removes Ku from the DNA after successful ligation of the break. Furthermore, co-depletion of the p97 adapter proteins Ufd1 and FAF1 exhibited a synergistic effect on Ku removal from the DNA. In contrast, this study reveals that p97 inhibition blocks end resection after CPT treatment but not after IR. These data implicate that Ku removal from DNA by p97 is not required for proper end resection. Nevertheless, upon IR, p97 was found to act at the step of Rad51 filament formation during HRR. After IR, the formation of Rad51 filaments was reduced by p97 inhibition or depletion, but notably not the preceding steps of ...
Analysis of the functions of the AAA-ATPase p97 in DNA double-strand break repair ; Analyse der Funktionen der AAA-ATPase p97 in DNA-Doppelstrangbruchreparatur
Ionizing radiation (IR) can cause DNA double-strand breaks (DSBs) in the cellular genome, which result in genomic instability, if left unrepaired. The two major repair pathways to cope with DSBs are non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). DSB repair by NHEJ is initiated by the Ku heterodimer that binds the open DNA ends and recruits all factors needed for ligation to the break. Each subunit of the Ku molecule fully encircles the DNA and is therefore trapped by ligation of the DSB. To restore the DNA integrity, Ku needs to be actively removed after successful repair by NHEJ. In addition, it was claimed that Ku blocks end resection and therefore needs to be removed from open DNA ends to enable HRR. If a DSB is repaired by HRR, the 5’ ends are extensively resected and the single-stranded DNA regions are covered by RPA. The DNA-bound RPA can be phosphorylated by DNA-PK, before it is replaced by Rad51 filaments. The Rad51 filaments mediate the search for the homologous sister chromatid for template-based repair. In this study, clear evidence was found for p97 removing sterically trapped Ku80 from the DNA after successful DSB repair by NHEJ in human cells. Inhibition or depletion of the AAA+-ATPase p97 significantly delays the removal of Ku80 from the DNA after IR. Together with data from pulsed-field gel-electrophoresis and in vitro studies, this data demonstrates that p97 removes Ku from the DNA after successful ligation of the break. Furthermore, co-depletion of the p97 adapter proteins Ufd1 and FAF1 exhibited a synergistic effect on Ku removal from the DNA. In contrast, this study reveals that p97 inhibition blocks end resection after CPT treatment but not after IR. These data implicate that Ku removal from DNA by p97 is not required for proper end resection. Nevertheless, upon IR, p97 was found to act at the step of Rad51 filament formation during HRR. After IR, the formation of Rad51 filaments was reduced by p97 inhibition or depletion, but notably not the preceding steps of ...
Analysis of the functions of the AAA-ATPase p97 in DNA double-strand break repair ; Analyse der Funktionen der AAA-ATPase p97 in DNA-Doppelstrangbruchreparatur
Weimann, Lena Christin (author) / Meyer, Hemmo
2019-09-03
Theses
Electronic Resource
English
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