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Analysis of p97-mediated Ku80 extraction in DNA double-strand break repair in cells
DNA double strand breaks (DSBs) are the most deleterious kind of DNA damage and threaten genomic integrity. Therefore, DSBs need to be repaired to avoid chromosomal aberrations, cell death, and malignant transformation. Sophisticated repair mechanisms have evolved and at least three competing pathways, non-homologous end-joining (NHEJ), homologous recombination repair (HRR), and alternative end-joining can repair DSBs. To ensure successful DSB repair, the complex multistep repair pathways are tightly controlled and the ubiquitin-proteasome system (UPS) has a fundamental role in this regulation. The AAA+-ATPase p97 is a critical factor of the UPS and facilitates extraction and unfolding of ubiquitinated substrate proteins. A set of bifunctional adaptor proteins, which can bind to ubiquitin and to p97, act as cofactors and form a variety of active p97 complexes. Previous studies have identified that p97 localizes to DSBs and is involved in DSB repair by NHEJ and HRR, which are impaired upon loss of p97. However, the underlying mechanisms and the substrates were unknown. Therefore, we aimed at clarifying the role of p97 and its cofactor proteins in DSB repair. In the present study, p97 was established as factor for Ku80 extraction, which was a promising candidate from a mass spectrometry approach performed in our lab. Ku80 is part of the ring-shaped heterodimer Ku that is essential for NHEJ. Ku rings slide onto open DNA ends and fully encircle the double helix. This special structure and mode of DNA binding prevents dissociation from chromatin and Ku becomes sterically trapped after religation of the DSBs. Hence, extraction of trapped Ku from chromatin requires substantial unfolding by an active mechanism. In this study, cell-based assays provided strong evidence that p97 extracts Ku80 from chromatin and that this is the major role of p97 in NHEJ. Sophisticated immunofluorescence techniques were established and enabled the visualization and kinetic analysis of DSB-bound Ku80. Additionally, the functionality of p97 ...
Analysis of p97-mediated Ku80 extraction in DNA double-strand break repair in cells
DNA double strand breaks (DSBs) are the most deleterious kind of DNA damage and threaten genomic integrity. Therefore, DSBs need to be repaired to avoid chromosomal aberrations, cell death, and malignant transformation. Sophisticated repair mechanisms have evolved and at least three competing pathways, non-homologous end-joining (NHEJ), homologous recombination repair (HRR), and alternative end-joining can repair DSBs. To ensure successful DSB repair, the complex multistep repair pathways are tightly controlled and the ubiquitin-proteasome system (UPS) has a fundamental role in this regulation. The AAA+-ATPase p97 is a critical factor of the UPS and facilitates extraction and unfolding of ubiquitinated substrate proteins. A set of bifunctional adaptor proteins, which can bind to ubiquitin and to p97, act as cofactors and form a variety of active p97 complexes. Previous studies have identified that p97 localizes to DSBs and is involved in DSB repair by NHEJ and HRR, which are impaired upon loss of p97. However, the underlying mechanisms and the substrates were unknown. Therefore, we aimed at clarifying the role of p97 and its cofactor proteins in DSB repair. In the present study, p97 was established as factor for Ku80 extraction, which was a promising candidate from a mass spectrometry approach performed in our lab. Ku80 is part of the ring-shaped heterodimer Ku that is essential for NHEJ. Ku rings slide onto open DNA ends and fully encircle the double helix. This special structure and mode of DNA binding prevents dissociation from chromatin and Ku becomes sterically trapped after religation of the DSBs. Hence, extraction of trapped Ku from chromatin requires substantial unfolding by an active mechanism. In this study, cell-based assays provided strong evidence that p97 extracts Ku80 from chromatin and that this is the major role of p97 in NHEJ. Sophisticated immunofluorescence techniques were established and enabled the visualization and kinetic analysis of DSB-bound Ku80. Additionally, the functionality of p97 ...
Analysis of p97-mediated Ku80 extraction in DNA double-strand break repair in cells
Wolf, Markus Andreas (author) / Meyer, Hemmo
2021-06-02
Theses
Electronic Resource
English
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