Ali Bakr1, Sabrina Köcher1, Jennifer Volquardsen1, Rudolph Reimer2, Kerstin Borgmann1, Ekkehard Dikomey1, Kai Rothkamm1, Wael Y Mansour3. 1. Laboratory of Radiobiology and Experimental Radio-oncology, University Medical Center Hamburg Eppendorf, Germany. 2. Heinrich-Pette-Institute Leibniz-Institute for Experimental Virology, Hamburg, Germany. 3. Laboratory of Radiobiology and Experimental Radio-oncology, University Medical Center Hamburg Eppendorf, Germany; Tumor Biology Department, National Cancer Institute, Cairo University, Egypt. Electronic address: w.mansour@uke.de.
Abstract
PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.
PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. MATERIALS AND METHODS:Humantumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.