Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1.
53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.
Bekker-Jensen
Simon
S
Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100, Copenhagen, Denmark.
Lukas
Claudia
C
Melander
Fredrik
F
Bartek
Jiri
J
Lukas
Jiri
J
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002843:Chromatin / Q000502:physiology; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D000071857:Tumor Suppressor p53-Binding Protein 1
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C092537:Ifi202b protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; D049452:Green Fluorescent Proteins