Abstract
53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified ‘hits’: a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153–importin-β interplay. Our data define the structure–function relationships within this emerging 53BP1-NUP153/importin-β pathway and implicate this mechanism in the maintenance of genome integrity.
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Abbreviations
- 53BP1:
-
p53-Binding Protein 1
- ANAPC10:
-
anaphase-promoting complex subunit 10
- ANAPC4:
-
anaphase-promoting complex subunit 4
- APC/C:
-
anaphase-promoting complex/cyclosome
- APH:
-
aphidicolin
- ASB18:
-
ankyrin repeat and SOCS box protein 18
- ATM:
-
ataxia telangiectasia mutated
- ATR:
-
ataxia telangiectasia and Rad3-related protein
- ATXN7:
-
ataxin-7
- BRCA1:
-
breast cancer type 1 susceptibility protein
- DDR:
-
DNA damage response
- DNA-PK:
-
DNA-dependent protein kinase
- DSB:
-
Double strand breaks
- ECS, elongin, Cullin:
-
SOCS-box containing complex
- FANCD2:
-
Fanconi anemia group D2 protein
- FG repeat:
-
phenylalanine-glycine repeat
- GFP:
-
green fluorescent protein
- γH2AX:
-
phosphorylated histone H2AX at serine 139
- HeLa:
-
human adenocarcinoma cell line
- HERC2:
-
HECT domain and RCC1-like domain-containing protein 2
- IR:
-
ionizing radiation
- MAP3K3:
-
mitogen-activated protein kinase kinase kinase 3
- MDC1:
-
mediator of DNA damage checkpoint protein 1
- Mec1:
-
Mitosis entry checkpoint protein 1
- MMSET:
-
multiple myeloma SET domain-containing protein
- NBS1:
-
Nijmegen breakage syndrome protein 1
- NPC:
-
nuclear pore complex
- NUP:
-
nucleoporin
- NUP153:
-
nucleoporin 153kDa protein
- NUP84:
-
nucleoporin 84kDa protein
- NUP93:
-
nucleoporin 93kDa protein
- PARP:
-
poly [ADP-ribose] polymerase
- PIAS4:
-
protein inhibitor of activated STAT protein 4
- Rad51:
-
DNA repair protein RAD51
- Rad53:
-
Serine/threonine-protein kinase RAD53
- RNF8:
-
RING finger protein 8
- RNF168:
-
RING finger protein 168
- SAGA:
-
Spt-Ada-Gcn5 acetyltransferase
- SCF, Skp, Cullin:
-
F-box containing complex
- SEC13L1:
-
SEC13-like protein 1
- SET8:
-
SET domain-containing protein 8
- Slx5:
-
synthetic lethal of unknown function protein 5
- Slx8:
-
synthetic lethal of unknown function protein 8
- TopBP1:
-
DNA topoisomerase 2-binding protein 1
- U2OS:
-
human osteosarcoma cell line
- UBA1:
-
ubiquitin-activating enzyme E1
- UBC9:
-
ubiquitin-conjugating enzyme 9
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Acknowledgements
We thank Dr. Ullman (University of Utah) and Dr. Durocher (University of Toronto) for reagents. This study was supported by Grant Agency of the Czech Republic (301/08/0353, P301/10/1525 and P305/10/P420), Institutional Grant (AV0Z5039906), the Danish Cancer Society, the Danish National Research Foundation, Novo Nordisk (R153-A12997), the Lundbeck Foundation (R-44-A4400) and the European Commission (projects TRIREME, Infla-Care, DDResponse and CZ.1.05/2.1.00/01.0030). PM was supported by Charles University in Prague. All original images from the screens are available at Mitocheck website (http://mitocheck.org/).
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Moudry, P., Lukas, C., Macurek, L. et al. Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1. Cell Death Differ 19, 798–807 (2012). https://doi.org/10.1038/cdd.2011.150
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DOI: https://doi.org/10.1038/cdd.2011.150
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