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. 2000 Dec 25;151(7):1381-90.
doi: 10.1083/jcb.151.7.1381.

p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks

L B Schultz  1 , N H Chehab, A Malikzay, T D Halazonetis
Affiliations

p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks

L B Schultz et al. J Cell Biol. .

Abstract

p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5-15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs.

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Figures

Figure 1
Figure 1
Alignment of the amino acid sequences of the BRCT domains of S. cerevisiae Rad9p, S. pombe Crb2p/Rhp9p (Crb2p), C. elegans T05F1 ORF, human 53BP1, and human KIAA0170 ORF (KIAA). The boxes indicate residues of 53BP1 and KIAA0170 ORF that are identical with Rad9p, Crb2p/Rhp9p, or T05F1 ORF residues. Residue positions are indicated on either side of the amino acid sequences. The termination codons are indicated by asterisks. Shaded numbers within the sequences refer to sequences of the indicated length that are not shown, because they are present only within one member.
Figure 5
Figure 5
Effect of wortmannin (Wort) and caffeine (Caff) on 53BP1 relocalization in response to irradiation. (A) Average number of 53BP1 foci per cell. (B) Fraction of cells with foci. Cells treated with 20 mM wortmannin, 2 mM caffeine, or untreated (Ctrl) cells were exposed to 1 Gy IR and 53BP1 relocalization was examined at the indicated times. 50–100 cells were counted per data point. Bars indicate standard errors in three different experiments.
Figure 2
Figure 2
Specificity of 53BP1-reactive monoclonal antibodies (MAb). Nuclear extracts of nontransfected cells and whole cell extracts from cells stably expressing HA-tagged full-length or truncated 53BP1 proteins were immunoblotted (IB) with a 53BP1-reactive monoclonal antibody (clone WI1) at a 1:20 dilution or an HA tag–reactive polyclonal antibody (PAb; clone Y11) used at a 1:1,000 dilution. Endog., endogenous.
Figure 3
Figure 3
Relocalization of 53BP1 in response to IR. (A) Immunofluorescence of nonirradiated (0 Gy) and irradiated (8 Gy) U2OS cells with 53BP1-reactive monoclonal antibodies (clones WI1 and WI4) or with no primary antibody (MAb −). α-53BP1, 53BP1 immunofluorescence; DAPI, DAPI fluorescence marking the cell nuclei; α-53BP1 + DAPI-P, merging of the α-53BP1 immunofluorescence and processed DAPI image (DAPI-P) that shows only the periphery of the nucleus. (B) Relocalization of HA-tagged full-length 53BP1 in stably transfected U2OS cells using an HA tag–reactive monoclonal antibody (clone B11). (C) Relocalization of endogenous 53BP1 in nontransfected U2OS cells exposed to etoposide (ETOP), neocarzinostatin (NCS), UV light, or HU. The cells exposed to UV light were examined 1 or 6 h after irradiation.
Figure 4
Figure 4
Dose dependence and time course of 53BP1 relocalization. (A and B) Dose dependence: (A) immunofluorescence images of U2OS cells exposed to the indicated dose of IR and examined 15-min later for 53BP1 relocalization; (B) average number of 53BP1 foci per cell calculated by counting 50–100 cells per data point. (C–E) Time course: (C) immunofluorescence images of U2OS cells exposed to 1 Gy IR and examined at the indicated times after irradiation; (D) average number of 53BP1 foci per cell; (E) fraction of cells with foci. 50–100 cells were counted per data point. Bars indicate standard errors in three different experiments.
Figure 6
Figure 6
Colocalization of 53BP1 with γ-H2AX and Mre11/NBS1 IR-induced foci. Nonirradiated (0 Gy) and irradiated (1 or 8 Gy) U2OS cells were stained by immunofluorescence with antibodies that recognize 53BP1 (clone WI1, 1:20 dilution), γ-H2AX (1:800 dilution of polyclonal antibody), Mre11 (1:1,000 dilution of polyclonal antibody), or NBS1 (1:1,000 dilution of polyclonal antibody) as indicated. As a control, cells were also stained with antibodies to PML (1:400 dilution of polyclonal antibody) to mark the nuclear domain 10 (ND10) nuclear structures.
Figure 7
Figure 7
53BP1 focus formation in cells deficient in ATM, NBS1, or DNA-PK. 53BP1 relocalization was studied in nontransformed fibroblasts from a normal individual (AG1522), from patients with A-T (AT1BR and AT5BI), and from patients with NBS (NBS7, NBS780816; and NBS8, NBS880823) and in MO59K and MO59J glioblastoma cells.
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