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

Schultz LB, Chehab NH, Malikzay A, Halazonetis TD - J. Cell Biol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

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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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.
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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.

Mentions: The availability of high-affinity antibodies allowed us to probe the intracellular localization of 53BP1 by immunofluorescence. In nonirradiated U2OS cells, 53BP1 showed diffuse staining of the nuclei with the exception of the nucleoli, which were not stained. After exposure to 8 Gy IR, 53BP1 relocalized to distinct nuclear foci. 53BP1 relocalization was evident with both antibody clones and no 53BP1 staining was observed in the absence of primary antibody (Fig. 3 A).


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

Schultz LB, Chehab NH, Malikzay A, Halazonetis TD - J. Cell Biol. (2000)

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.
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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.
Mentions: The availability of high-affinity antibodies allowed us to probe the intracellular localization of 53BP1 by immunofluorescence. In nonirradiated U2OS cells, 53BP1 showed diffuse staining of the nuclei with the exception of the nucleoli, which were not stained. After exposure to 8 Gy IR, 53BP1 relocalized to distinct nuclear foci. 53BP1 relocalization was evident with both antibody clones and no 53BP1 staining was observed in the absence of primary antibody (Fig. 3 A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

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.

Show MeSH
Related in: MedlinePlus