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Regulation of 53BP1 protein stability by RNF8 and RNF168 is important for efficient DNA double-strand break repair.

Hu Y, Wang C, Huang K, Xia F, Parvin JD, Mondal N - PLoS ONE (2014)

Bottom Line: In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168.Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form.Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.

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53BP1 is degraded except when bound to a damage site.A. upper panel shows the workflow of the experiment. 1 h after 10 Gy X-ray irradiation was applied to the HeLa cells, DMSO or MG132 (20 µM) was added to the media. At time points 1.5 h, 2 h, 3 h, and 5 h post-IR, cells were either fixed for microscopy (A, bottom) or lysed for immunoblot analysis (C). B. in each sample, the percentage of the cells that have diffuse 53BP1 stain (blue), 53BP1 foci (orange) or diffuse 53BP1 stain with foci (green) was quantified (mean ±SEM; N = 3). C. cell lysates taken from panel A were subjected to immunoblot for 53BP1 and RIF1 stain. Samples were treated with DMSO vehicle (D, even lanes) or MG132 (M, lanes 3, 5, 7, 9). Tubulin was a loading control.
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pone-0110522-g006: 53BP1 is degraded except when bound to a damage site.A. upper panel shows the workflow of the experiment. 1 h after 10 Gy X-ray irradiation was applied to the HeLa cells, DMSO or MG132 (20 µM) was added to the media. At time points 1.5 h, 2 h, 3 h, and 5 h post-IR, cells were either fixed for microscopy (A, bottom) or lysed for immunoblot analysis (C). B. in each sample, the percentage of the cells that have diffuse 53BP1 stain (blue), 53BP1 foci (orange) or diffuse 53BP1 stain with foci (green) was quantified (mean ±SEM; N = 3). C. cell lysates taken from panel A were subjected to immunoblot for 53BP1 and RIF1 stain. Samples were treated with DMSO vehicle (D, even lanes) or MG132 (M, lanes 3, 5, 7, 9). Tubulin was a loading control.

Mentions: Previous reports had suggested that 53BP1 is recruited to damage sites in an RNF8/RNF168 dependent manner [19], [22]. We speculated that after 53BP1 stably localizes and binds to DSBs at chromatin at time points as early as four minutes post-irradiation [22], degradation of 53BP1 rather than the protein movement to the sites of DSBs, leads to prominent 53BP1 focus formation. To differentiate between movement of the protein versus degradation of bulk 53BP1, we irradiated HeLa cells and after foci formed we blocked proteasome-mediated degradation. If 53BP1 protein relocated within the nucleus, then foci would remain even if the proteasome were blocked. If, on the other hand, the 53BP1 bound to the DNA damage site was stabilized then the foci would be surrounded by diffuse 53BP1 as new protein was synthesized. MG132 was added to the cells 1 hour post-irradiation and a series of times points were analyzed to observe the 53BP1 foci at the damage sites. (Refer to time-line in Figure 6A, top.) In the absence of the MG132, 53BP1 containing IRIF were apparent at all post-IR time-points analyzed. By comparison, in the cells in which MG132 was added to the medium one hour post-IR, foci were still apparent, but these IRIF were in the presence of diffuse 53BP1 stain at late time points (Figure 6A, bottom). As an indication of the diffuse 53BP1 localization, in the presence of MG132 the nucleoli become apparent as holes in the diffuse pattern. The results from this experiment were quantified in Figure 6B and show that MG132 treated cells primarily had diffuse nuclear 53BP1 stain or diffuse stain with foci, suggesting that degradation event in the nucleoplasm happened prior to MG132 treatment, and the diffuse stain was due to the appearance of newly synthesized 53BP1. When MG132 was added to the cells prior to IR, no foci were apparent (Figure 3B). These results are most consistent with a model in which 53BP1 is continuously synthesized at a high rate, and post-IR it is rapidly degraded via the ubiquitin-proteasome system (Figure 5A, B). Only at sites of DNA damage is it protected from ubiquitin-dependent degradation. Immunoblot results of 53BP1 protein from experiment in Figure 6A, top were consistent with the notion that 53BP1 accumulated to high levels when in the presence of MG132 (Figure 6C). By comparison, the concentration of the downstream effector RIF1 did not change following DNA damage (Figure 6C). Together, these data suggest that following ionizing radiation, 53BP1 is rapidly synthesized and rapidly degraded except when bound to repair sites.


Regulation of 53BP1 protein stability by RNF8 and RNF168 is important for efficient DNA double-strand break repair.

Hu Y, Wang C, Huang K, Xia F, Parvin JD, Mondal N - PLoS ONE (2014)

53BP1 is degraded except when bound to a damage site.A. upper panel shows the workflow of the experiment. 1 h after 10 Gy X-ray irradiation was applied to the HeLa cells, DMSO or MG132 (20 µM) was added to the media. At time points 1.5 h, 2 h, 3 h, and 5 h post-IR, cells were either fixed for microscopy (A, bottom) or lysed for immunoblot analysis (C). B. in each sample, the percentage of the cells that have diffuse 53BP1 stain (blue), 53BP1 foci (orange) or diffuse 53BP1 stain with foci (green) was quantified (mean ±SEM; N = 3). C. cell lysates taken from panel A were subjected to immunoblot for 53BP1 and RIF1 stain. Samples were treated with DMSO vehicle (D, even lanes) or MG132 (M, lanes 3, 5, 7, 9). Tubulin was a loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206297&req=5

pone-0110522-g006: 53BP1 is degraded except when bound to a damage site.A. upper panel shows the workflow of the experiment. 1 h after 10 Gy X-ray irradiation was applied to the HeLa cells, DMSO or MG132 (20 µM) was added to the media. At time points 1.5 h, 2 h, 3 h, and 5 h post-IR, cells were either fixed for microscopy (A, bottom) or lysed for immunoblot analysis (C). B. in each sample, the percentage of the cells that have diffuse 53BP1 stain (blue), 53BP1 foci (orange) or diffuse 53BP1 stain with foci (green) was quantified (mean ±SEM; N = 3). C. cell lysates taken from panel A were subjected to immunoblot for 53BP1 and RIF1 stain. Samples were treated with DMSO vehicle (D, even lanes) or MG132 (M, lanes 3, 5, 7, 9). Tubulin was a loading control.
Mentions: Previous reports had suggested that 53BP1 is recruited to damage sites in an RNF8/RNF168 dependent manner [19], [22]. We speculated that after 53BP1 stably localizes and binds to DSBs at chromatin at time points as early as four minutes post-irradiation [22], degradation of 53BP1 rather than the protein movement to the sites of DSBs, leads to prominent 53BP1 focus formation. To differentiate between movement of the protein versus degradation of bulk 53BP1, we irradiated HeLa cells and after foci formed we blocked proteasome-mediated degradation. If 53BP1 protein relocated within the nucleus, then foci would remain even if the proteasome were blocked. If, on the other hand, the 53BP1 bound to the DNA damage site was stabilized then the foci would be surrounded by diffuse 53BP1 as new protein was synthesized. MG132 was added to the cells 1 hour post-irradiation and a series of times points were analyzed to observe the 53BP1 foci at the damage sites. (Refer to time-line in Figure 6A, top.) In the absence of the MG132, 53BP1 containing IRIF were apparent at all post-IR time-points analyzed. By comparison, in the cells in which MG132 was added to the medium one hour post-IR, foci were still apparent, but these IRIF were in the presence of diffuse 53BP1 stain at late time points (Figure 6A, bottom). As an indication of the diffuse 53BP1 localization, in the presence of MG132 the nucleoli become apparent as holes in the diffuse pattern. The results from this experiment were quantified in Figure 6B and show that MG132 treated cells primarily had diffuse nuclear 53BP1 stain or diffuse stain with foci, suggesting that degradation event in the nucleoplasm happened prior to MG132 treatment, and the diffuse stain was due to the appearance of newly synthesized 53BP1. When MG132 was added to the cells prior to IR, no foci were apparent (Figure 3B). These results are most consistent with a model in which 53BP1 is continuously synthesized at a high rate, and post-IR it is rapidly degraded via the ubiquitin-proteasome system (Figure 5A, B). Only at sites of DNA damage is it protected from ubiquitin-dependent degradation. Immunoblot results of 53BP1 protein from experiment in Figure 6A, top were consistent with the notion that 53BP1 accumulated to high levels when in the presence of MG132 (Figure 6C). By comparison, the concentration of the downstream effector RIF1 did not change following DNA damage (Figure 6C). Together, these data suggest that following ionizing radiation, 53BP1 is rapidly synthesized and rapidly degraded except when bound to repair sites.

Bottom Line: In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168.Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form.Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.

Show MeSH
Related in: MedlinePlus