Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of 53BP1 degradation causes failure to recruit RIF1 to the DSB sites.A. HeLa cells were treated with or without MG132 (20 µM) before exposure to 10 Gy X- irradiation. 4 h post-IR, cells were fixed for immunofluorescence microscopy as indicated. B. different treatments were applied to HeLa cells and immunoblot for RIF1 was done. Ctrl, no treatment (lane 1) or4 h post 10 Gy-IR (lanes 2–7). Additional treatments included caffeine (10 mM; lane 3) MG132 (20 µM; lane 4), siRNA specific for RNF8 (lane 5), siRNA specific for RNF168 (lane 6), and mixed siRNA specific for both RNF8 and RNF168 (lane 7). C. MG132 (20 µM) was included in medium 30 minutes prior to exposure to 10 Gy X-ray irradiation. At 4 h post-IR, HeLa cells were extracted in situ with cell extraction buffer (Material and Methods) on ice for 15 min (+NP40) or not extracted. Cells were fixed and stained for immunofluorescence microscopy as above.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206297&req=5

pone-0110522-g007: Inhibition of 53BP1 degradation causes failure to recruit RIF1 to the DSB sites.A. HeLa cells were treated with or without MG132 (20 µM) before exposure to 10 Gy X- irradiation. 4 h post-IR, cells were fixed for immunofluorescence microscopy as indicated. B. different treatments were applied to HeLa cells and immunoblot for RIF1 was done. Ctrl, no treatment (lane 1) or4 h post 10 Gy-IR (lanes 2–7). Additional treatments included caffeine (10 mM; lane 3) MG132 (20 µM; lane 4), siRNA specific for RNF8 (lane 5), siRNA specific for RNF168 (lane 6), and mixed siRNA specific for both RNF8 and RNF168 (lane 7). C. MG132 (20 µM) was included in medium 30 minutes prior to exposure to 10 Gy X-ray irradiation. At 4 h post-IR, HeLa cells were extracted in situ with cell extraction buffer (Material and Methods) on ice for 15 min (+NP40) or not extracted. Cells were fixed and stained for immunofluorescence microscopy as above.

Mentions: RIF1 is the only known DNA damage repair factor that requires 53BP1 for its recruitment to damage sites [45], and which indirectly depends on RNF8 and RNF168 [46]. We tested if inhibition of 53BP1 protein degradation affects RIF1 association with IRIF. In contrast to 53BP1, irradiation did not affect RIF1 protein levels detected from immunoblots of lysates from HeLa cells (Figure 7B, lane 2). Inclusion of MG132 abolished RIF1 association with IRIF in HeLa cells (Figure 7A) without changing its protein level (Figure 7B, lane 4). Similarly, caffeine or RNF8 and/or RNF168 depletion did not affect RIF1 abundance (Figure 7B, lane 3, 5–7 and [45]).


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)

Inhibition of 53BP1 degradation causes failure to recruit RIF1 to the DSB sites.A. HeLa cells were treated with or without MG132 (20 µM) before exposure to 10 Gy X- irradiation. 4 h post-IR, cells were fixed for immunofluorescence microscopy as indicated. B. different treatments were applied to HeLa cells and immunoblot for RIF1 was done. Ctrl, no treatment (lane 1) or4 h post 10 Gy-IR (lanes 2–7). Additional treatments included caffeine (10 mM; lane 3) MG132 (20 µM; lane 4), siRNA specific for RNF8 (lane 5), siRNA specific for RNF168 (lane 6), and mixed siRNA specific for both RNF8 and RNF168 (lane 7). C. MG132 (20 µM) was included in medium 30 minutes prior to exposure to 10 Gy X-ray irradiation. At 4 h post-IR, HeLa cells were extracted in situ with cell extraction buffer (Material and Methods) on ice for 15 min (+NP40) or not extracted. Cells were fixed and stained for immunofluorescence microscopy as above.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110522-g007: Inhibition of 53BP1 degradation causes failure to recruit RIF1 to the DSB sites.A. HeLa cells were treated with or without MG132 (20 µM) before exposure to 10 Gy X- irradiation. 4 h post-IR, cells were fixed for immunofluorescence microscopy as indicated. B. different treatments were applied to HeLa cells and immunoblot for RIF1 was done. Ctrl, no treatment (lane 1) or4 h post 10 Gy-IR (lanes 2–7). Additional treatments included caffeine (10 mM; lane 3) MG132 (20 µM; lane 4), siRNA specific for RNF8 (lane 5), siRNA specific for RNF168 (lane 6), and mixed siRNA specific for both RNF8 and RNF168 (lane 7). C. MG132 (20 µM) was included in medium 30 minutes prior to exposure to 10 Gy X-ray irradiation. At 4 h post-IR, HeLa cells were extracted in situ with cell extraction buffer (Material and Methods) on ice for 15 min (+NP40) or not extracted. Cells were fixed and stained for immunofluorescence microscopy as above.
Mentions: RIF1 is the only known DNA damage repair factor that requires 53BP1 for its recruitment to damage sites [45], and which indirectly depends on RNF8 and RNF168 [46]. We tested if inhibition of 53BP1 protein degradation affects RIF1 association with IRIF. In contrast to 53BP1, irradiation did not affect RIF1 protein levels detected from immunoblots of lysates from HeLa cells (Figure 7B, lane 2). Inclusion of MG132 abolished RIF1 association with IRIF in HeLa cells (Figure 7A) without changing its protein level (Figure 7B, lane 4). Similarly, caffeine or RNF8 and/or RNF168 depletion did not affect RIF1 abundance (Figure 7B, lane 3, 5–7 and [45]).

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