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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 turnover is accelerated upon irradiation.A. Cycloheximide (100 µg/mL) was added to MCF7 cells with or without irradiation (10 Gy) and total cell lysates were prepared in SDS containing buffer according to the indicated time course (0, 0.5 and 4 h) and analyzed by immunoblotting as indicated. B. 53BP1 protein signal in immunoblot in A was measured by densitometry in each sample.
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pone-0110522-g005: 53BP1 turnover is accelerated upon irradiation.A. Cycloheximide (100 µg/mL) was added to MCF7 cells with or without irradiation (10 Gy) and total cell lysates were prepared in SDS containing buffer according to the indicated time course (0, 0.5 and 4 h) and analyzed by immunoblotting as indicated. B. 53BP1 protein signal in immunoblot in A was measured by densitometry in each sample.

Mentions: Since 53BP1 protein abundance changed following irradiation, we speculated that irradiation shortened the 53BP1 protein half-life. Using MCF7 (Figure 5) or HeLa cells (Figure S2 in File S1), we blocked new protein synthesis by the addition of cycloheximide to tissue culture media and made whole cell lysates in 2% SDS containing buffer. In the absence of IR, protein levels were stable (Figure 5A, lanes 1–3). By contrast, following IR, 53BP1 turnover was apparent as early as 30 min post-IR, and the protein level decreased to 4% 4 hours post IR (Figure 5A, B). By contrast, RAD51 half-life was not affected by IR (Figure 5A). The results together implicated that ionizing radiation accelerates 53BP1 protein turnover via the proteasome-dependent pathway.


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 turnover is accelerated upon irradiation.A. Cycloheximide (100 µg/mL) was added to MCF7 cells with or without irradiation (10 Gy) and total cell lysates were prepared in SDS containing buffer according to the indicated time course (0, 0.5 and 4 h) and analyzed by immunoblotting as indicated. B. 53BP1 protein signal in immunoblot in A was measured by densitometry in each sample.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110522-g005: 53BP1 turnover is accelerated upon irradiation.A. Cycloheximide (100 µg/mL) was added to MCF7 cells with or without irradiation (10 Gy) and total cell lysates were prepared in SDS containing buffer according to the indicated time course (0, 0.5 and 4 h) and analyzed by immunoblotting as indicated. B. 53BP1 protein signal in immunoblot in A was measured by densitometry in each sample.
Mentions: Since 53BP1 protein abundance changed following irradiation, we speculated that irradiation shortened the 53BP1 protein half-life. Using MCF7 (Figure 5) or HeLa cells (Figure S2 in File S1), we blocked new protein synthesis by the addition of cycloheximide to tissue culture media and made whole cell lysates in 2% SDS containing buffer. In the absence of IR, protein levels were stable (Figure 5A, lanes 1–3). By contrast, following IR, 53BP1 turnover was apparent as early as 30 min post-IR, and the protein level decreased to 4% 4 hours post IR (Figure 5A, B). By contrast, RAD51 half-life was not affected by IR (Figure 5A). The results together implicated that ionizing radiation accelerates 53BP1 protein turnover via the proteasome-dependent pathway.

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