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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 degradation upon irradiation is regulated by RNF8 and RNF168.A. HeLa cells transfected with two different siRNAs (indicated in the grid) were treated with 10 Gy X-rays. 4 hours post-IR cell lysates were prepared for 53BP1 immunoblot analysis. RHA served as a loading control. B. Immunofluorescence microscopy analysis of cells from panel A were stained for 53BP1 (green) and γ-H2AX (red). C. distribution of pixel intensity was analyzed from microscopic images in panel B.
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pone-0110522-g004: 53BP1 degradation upon irradiation is regulated by RNF8 and RNF168.A. HeLa cells transfected with two different siRNAs (indicated in the grid) were treated with 10 Gy X-rays. 4 hours post-IR cell lysates were prepared for 53BP1 immunoblot analysis. RHA served as a loading control. B. Immunofluorescence microscopy analysis of cells from panel A were stained for 53BP1 (green) and γ-H2AX (red). C. distribution of pixel intensity was analyzed from microscopic images in panel B.

Mentions: RNF8 and RNF168 are E3 ubiquitin ligases that mediate the conjugation of ubiquitin multimers on histone H2A via the degradation-independent lysine-63 side-chain of ubiquitin and via this activity recruit other proteins, such as 53BP1 and BRCA1, to the sites of DNA damage [17], [19]–[22], [26]. We tested the possibility that these two enzymes are involved in 53BP1 protein degradation. Indeed, depletion of RNF8 or of RNF168 from HeLa cells and following irradiation-induced DNA damage, 53BP1 degradation was blocked (Figure 4A, lanes 6, 8, 10). Depletion of BRCA1, another E3 ubiquitin ligase involved in the DNA damage response, did not affect the 53BP1 protein level, indicating RNF8/RNF168 had a specific role in the control of 53BP1 protein levels upon irradiation (Figure 4A, lane 4). Consistent with the model that 53BP1 is degraded dependent on RNF8 and RNF168, the 53BP1 protein remained diffusely localized in the nucleus in HeLa cells in which these factors were depleted (Figure 4B). The distribution of pixel intensity in immunofluorescence images showed that depletion of RNF8 and/or RNF168 had a similar distribution pattern as no irradiation, though with more intense pixels (Figure 4C), suggesting that RNF8 and RNF168 regulate proteasome-dependent degradation and IRIF formation of 53BP1 in response to irradiation-induced DNA damage.


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 degradation upon irradiation is regulated by RNF8 and RNF168.A. HeLa cells transfected with two different siRNAs (indicated in the grid) were treated with 10 Gy X-rays. 4 hours post-IR cell lysates were prepared for 53BP1 immunoblot analysis. RHA served as a loading control. B. Immunofluorescence microscopy analysis of cells from panel A were stained for 53BP1 (green) and γ-H2AX (red). C. distribution of pixel intensity was analyzed from microscopic images in panel B.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110522-g004: 53BP1 degradation upon irradiation is regulated by RNF8 and RNF168.A. HeLa cells transfected with two different siRNAs (indicated in the grid) were treated with 10 Gy X-rays. 4 hours post-IR cell lysates were prepared for 53BP1 immunoblot analysis. RHA served as a loading control. B. Immunofluorescence microscopy analysis of cells from panel A were stained for 53BP1 (green) and γ-H2AX (red). C. distribution of pixel intensity was analyzed from microscopic images in panel B.
Mentions: RNF8 and RNF168 are E3 ubiquitin ligases that mediate the conjugation of ubiquitin multimers on histone H2A via the degradation-independent lysine-63 side-chain of ubiquitin and via this activity recruit other proteins, such as 53BP1 and BRCA1, to the sites of DNA damage [17], [19]–[22], [26]. We tested the possibility that these two enzymes are involved in 53BP1 protein degradation. Indeed, depletion of RNF8 or of RNF168 from HeLa cells and following irradiation-induced DNA damage, 53BP1 degradation was blocked (Figure 4A, lanes 6, 8, 10). Depletion of BRCA1, another E3 ubiquitin ligase involved in the DNA damage response, did not affect the 53BP1 protein level, indicating RNF8/RNF168 had a specific role in the control of 53BP1 protein levels upon irradiation (Figure 4A, lane 4). Consistent with the model that 53BP1 is degraded dependent on RNF8 and RNF168, the 53BP1 protein remained diffusely localized in the nucleus in HeLa cells in which these factors were depleted (Figure 4B). The distribution of pixel intensity in immunofluorescence images showed that depletion of RNF8 and/or RNF168 had a similar distribution pattern as no irradiation, though with more intense pixels (Figure 4C), suggesting that RNF8 and RNF168 regulate proteasome-dependent degradation and IRIF formation of 53BP1 in response to irradiation-induced DNA damage.

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