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TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

Broderick R, Nieminuszczy J, Blackford AN, Winczura A, Niedzwiedz W - Nat Commun (2015)

Bottom Line: Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci.Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion.These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

ABSTRACT
During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

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TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus(a) Schematic representation of GFP-TOPBP1 truncation mutants used in panel b. (b) TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus. HEK 293FT control cells, or cells transiently expressing WT GFP-TOPBP1 and indicated truncation mutants were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. (c) TOPBP1-TOP2A interaction requires BRCT 7/8. HEK 293FT control cells, or cells transiently expressing GFP-TOPBP1 WT or the indicated truncation mutant were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation.
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Figure 5: TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus(a) Schematic representation of GFP-TOPBP1 truncation mutants used in panel b. (b) TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus. HEK 293FT control cells, or cells transiently expressing WT GFP-TOPBP1 and indicated truncation mutants were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. (c) TOPBP1-TOP2A interaction requires BRCT 7/8. HEK 293FT control cells, or cells transiently expressing GFP-TOPBP1 WT or the indicated truncation mutant were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation.

Mentions: To gain further insight into the potential functional relevance of the TOPBP1-TOP2A interaction we designed a series of GFP-tagged TOPBP1 truncation constructs that sequentially removed single or tandem BRCT domains (Fig. 5a). We confirmed the efficacy of this approach by assaying their ability to interact with previously reported TOPBP1 binding partners (Supplementary Fig. 5a). Significantly, a TOPBP1 truncation mutant lacking the ATR-activation domain as well as the BRCT7 and 8 domains displayed no detectable binding to the endogenous TOP2A (Fig. 5b). To further map the minimal binding region we generated TOPBP1 construct lacking only BRCT7 and 8 and found that this mutant was also defective in its interaction with TOP2A (Fig. 5c and Supplementary Fig. 5b, BLM and FANCJ were used as internal controls for this construct). This suggests that the extreme C-terminus of TOPBP1 mediates its interaction with TOP2A.


TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

Broderick R, Nieminuszczy J, Blackford AN, Winczura A, Niedzwiedz W - Nat Commun (2015)

TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus(a) Schematic representation of GFP-TOPBP1 truncation mutants used in panel b. (b) TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus. HEK 293FT control cells, or cells transiently expressing WT GFP-TOPBP1 and indicated truncation mutants were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. (c) TOPBP1-TOP2A interaction requires BRCT 7/8. HEK 293FT control cells, or cells transiently expressing GFP-TOPBP1 WT or the indicated truncation mutant were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation.
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Figure 5: TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus(a) Schematic representation of GFP-TOPBP1 truncation mutants used in panel b. (b) TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus. HEK 293FT control cells, or cells transiently expressing WT GFP-TOPBP1 and indicated truncation mutants were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. (c) TOPBP1-TOP2A interaction requires BRCT 7/8. HEK 293FT control cells, or cells transiently expressing GFP-TOPBP1 WT or the indicated truncation mutant were nocodazole treated (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation.
Mentions: To gain further insight into the potential functional relevance of the TOPBP1-TOP2A interaction we designed a series of GFP-tagged TOPBP1 truncation constructs that sequentially removed single or tandem BRCT domains (Fig. 5a). We confirmed the efficacy of this approach by assaying their ability to interact with previously reported TOPBP1 binding partners (Supplementary Fig. 5a). Significantly, a TOPBP1 truncation mutant lacking the ATR-activation domain as well as the BRCT7 and 8 domains displayed no detectable binding to the endogenous TOP2A (Fig. 5b). To further map the minimal binding region we generated TOPBP1 construct lacking only BRCT7 and 8 and found that this mutant was also defective in its interaction with TOP2A (Fig. 5c and Supplementary Fig. 5b, BLM and FANCJ were used as internal controls for this construct). This suggests that the extreme C-terminus of TOPBP1 mediates its interaction with TOP2A.

Bottom Line: Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci.Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion.These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

ABSTRACT
During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

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