Limits...
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.

Show MeSH

Related in: MedlinePlus

TOPBP1 is a novel TOP2A interacting protein that localises to UFBs(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 μg ml−1 for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4374157&req=5

Figure 1: TOPBP1 is a novel TOP2A interacting protein that localises to UFBs(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 μg ml−1 for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 μm.

Mentions: To understand the mechanism of TOP2A recruitment to UFBs we undertook an unbiased proteomic approach to map TOP2A’s protein-protein interactions. To this end, we performed immuno-precipitation (IP) experiments followed by mass spectrometry (IP/MS) analysis of TOP2A-associated proteins from HEK293 cells enriched in G2/M phase. Among known interactors including TOP1 17, XRCC5 18, the FACT complex subunit SSRP1 19, Aurora B kinase 20, HDAC1 21, DDX21 19 and CDC5L 22 we also identified TOPBP1 as a novel putative TOP2A binding partner (Fig. 1a). Given that TOPBP1/Dpb11 has been recently shown to localise to ultra-fine anaphase bridges we set out to validate this interaction 16. First, we immunoprecipitated GFP-TOPBP1 from nocodazole-treated or asynchronous U2OS cells stably expressing this fusion protein at a level similar to that of the endogenous protein (Fig. 1b). We could readily detect endogenous TOP2A by Western Blotting of GFP-TOPBP1 immunoprecipitates in G2/M enriched cells (Fig. 1b). Accordingly, endogenous TOPBP1 was detected in reciprocal GFP-TOP2A immunoprecipitates from cell extracts (Fig. 1c), thus confirming that the two proteins likely exist in the same complex in cells. Enrichment in G2/M phase in all cases was determined by FACS analysis (Supplementary Fig. 1a). Thus far TOPBP1 localisation to ultra-fine anaphase bridges (UFBs) has been reported only in yeast Saccharomyces cerevisiae and the transformed avian B-lymphoblastoid cell line DT40 16. Therefore, we decided to test if this was also the case in human transformed and importantly, primary cells. We found by antibody staining and immunofluorescence microscopy that in U2OS cells undergoing mitosis TOPBP1 protein localised to “thread-like” structures forming a bridge linking the daughter DNA masses in anaphase and early telophase cells (Fig. 1d). To verify that these were indeed UFBs, we counterstained the cells with an antibody directed against BLM, a known marker of UFBs, where a subset of TOPBP1 bridges also stained for BLM on the same bridge (Fig. 1e). Next, we validated that this localisation was not an antibody-based artifact by reproducing this observation in U2OS cells stably expressing GFP-TOPBP1 (Supplementary Fig. 1b). We also confirmed this observation in another human cell line (HeLa cells, Fig. 1f) and importantly, in a human primary cell line (human dermal fibroblasts (HDF), Fig 1g). Taken together, this data supports the notion that TOPBP1 plays an evolutionarily conserved role in sister chromatid disjunction, and importantly demonstrates that this localisation is likely to represent a physiological process associated with normal cell division. Interestingly, TOPBP1 seemed to stain only a part of the bridge and its co-localisation with BLM is rare (Fig. 1e).


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 is a novel TOP2A interacting protein that localises to UFBs(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 μg ml−1 for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4374157&req=5

Figure 1: TOPBP1 is a novel TOP2A interacting protein that localises to UFBs(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 μg ml−1 for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 μg ml−1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 μm.
Mentions: To understand the mechanism of TOP2A recruitment to UFBs we undertook an unbiased proteomic approach to map TOP2A’s protein-protein interactions. To this end, we performed immuno-precipitation (IP) experiments followed by mass spectrometry (IP/MS) analysis of TOP2A-associated proteins from HEK293 cells enriched in G2/M phase. Among known interactors including TOP1 17, XRCC5 18, the FACT complex subunit SSRP1 19, Aurora B kinase 20, HDAC1 21, DDX21 19 and CDC5L 22 we also identified TOPBP1 as a novel putative TOP2A binding partner (Fig. 1a). Given that TOPBP1/Dpb11 has been recently shown to localise to ultra-fine anaphase bridges we set out to validate this interaction 16. First, we immunoprecipitated GFP-TOPBP1 from nocodazole-treated or asynchronous U2OS cells stably expressing this fusion protein at a level similar to that of the endogenous protein (Fig. 1b). We could readily detect endogenous TOP2A by Western Blotting of GFP-TOPBP1 immunoprecipitates in G2/M enriched cells (Fig. 1b). Accordingly, endogenous TOPBP1 was detected in reciprocal GFP-TOP2A immunoprecipitates from cell extracts (Fig. 1c), thus confirming that the two proteins likely exist in the same complex in cells. Enrichment in G2/M phase in all cases was determined by FACS analysis (Supplementary Fig. 1a). Thus far TOPBP1 localisation to ultra-fine anaphase bridges (UFBs) has been reported only in yeast Saccharomyces cerevisiae and the transformed avian B-lymphoblastoid cell line DT40 16. Therefore, we decided to test if this was also the case in human transformed and importantly, primary cells. We found by antibody staining and immunofluorescence microscopy that in U2OS cells undergoing mitosis TOPBP1 protein localised to “thread-like” structures forming a bridge linking the daughter DNA masses in anaphase and early telophase cells (Fig. 1d). To verify that these were indeed UFBs, we counterstained the cells with an antibody directed against BLM, a known marker of UFBs, where a subset of TOPBP1 bridges also stained for BLM on the same bridge (Fig. 1e). Next, we validated that this localisation was not an antibody-based artifact by reproducing this observation in U2OS cells stably expressing GFP-TOPBP1 (Supplementary Fig. 1b). We also confirmed this observation in another human cell line (HeLa cells, Fig. 1f) and importantly, in a human primary cell line (human dermal fibroblasts (HDF), Fig 1g). Taken together, this data supports the notion that TOPBP1 plays an evolutionarily conserved role in sister chromatid disjunction, and importantly demonstrates that this localisation is likely to represent a physiological process associated with normal cell division. Interestingly, TOPBP1 seemed to stain only a part of the bridge and its co-localisation with BLM is rare (Fig. 1e).

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.

Show MeSH
Related in: MedlinePlus