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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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TOP2A localises to UFBs(a) Schematic describing the proximity ligation assay (PLA) employed to visualise TOPBP1-TOP2A co-localisation on UFBs. (b) Representative image of PLA assay carried out using antibodies raised against endogenous TOPBP1 and TOP2A in U2OS cells stably expressing GFP-TOPBP1 WT. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. PLA signal co-localisation with a UFB is highlighted in the zoomed section. Scale bar = 6.4 μm. (c) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 UFBs. “Control” represents signal obtained for PLA assay when using only one of the antibodies (TOP2A antibody). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis. (d) Representative image of PLA assay carried out using antibodies raised against GFP and TOP2A in U2OS cells transiently expressing GFP-TOPBP1 WT or BRCT7/8 truncation mutant. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. Scale bar = 6.4 μm. (e) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 WT and BRCT7/8 truncation mutant. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis and the Chi-square test was used to determine statistical significance.
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Figure 6: TOP2A localises to UFBs(a) Schematic describing the proximity ligation assay (PLA) employed to visualise TOPBP1-TOP2A co-localisation on UFBs. (b) Representative image of PLA assay carried out using antibodies raised against endogenous TOPBP1 and TOP2A in U2OS cells stably expressing GFP-TOPBP1 WT. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. PLA signal co-localisation with a UFB is highlighted in the zoomed section. Scale bar = 6.4 μm. (c) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 UFBs. “Control” represents signal obtained for PLA assay when using only one of the antibodies (TOP2A antibody). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis. (d) Representative image of PLA assay carried out using antibodies raised against GFP and TOP2A in U2OS cells transiently expressing GFP-TOPBP1 WT or BRCT7/8 truncation mutant. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. Scale bar = 6.4 μm. (e) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 WT and BRCT7/8 truncation mutant. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis and the Chi-square test was used to determine statistical significance.

Mentions: Next to address whether TOPBP1 promotes recruitment of TOP2A to UFBs in order to promote their decatenation, it was important to establish if TOP2A and TOPBP1 co-localise on these structures. To this end, we optimised the recently developed Proximity Ligation Assay (PLA) that allows detection of protein-protein interactions at the single molecule level by immunofluorescence microscopy 35. Using this approach and employing antibodies directed against endogenous TOPBP1 and TOP2A we were able to visualise co-localisation of TOPBP1 and TOP2A on UFBs (Fig. 6a and b; Supplementary Fig. 7a; these cells stably express GFP-TOPBP1, which serves as a marker of UFBs). Importantly, the majority of GFP-TOPBP1 UFBs observed were also associated with the PLA signal (co-localising TOP2A-TOPBP1) (Fig. 6c). No PLA signal was observed in the negative control (Fig. 6c). Moreover, and in support of our hypothesis the TOPBP1 BRCT7/8 truncation mutant unable to bind TOP2A (Fig. 5c) showed defective co-localisation of TOPBP1-TOP2A on UFBs as assayed by the PLA technique (Fig. 6d and e). To our knowledge this is the first time that TOP2A localisation to UFBs has been observed. Given the above and the facts that (i) these two proteins co-immunoprecipitate in cell enriched in G2/M phase of the cell cycle and (ii) TOPBP1 depletion leads to the accumulation of C-UFBs we hypothesised that TOPBP1 promotes recruitment of TOP2A to UFBs thereby aiding their resolution.


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)

TOP2A localises to UFBs(a) Schematic describing the proximity ligation assay (PLA) employed to visualise TOPBP1-TOP2A co-localisation on UFBs. (b) Representative image of PLA assay carried out using antibodies raised against endogenous TOPBP1 and TOP2A in U2OS cells stably expressing GFP-TOPBP1 WT. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. PLA signal co-localisation with a UFB is highlighted in the zoomed section. Scale bar = 6.4 μm. (c) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 UFBs. “Control” represents signal obtained for PLA assay when using only one of the antibodies (TOP2A antibody). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis. (d) Representative image of PLA assay carried out using antibodies raised against GFP and TOP2A in U2OS cells transiently expressing GFP-TOPBP1 WT or BRCT7/8 truncation mutant. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. Scale bar = 6.4 μm. (e) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 WT and BRCT7/8 truncation mutant. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis and the Chi-square test was used to determine statistical significance.
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Figure 6: TOP2A localises to UFBs(a) Schematic describing the proximity ligation assay (PLA) employed to visualise TOPBP1-TOP2A co-localisation on UFBs. (b) Representative image of PLA assay carried out using antibodies raised against endogenous TOPBP1 and TOP2A in U2OS cells stably expressing GFP-TOPBP1 WT. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. PLA signal co-localisation with a UFB is highlighted in the zoomed section. Scale bar = 6.4 μm. (c) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 UFBs. “Control” represents signal obtained for PLA assay when using only one of the antibodies (TOP2A antibody). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis. (d) Representative image of PLA assay carried out using antibodies raised against GFP and TOP2A in U2OS cells transiently expressing GFP-TOPBP1 WT or BRCT7/8 truncation mutant. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. Scale bar = 6.4 μm. (e) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 WT and BRCT7/8 truncation mutant. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis and the Chi-square test was used to determine statistical significance.
Mentions: Next to address whether TOPBP1 promotes recruitment of TOP2A to UFBs in order to promote their decatenation, it was important to establish if TOP2A and TOPBP1 co-localise on these structures. To this end, we optimised the recently developed Proximity Ligation Assay (PLA) that allows detection of protein-protein interactions at the single molecule level by immunofluorescence microscopy 35. Using this approach and employing antibodies directed against endogenous TOPBP1 and TOP2A we were able to visualise co-localisation of TOPBP1 and TOP2A on UFBs (Fig. 6a and b; Supplementary Fig. 7a; these cells stably express GFP-TOPBP1, which serves as a marker of UFBs). Importantly, the majority of GFP-TOPBP1 UFBs observed were also associated with the PLA signal (co-localising TOP2A-TOPBP1) (Fig. 6c). No PLA signal was observed in the negative control (Fig. 6c). Moreover, and in support of our hypothesis the TOPBP1 BRCT7/8 truncation mutant unable to bind TOP2A (Fig. 5c) showed defective co-localisation of TOPBP1-TOP2A on UFBs as assayed by the PLA technique (Fig. 6d and e). To our knowledge this is the first time that TOP2A localisation to UFBs has been observed. Given the above and the facts that (i) these two proteins co-immunoprecipitate in cell enriched in G2/M phase of the cell cycle and (ii) TOPBP1 depletion leads to the accumulation of C-UFBs we hypothesised that TOPBP1 promotes recruitment of TOP2A to UFBs thereby aiding their resolution.

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