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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 inhibition or depletion results in increased TOPBP1 localisation to UFBs(a and b) Quantification of the percentage of ana/telophase cells staining positive for TOPBP1 localisation to UFBs in U2OS cells treated for 24h with DMSO as a control, or with 0.4 μM aphidicolin (APH) or 10 μM razoxane (RAZ). Bars represent mean values from three independent experiments +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (c) Quantification of the percentage of U2OS ana/telophase cells staining positive for BLM-UFBs in cells treated for 24h with DMSO as a control or with 0.4 μM aphidicolin (APH). Bars represent mean values from three independent experiments +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (d) Quantification of the percentage of U2OS cells positive for TOPBP1-UFBs in cells treated with siRNA targeting TOP2A or in control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (e and f) Quantification of the percentage of U2OS cells positive for BLM-UFBs and DAPI-positive anaphase bridges, respectively cells treated with siRNA directed against TOP2A or control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance.
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Figure 3: TOP2A inhibition or depletion results in increased TOPBP1 localisation to UFBs(a and b) Quantification of the percentage of ana/telophase cells staining positive for TOPBP1 localisation to UFBs in U2OS cells treated for 24h with DMSO as a control, or with 0.4 μM aphidicolin (APH) or 10 μM razoxane (RAZ). Bars represent mean values from three independent experiments +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (c) Quantification of the percentage of U2OS ana/telophase cells staining positive for BLM-UFBs in cells treated for 24h with DMSO as a control or with 0.4 μM aphidicolin (APH). Bars represent mean values from three independent experiments +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (d) Quantification of the percentage of U2OS cells positive for TOPBP1-UFBs in cells treated with siRNA targeting TOP2A or in control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (e and f) Quantification of the percentage of U2OS cells positive for BLM-UFBs and DAPI-positive anaphase bridges, respectively cells treated with siRNA directed against TOP2A or control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance.

Mentions: UFBs can arise as a result of either decatenation or replication problems 1,9. Given that we identify TOPBP1 as a novel TOP2A interactor, and that inhibition of TOP2A leads to an increase in centromeric associated UFBs 3,9 we hypothesised that TOPBP1 may localise to this sub-type of UFBs. To test this, we analysed the frequency of TOPBP1-coated UFBs in cells treated with aphidicolin, a potent DNA replication inhibitor, and razoxane, a specific topoisomerase II inhibitor 3,9. Upon treatment with razoxane, but not aphidicolin, we could detect an increase in the percentage of ana/telophase cells with TOPBP1-UFBs (Fig. 3a and b). As an additional control to validate the efficacy of aphidicolin and razoxane treatment, we analysed the incidence of BLM-UFBs under these experimental conditions. In line with previous reports and in support of the notion above we observed a significant increase in BLM-positive UFBs upon drug treatment (Fig. 3c and Supplementary Fig. 4a and b) 3,9. Moreover, treatment with another TOP2A inhibitor ICRF-193 also resulted in an increased frequency of TOPBP1 UFBs with a concomitant increase in BLM UFBs (Supplementary Fig. 4c and d). This suggests that TOPBP1 is specifically recruited to UFBs arising as an effect of TOP2A inhibition. Moreover, the amount of TOPBP1 UFBs co-staining with BLM also increased following razoxane, but not aphidicolin treatment (Supplementary Fig. 4e and f). TOP2A depletion and/or inhibition specifically increases the frequency of C-UFBs 1,3, therefore if our hypothesis was correct then depletion of TOP2A should also result in an increased frequency of TOPBP1-UFBs, and indeed this is exactly what we observed (Fig. 3d and Supplementary Fig. 4g). Importantly, this was also accompanied by an increase in BLM-UFBs as well as DAPI-positive anaphase bridges (Fig. 3e and f), a previously characterised phenotype of TOP2A deficiency 3,9,16. Since treatment with the replication inhibitor aphidicolin did not increase the frequency of TOPBP1 UFBs but TOP2A depletion or inhibition did, these data suggest that TOPBP1 predominately associates with UFBs arising from centromeric loci (C-UFBs). These are most likely derived from problems associated with decatenation of sister chromatids and would require TOP2A for 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 inhibition or depletion results in increased TOPBP1 localisation to UFBs(a and b) Quantification of the percentage of ana/telophase cells staining positive for TOPBP1 localisation to UFBs in U2OS cells treated for 24h with DMSO as a control, or with 0.4 μM aphidicolin (APH) or 10 μM razoxane (RAZ). Bars represent mean values from three independent experiments +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (c) Quantification of the percentage of U2OS ana/telophase cells staining positive for BLM-UFBs in cells treated for 24h with DMSO as a control or with 0.4 μM aphidicolin (APH). Bars represent mean values from three independent experiments +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (d) Quantification of the percentage of U2OS cells positive for TOPBP1-UFBs in cells treated with siRNA targeting TOP2A or in control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (e and f) Quantification of the percentage of U2OS cells positive for BLM-UFBs and DAPI-positive anaphase bridges, respectively cells treated with siRNA directed against TOP2A or control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance.
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Figure 3: TOP2A inhibition or depletion results in increased TOPBP1 localisation to UFBs(a and b) Quantification of the percentage of ana/telophase cells staining positive for TOPBP1 localisation to UFBs in U2OS cells treated for 24h with DMSO as a control, or with 0.4 μM aphidicolin (APH) or 10 μM razoxane (RAZ). Bars represent mean values from three independent experiments +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (c) Quantification of the percentage of U2OS ana/telophase cells staining positive for BLM-UFBs in cells treated for 24h with DMSO as a control or with 0.4 μM aphidicolin (APH). Bars represent mean values from three independent experiments +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (d) Quantification of the percentage of U2OS cells positive for TOPBP1-UFBs in cells treated with siRNA targeting TOP2A or in control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (e and f) Quantification of the percentage of U2OS cells positive for BLM-UFBs and DAPI-positive anaphase bridges, respectively cells treated with siRNA directed against TOP2A or control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance.
Mentions: UFBs can arise as a result of either decatenation or replication problems 1,9. Given that we identify TOPBP1 as a novel TOP2A interactor, and that inhibition of TOP2A leads to an increase in centromeric associated UFBs 3,9 we hypothesised that TOPBP1 may localise to this sub-type of UFBs. To test this, we analysed the frequency of TOPBP1-coated UFBs in cells treated with aphidicolin, a potent DNA replication inhibitor, and razoxane, a specific topoisomerase II inhibitor 3,9. Upon treatment with razoxane, but not aphidicolin, we could detect an increase in the percentage of ana/telophase cells with TOPBP1-UFBs (Fig. 3a and b). As an additional control to validate the efficacy of aphidicolin and razoxane treatment, we analysed the incidence of BLM-UFBs under these experimental conditions. In line with previous reports and in support of the notion above we observed a significant increase in BLM-positive UFBs upon drug treatment (Fig. 3c and Supplementary Fig. 4a and b) 3,9. Moreover, treatment with another TOP2A inhibitor ICRF-193 also resulted in an increased frequency of TOPBP1 UFBs with a concomitant increase in BLM UFBs (Supplementary Fig. 4c and d). This suggests that TOPBP1 is specifically recruited to UFBs arising as an effect of TOP2A inhibition. Moreover, the amount of TOPBP1 UFBs co-staining with BLM also increased following razoxane, but not aphidicolin treatment (Supplementary Fig. 4e and f). TOP2A depletion and/or inhibition specifically increases the frequency of C-UFBs 1,3, therefore if our hypothesis was correct then depletion of TOP2A should also result in an increased frequency of TOPBP1-UFBs, and indeed this is exactly what we observed (Fig. 3d and Supplementary Fig. 4g). Importantly, this was also accompanied by an increase in BLM-UFBs as well as DAPI-positive anaphase bridges (Fig. 3e and f), a previously characterised phenotype of TOP2A deficiency 3,9,16. Since treatment with the replication inhibitor aphidicolin did not increase the frequency of TOPBP1 UFBs but TOP2A depletion or inhibition did, these data suggest that TOPBP1 predominately associates with UFBs arising from centromeric loci (C-UFBs). These are most likely derived from problems associated with decatenation of sister chromatids and would require TOP2A for 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.

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