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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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BRCT 5 of TOPBP1 mediates its recruitment to UFBs(a) Schematic representation of GFP-TOPBP1 mutant constructs used in this study (WT TOPBP1, BRCT1 - K154A; BRCT5 - K704A). (b, c and d) Quantification of GFP-TOPBP1 WT, K154A and K704A mutant localisation to UFBs in pools of U2OS cells stably expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was knocked down using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (e, f and g) Analysis of localisation of GFP-TOPBP1 WT, K704A and K704E mutant localisation to UFBs in single U2OS clones expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was depleted using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance.
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Figure 2: BRCT 5 of TOPBP1 mediates its recruitment to UFBs(a) Schematic representation of GFP-TOPBP1 mutant constructs used in this study (WT TOPBP1, BRCT1 - K154A; BRCT5 - K704A). (b, c and d) Quantification of GFP-TOPBP1 WT, K154A and K704A mutant localisation to UFBs in pools of U2OS cells stably expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was knocked down using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (e, f and g) Analysis of localisation of GFP-TOPBP1 WT, K704A and K704E mutant localisation to UFBs in single U2OS clones expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was depleted using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance.

Mentions: TOPBP1 carries out most of its functions via nine BRCT domains that mediate phosphorylation-dependent protein-protein interactions in several distinct complexes 23. Importantly, only the BRCT domains 1/2 and 4/5 are conserved between yeast and human 24. As TOPBP1’s yeast orthologue has been shown to localise to UFBs 16, we hypothesised that these conserved BRCT domains may mediate its localisation to UFBs in human cells. To test this, we generated pools of U2OS cells stably expressing GFP-tagged TOPBP1 BRCT1 and BRCT5 mutant proteins, with highly conserved lysine residues binding the phosphate group of the modified ligand, mutated to alanine (Fig. 2a), as we considered these domains as most likely to mediate TOPBP1’s interactions/recruitment 25-27. We found that only the K704A TOPBP1 BRCT5 mutant protein was defective in its localisation to UFBs in the absence of endogenous TOPBP1 with no defect observed for the GFP-TOPBP1 WT or the GFP-TOPBP1 K154A expressing cells (Fig. 2b-d and Supplementary Fig. 2a-c). To validate this data we analysed single clones stably expressing the TOPBP1 mutant proteins. Again we found that only the BRCT domain 5 (K704A) mutant of TOPBP1 was defective in its localisation to UFBs in the absence of endogenous TOPBP1 with no defect observed for the cells expressing GFP-TOPBP1 WT or GFP TOPBP1 K154A (Fig. 2e and f and Supplementary Fig. 2d-f). Moreover, and in support of the data above mutating the BRCT5 Lys704 to glutamic acid (K704E) also resulted in a profound defect in the ability of this mutant protein to localise to UFBs in the absence of endogenous TOPBP1 (Fig. 2g and Supplementary Fig. 2g). In addition, given that BRCT domains interact with phosphorylated proteins, this suggested that a phosphorylated residue in a TOPBP1 interacting protein might mediate such localisation.


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)

BRCT 5 of TOPBP1 mediates its recruitment to UFBs(a) Schematic representation of GFP-TOPBP1 mutant constructs used in this study (WT TOPBP1, BRCT1 - K154A; BRCT5 - K704A). (b, c and d) Quantification of GFP-TOPBP1 WT, K154A and K704A mutant localisation to UFBs in pools of U2OS cells stably expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was knocked down using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (e, f and g) Analysis of localisation of GFP-TOPBP1 WT, K704A and K704E mutant localisation to UFBs in single U2OS clones expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was depleted using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance.
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Figure 2: BRCT 5 of TOPBP1 mediates its recruitment to UFBs(a) Schematic representation of GFP-TOPBP1 mutant constructs used in this study (WT TOPBP1, BRCT1 - K154A; BRCT5 - K704A). (b, c and d) Quantification of GFP-TOPBP1 WT, K154A and K704A mutant localisation to UFBs in pools of U2OS cells stably expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was knocked down using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (e, f and g) Analysis of localisation of GFP-TOPBP1 WT, K704A and K704E mutant localisation to UFBs in single U2OS clones expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was depleted using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance.
Mentions: TOPBP1 carries out most of its functions via nine BRCT domains that mediate phosphorylation-dependent protein-protein interactions in several distinct complexes 23. Importantly, only the BRCT domains 1/2 and 4/5 are conserved between yeast and human 24. As TOPBP1’s yeast orthologue has been shown to localise to UFBs 16, we hypothesised that these conserved BRCT domains may mediate its localisation to UFBs in human cells. To test this, we generated pools of U2OS cells stably expressing GFP-tagged TOPBP1 BRCT1 and BRCT5 mutant proteins, with highly conserved lysine residues binding the phosphate group of the modified ligand, mutated to alanine (Fig. 2a), as we considered these domains as most likely to mediate TOPBP1’s interactions/recruitment 25-27. We found that only the K704A TOPBP1 BRCT5 mutant protein was defective in its localisation to UFBs in the absence of endogenous TOPBP1 with no defect observed for the GFP-TOPBP1 WT or the GFP-TOPBP1 K154A expressing cells (Fig. 2b-d and Supplementary Fig. 2a-c). To validate this data we analysed single clones stably expressing the TOPBP1 mutant proteins. Again we found that only the BRCT domain 5 (K704A) mutant of TOPBP1 was defective in its localisation to UFBs in the absence of endogenous TOPBP1 with no defect observed for the cells expressing GFP-TOPBP1 WT or GFP TOPBP1 K154A (Fig. 2e and f and Supplementary Fig. 2d-f). Moreover, and in support of the data above mutating the BRCT5 Lys704 to glutamic acid (K704E) also resulted in a profound defect in the ability of this mutant protein to localise to UFBs in the absence of endogenous TOPBP1 (Fig. 2g and Supplementary Fig. 2g). In addition, given that BRCT domains interact with phosphorylated proteins, this suggested that a phosphorylated residue in a TOPBP1 interacting protein might mediate such localisation.

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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