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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 promotes UFB resolution(a) Western blot depicting the levels of expression of GFP-TOPBP1 WT and GFP-TOPBP1 ΔBRCT7/8 mutant transiently expressed in U2OS cells (GFP). α-Tubulin serves as a loading control. (b and c) Quantification of the frequency of BLM-UFBs or DAPI-positive anaphase bridges in mock-transfected U2OS cells (Mock) or cells transiently expressing GFP-TOPBP1 WT or ΔBRCT7/8 mutant (WT or ΔBRCT7/8). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment. Chi-square test was used to evaluate statistical significance. (d) Schematic model of TOPBP1-mediated UFB resolution: TOPBP1 binds to C-UFBs via its BRCT5 domain and recruits TOP2A via its C-terminus to aid their resolution.
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Figure 7: TOPBP1-TOP2A interaction promotes UFB resolution(a) Western blot depicting the levels of expression of GFP-TOPBP1 WT and GFP-TOPBP1 ΔBRCT7/8 mutant transiently expressed in U2OS cells (GFP). α-Tubulin serves as a loading control. (b and c) Quantification of the frequency of BLM-UFBs or DAPI-positive anaphase bridges in mock-transfected U2OS cells (Mock) or cells transiently expressing GFP-TOPBP1 WT or ΔBRCT7/8 mutant (WT or ΔBRCT7/8). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment. Chi-square test was used to evaluate statistical significance. (d) Schematic model of TOPBP1-mediated UFB resolution: TOPBP1 binds to C-UFBs via its BRCT5 domain and recruits TOP2A via its C-terminus to aid their resolution.

Mentions: If the above were correct then one would expect that cells expressing a mutant protein defective for TOPBP1-TOP2A interaction would display aberrant resolution of UFBs and increased incidence of DAPI-positive anaphase bridges, as observed in TOP2A-depleted cells (Fig. 3e and f and 36). We tested this hypothesis using TOPBP1 mutant defective in TOP2A interaction, but proficient in UFB localisation (Fig. 5c and 6d). In support of our line of thinking, cells expressing this TOPBP1 mutant protein phenocopied TOP2A depletion showing a significant increase in the incidence of both BLM-UFBs as well as DAPI positive anaphase bridges, as compared to mock transfected control cells and cells expressing GFP-TOPBP1 WT (Fig. 7a-c). Moreover, the majority of these induced BLM UFBs emanated from centromere loci as assayed by CENPB staining (Supplementary Fig. 7b). Taken together our data support a model whereby TOPBP1-TOP2A interaction promotes recruitment of TOP2A to centromeric UFBs in order to aid their resolution (Fig. 7d).


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 promotes UFB resolution(a) Western blot depicting the levels of expression of GFP-TOPBP1 WT and GFP-TOPBP1 ΔBRCT7/8 mutant transiently expressed in U2OS cells (GFP). α-Tubulin serves as a loading control. (b and c) Quantification of the frequency of BLM-UFBs or DAPI-positive anaphase bridges in mock-transfected U2OS cells (Mock) or cells transiently expressing GFP-TOPBP1 WT or ΔBRCT7/8 mutant (WT or ΔBRCT7/8). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment. Chi-square test was used to evaluate statistical significance. (d) Schematic model of TOPBP1-mediated UFB resolution: TOPBP1 binds to C-UFBs via its BRCT5 domain and recruits TOP2A via its C-terminus to aid their resolution.
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Figure 7: TOPBP1-TOP2A interaction promotes UFB resolution(a) Western blot depicting the levels of expression of GFP-TOPBP1 WT and GFP-TOPBP1 ΔBRCT7/8 mutant transiently expressed in U2OS cells (GFP). α-Tubulin serves as a loading control. (b and c) Quantification of the frequency of BLM-UFBs or DAPI-positive anaphase bridges in mock-transfected U2OS cells (Mock) or cells transiently expressing GFP-TOPBP1 WT or ΔBRCT7/8 mutant (WT or ΔBRCT7/8). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment. Chi-square test was used to evaluate statistical significance. (d) Schematic model of TOPBP1-mediated UFB resolution: TOPBP1 binds to C-UFBs via its BRCT5 domain and recruits TOP2A via its C-terminus to aid their resolution.
Mentions: If the above were correct then one would expect that cells expressing a mutant protein defective for TOPBP1-TOP2A interaction would display aberrant resolution of UFBs and increased incidence of DAPI-positive anaphase bridges, as observed in TOP2A-depleted cells (Fig. 3e and f and 36). We tested this hypothesis using TOPBP1 mutant defective in TOP2A interaction, but proficient in UFB localisation (Fig. 5c and 6d). In support of our line of thinking, cells expressing this TOPBP1 mutant protein phenocopied TOP2A depletion showing a significant increase in the incidence of both BLM-UFBs as well as DAPI positive anaphase bridges, as compared to mock transfected control cells and cells expressing GFP-TOPBP1 WT (Fig. 7a-c). Moreover, the majority of these induced BLM UFBs emanated from centromere loci as assayed by CENPB staining (Supplementary Fig. 7b). Taken together our data support a model whereby TOPBP1-TOP2A interaction promotes recruitment of TOP2A to centromeric UFBs in order to aid their resolution (Fig. 7d).

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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