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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 depletion impedes C-UFBs resolution(a) Quantification of BLM-positive UFBs in control U2OS cells or cells stably expressing GFP-TOPBP1 WT or GFP-TOPBP1 K704E, respectively in the presence or absence of endogenous TOPBP1. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (b) Quantification of UFBs emanating from centromeric loci as assayed by CENPB staining in TOPBP1-depleted cells. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment (c) Representative image of an anaphase cell with a BLM-positive UFB (Red) which emanates from a CENPB focus (Green). DAPI (Blue) serves as a DNA stain. Scale bar = 6.4 μm.
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Figure 4: TOPBP1 depletion impedes C-UFBs resolution(a) Quantification of BLM-positive UFBs in control U2OS cells or cells stably expressing GFP-TOPBP1 WT or GFP-TOPBP1 K704E, respectively in the presence or absence of endogenous TOPBP1. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (b) Quantification of UFBs emanating from centromeric loci as assayed by CENPB staining in TOPBP1-depleted cells. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment (c) Representative image of an anaphase cell with a BLM-positive UFB (Red) which emanates from a CENPB focus (Green). DAPI (Blue) serves as a DNA stain. Scale bar = 6.4 μm.

Mentions: To further explore the role of TOPBP1 in promoting resolution of C-UFBs, it was important to establish the consequences of TOPBP1 depletion on their resolution. To this end, we depleted TOPBP1 by siRNA and analysed the frequency of BLM-positive UFBs. Upon TOPBP1 depletion we detected a significant increase in the number of cells with BLM UFBs (Fig. 4a). One prediction from this notion would be that the TOPBP1 mutant defective in its association with UFBs (BRCT5 mutant) should display the same phenotype. In line with this, we detected a statistically significant increase in the number of BLM-positive UFBs in cells expressing this mutant protein, while cells expressing wild-type GFP-TOPBP1 (in the absence of endogenous TOPBP1) show no such increase under the same conditions (Fig. 4a), further validating the functionality of this fusion protein. Next, we addressed whether a physical link between these induced UFBs and centromeric DNA can be established. To do so, we stained cells for BLM and a centromeric marker, CENPB. Strikingly, the majority of the UFBs induced in the absence of TOPBP1 stained positive for CENPB (Fig. 4b and c) thus supporting the idea that TOPBP1 associates predominately with C-UFBs. Taken together, these data support a role for TOPBP1 in promoting chromosome disjunction by aiding resolution of centromere-associated UFBs. Moreover, BLM recruitment to UFBs seems to be TOPBP1-independent.


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 depletion impedes C-UFBs resolution(a) Quantification of BLM-positive UFBs in control U2OS cells or cells stably expressing GFP-TOPBP1 WT or GFP-TOPBP1 K704E, respectively in the presence or absence of endogenous TOPBP1. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (b) Quantification of UFBs emanating from centromeric loci as assayed by CENPB staining in TOPBP1-depleted cells. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment (c) Representative image of an anaphase cell with a BLM-positive UFB (Red) which emanates from a CENPB focus (Green). DAPI (Blue) serves as a DNA stain. Scale bar = 6.4 μm.
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Figure 4: TOPBP1 depletion impedes C-UFBs resolution(a) Quantification of BLM-positive UFBs in control U2OS cells or cells stably expressing GFP-TOPBP1 WT or GFP-TOPBP1 K704E, respectively in the presence or absence of endogenous TOPBP1. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (b) Quantification of UFBs emanating from centromeric loci as assayed by CENPB staining in TOPBP1-depleted cells. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment (c) Representative image of an anaphase cell with a BLM-positive UFB (Red) which emanates from a CENPB focus (Green). DAPI (Blue) serves as a DNA stain. Scale bar = 6.4 μm.
Mentions: To further explore the role of TOPBP1 in promoting resolution of C-UFBs, it was important to establish the consequences of TOPBP1 depletion on their resolution. To this end, we depleted TOPBP1 by siRNA and analysed the frequency of BLM-positive UFBs. Upon TOPBP1 depletion we detected a significant increase in the number of cells with BLM UFBs (Fig. 4a). One prediction from this notion would be that the TOPBP1 mutant defective in its association with UFBs (BRCT5 mutant) should display the same phenotype. In line with this, we detected a statistically significant increase in the number of BLM-positive UFBs in cells expressing this mutant protein, while cells expressing wild-type GFP-TOPBP1 (in the absence of endogenous TOPBP1) show no such increase under the same conditions (Fig. 4a), further validating the functionality of this fusion protein. Next, we addressed whether a physical link between these induced UFBs and centromeric DNA can be established. To do so, we stained cells for BLM and a centromeric marker, CENPB. Strikingly, the majority of the UFBs induced in the absence of TOPBP1 stained positive for CENPB (Fig. 4b and c) thus supporting the idea that TOPBP1 associates predominately with C-UFBs. Taken together, these data support a role for TOPBP1 in promoting chromosome disjunction by aiding resolution of centromere-associated UFBs. Moreover, BLM recruitment to UFBs seems to be TOPBP1-independent.

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