1. The BUB3-BUB1 Complex Promotes Telomere DNA Replication
Feng Li, Hyeung Kim, Zhejian Ji, Tianpeng Zhang, Bohong Chen, Yuanlong Ge, Yang Hu, Xuyang Feng, Xin Han, Huimin Xu, Youwei Zhang, Hongtao Yu, Dan Liu, Wenbin Ma, Zhou Songyang 
Molecular Cell 
Volume 70, Issue 3, Pages e4 (May 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 70, 395-407.e4DOI: (10.1016/j.molcel.2018.03.032)
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3. Figure 1 BUB3 Localizes to Telomeres during S Phase
(A) Asynchronous HeLa cells were co-stained with antibodies against BUB3 and the telomere-binding protein RAP1 or TRF2. DAPI was used to stain the nuclei. Arrowheads indicate co-localized signals.
(B) Data from (A) were quantitated to calculate the percentage of cells with overlapping signals. ≥100 cells were examined for each experiment. Error bars represent SD (3 independent experiments). p values were calculated using the Student’s t test.
(C) HeLa cells synchronized by double thymidine block were stained using antibodies against BUB3 and RAP1 at different time points following cell cycle release. Arrowheads indicate overlapping signals.
(D) The percentages of RAP1-BUB3 co-staining foci were calculated from (C). Cell cycle profiles were determined by FACS. ≥100 cells from each cell cycle stage were examined. Error bars represent SD (3 independent experiments). p values were calculated using the Student’s t test.
(E) Bacterially purified GST-tagged BUB3 and TRF2 were used for in vitro binding assays. The precipitates were resolved by SDS-PAGE and visualized by Coomassie staining.
(F) 293T cells transiently co-expressing GFP-TRF2 and GST-tagged BUB3 were synchronized by double thymidine block and collected at different time points for immunoprecipitation (IP) with an anti-GST antibody. The immunoprecipitates were probed with as indicated. A, asynchronous cells.
(G) HeLa cells were transfected with a scramble siRNA oligo (siControl) or an oligo against TRF2 (siTRF2) and stained after 48 hr using antibodies against BUB3 and telomeric proteins TRF2 and POT1. DAPI was used to stain the nuclei.
(H) Quantification of data from (G). ≥100 cells were examined for each experiment. Error bars represent SD (three independent experiments). p values were calculated using the Student’s t test.
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4. Figure 2 RNAi KD of BUB3 Results in Telomere Abnormalities
HeLa cells were transfected with two different siRNA oligos against BUB3 (siBUB3-1 and siBUB3-3). A scramble siRNA was used as control (siCtrl).
(A and B) Western blotting and qRT-PCR were performed to determine the mRNA (A) and protein (B) levels of endogenous BUB3 48 hr after transfection. qRT-PCR results were normalized to GAPDH. An antibody against GAPDH was used for western loading control.
(C) Time course of double thymidine block and Q-FISH following siRNA treatment.
(D) BUB3 KD cells were examined by metaphase-FISH using a fluorescein isothiocyanate (FITC)-labeled telomere probe (green). Box with solid lines, multiple telomere signals (MTSs) (enlarged image in upper right); box with dotted lines, telomere loss (enlarged picture in lower right). DAPI was used to stain the nuclei.
(E and F) Quantifications of telomere loss (E) and MTS (F) in the BUB3 KD cells were graphed. ≥1,000 chromosomes were examined for each experiment. Error bars represent SD (3 independent experiments). p values were calculated using the Student’s t test.
(G) BUB3 KD cells from (A) and (B) were analyzed by Q-FISH using a FITC-labeled telomere probe (green). DAPI was used to stain the nuclei. Fluorescence intensities (i.e., telomere signals) were plotted on the x axis and the frequency of signal intensities plotted on the y axis. n, total number of telomere signals detected; red lines, mean telomere signal intensity. p values were calculated using the Student’s t test.
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5. Figure 3 BUB3 and BUB1 Work Together in Telomere Regulation
(A) 293T cells in which endogenous BUB1 was homozygously tagged at the N terminus with a SFB-GFP cassette were generated. gRNA1 and gRNA2 target, respectively, the N terminus of BUB1 and sequences outside the SFB-GFP cassette on the PITCh donor vector. Cas9 cleaves both genomic DNA and the PITCh vector, and precise knockin is mediated by MMEJ using the two 20-bp micro-homology sequences immediately flanking the tag cassette. 293T cells expressing GFP-SFB were used as negative controls in the following experiments.
(B) BUB1-N-SFB-GFP knockin 293T cells were immunoprecipitated using anti-Flag antibodies and western blotted with the indicated antibodies.
(C) For telomere-ChIP, cells were immunoprecipitated with an anti-Flag antibody and the co-precipitated DNA was analyzed by slot blots and hybridization to telomere (TTAGGG)3 and Alu repeat probes.
(D) Telomeric signals from (C) were normalized against Alu signals. Error bars represent SD (n = 3). p values were calculated using the Student’s t test.
(E and F) Cells transiently expressing siRNAs against BUB3 and BUB1 individually or together were examined by metaphase-FISH using a FITC-labeled telomere probe (green). DAPI was used to stain the nuclei. Example images of MTSs (E) and telomere loss (F) are shown (upper left). ≥35 metaphase spreads were examined for each cell line. The number of MTSs and telomere loss events per metaphase was plotted on the y axis. Values for the mean and SEM (error bars) are listed. p values were determined by Student’s t test.
(G) The same KD cells were analyzed by Q-FISH using a FITC-labeled telomere probe (green). DAPI was used to stain the nuclei. Fluorescence intensities (telomere signals) were plotted on the x axis and the frequency of signal intensities on the y axis. Red lines indicate mean telomere signal intensity. n, total number of telomere signals. P-p values were calculated using the Student’s t test.
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6. Figure 4
Inhibition of BUB3 and BUB1 Leads to Replication Fork Stalling at Telomeres and Inefficient Telomere Replication
(A) HeLa cells transfected with control siRNAs or those against BUB3 or BUB1 were co-stained with antibodies against TIN2 (red) and PCNA (green). Arrowheads indicate co-localized foci. DAPI was used to stain the nuclei.
(B) Data from (A) were graphed. ≥100 cells were examined for each cell line. Cells containing ≥3 co-localized foci were scored as positive. Error bars indicate SEs, and p values were calculated with the Student’s t test (n = 3).
(C–E) Schematic representation of the strategy to monitor replication rates of total genomic (EdU) versus telomeric (BrdU/CsCl) DNA (C). Cells from (A) were synchronized with double thymidine block and released before being incubated (12 hr) with EdU or BrdU. Newly synthesized DNA that has incorporated EdU will inform the rate of bulk genomic DNA replication, and harvested cells were labeled with FITC and stained for propidium iodide (PI) before FACS (D). Replicated telomeres (BrdU-labeled) can be fractionated by CsCl ultracentrifugation and detected by slot blot hybridization using a radio-labeled telomere probe to estimate rates of telomere replication (E).
(F) Data from (E) were quantified and graphed for BUB3 (left) and BUB1 (right) KD cells, with gradient density on the x axis and telomere signal intensity on the y axis. Positions that correspond to unreplicated and lagging versus leading strands are indicated.
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7. Figure 5
Kinase and BUB3-Binding Activities of BUB1 Are Critical to Telomere Replication
(A) DMSO (control) or 2OH-BNPP1 (10 μM)-treated HeLa cells were incubated with nocodazole (300 nM) for 16 hr and visualized under a fluorescence microscope. ≥100 cells were examined under each condition to determine the mitotic index. Error bars indicate SE (n = 3). p values were calculated using the Student’s t test.
(B and C) HeLa cells synchronized by double thymidine block were treated with DMSO or 2OH-BNPP1 (10 μM) for 8 hr after release into cell cycle before metaphase-FISH analysis. MTS (B) and telomere loss (C) were quantified and graphed. ≥35 metaphase spreads were examined for each condition. Mean and SEM (error bars) are indicated. p values were determined by Student’s t test.
(D) Cells stably expressing HA-tagged full-length or mutant BUB1 were transfected with siRNAs against BUB1. Parental cells transfected with the scramble (siCtrl) or BUB1 (siBUB1) oligo alone served as controls. Cells were co-stained with antibodies against TRF2 (red) and PCNA (green). Arrowheads indicate co-localized foci.
(E) (Top) Data from (D) were quantified and graphed. ≥100 cells were examined for each cell line. Cells containing ≥3 co-localized foci were scored as positive. Error bars indicate SEs, and p values were calculated with the Student’s t test (n = 3). (Bottom) A diagram of various BUB1 constructs is shown. The BUB3-binding domain is deleted from BUB1-M1 (M1). The K821A mutation blocks ATP binding in the kinase-dead mutant BUB1-K821A (K821A).
(F and G) The same cells from (D) were used in metaphase-FISH assays to determine MTS (F) and telomere loss (G). ≥35 metaphase spreads were examined for each cell line. The number of MTSs or telomere loss events per metaphase was determined and plotted on the y axis. Mean and SEM (error bars) are indicated. p values were determined by Student’s t test.
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8. Figure 6
BUB1 and BUB3 Function through TRF1 and BLM to Facilitate Telomere Replication
(A) HeLa cells transfected with siRNAs against BUB1, TRF1, and TRF2 either individually or in combination were co-stained with antibodies against TIN2 (red) and PCNA (green). Arrowheads indicate co-localized foci.
(B) Data from (A) were quantified and graphed. ≥100 cells were examined for each cell line. Cells containing ≥3 co-localized foci were scored as positive. Error bars indicate SEs, and p values were calculated with the Student’s t test (n = 3).
(C) 293T cells co-expressing differently tagged TRF1 (Flag and GST) were transfected with siRNAs against BUB1. The cells were then used for GST pull-down and subsequent western blotting with the indicated antibodies. GST-tagged GFP was used as a negative control and GAPDH as a loading control.
(D) Cells transfected with siRNAs against BUB1 and BLM singly or in combination were examined by western blotting. GAPDH was used as loading control.
(E and F) Cells from (D) were analyzed by metaphase-FISH to determine MTS (E) and telomere loss (F). ≥35 metaphase spreads were examined for each cell line. Mean and SEM (error bars) are indicated. p values were determined by Student’s t test.
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9. Figure 7
BUB1-Mediated Phosphorylation of TRF1 on S296 Is Essential for Recruiting BLM to Promote Telomere Replication
(A) Recombinant BUB3/BUB1 proteins were incubated with recombinant epitope-tagged TRF1, TRF2, RAP1, or bulk histones in the presence of γ-32P-ATP. The reactions were quenched and resolved by SDS-PAGE and analyzed by autoradiography. Positions that correspond to phosphorylated BUB1 (autophosphorylation) and TRF1 are indicated (left). An image of the gel stained with Coomassie brilliant blue (CBB) is shown (right).
(B) Amino acid sequence alignment of BUB1 kinase substrates H2A, CDC20, and TRF1. The basic patches representing putative amino acids around the Bub1 phosphorylation sites are highlighted in blue.
(C) 293T cells expressing Flag-tagged TRF1 (wild-type [WT] or S296A) were used for IP and western blotting with the indicated antibodies. Flag-tagged GFP was used as a negative control and GAPDH as a loading control.
(D and E) 293T cells overexpressing GFP control, TRF1, or TRF1-S296A were examined using metaphase-FISH assays to determine MTS (D) and telomere loss (E). ≥35 metaphase spreads were examined for each cell line. The number of MTSs or telomere loss events per metaphase was determined and plotted on the y axis. Mean and SEM (error bars) are indicated. p values were determined by Student’s t test.
(F) BUB1 and BUB3 function together to regulate telomere replication by helping to resolve telomere replication blocks mediated by TRF2 and promoting telomere replication progression through modulating TRF1 and BLM.
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