Killing a zombie: a full deletion of the
            <scp>BUB</scp>
            1 gene in
            <scp>HAP</scp>
            1 cells

Raaijmakers, Jonne A.; Medema, René H.

doi:10.15252/embj.2019102423

Cited by 16 publications

(27 citation statements)

References 11 publications

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“…Although these two pools have previously been suggested to function independently (Silio et al , ), we favour a more integrated model whereby corona‐MAD1 supports the SAC by contributing to the pool of MAD1 on the KMN network, as suggested previously by others (Zhang et al , ). We favour this hypothesis for a number of reasons: (i) corona MAD1 has been shown to facilitate MAD1‐BUB1 interaction (Zhang et al , ), (ii) BUB1 is critical for cells to mount a prolonged and efficient checkpoint response (Meraldi & Sorger, ; Rodriguez‐Rodriguez et al , ; Raaijmakers & Medema, ; Zhang et al , ), (iii) BUB1 is the likely catalytic centre of MCC production since it can co‐localise MAD1/MAD2 together with CDC20 (Di Fiore et al , ; Vleugel et al , 2015a; Faesen et al , ; Ji et al , ), and (iv) the corona MAD1‐pT716 that can catalyse MCC production is present in the vicinity of the KMN network where BUB1 is localised (Figs A and EV4C).…”

Section: Discussionmentioning

confidence: 99%

Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint

et al. 2020

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Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N‐terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long‐sought‐after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona‐localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona‐MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.

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Section: Discussionmentioning

confidence: 99%

Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint

et al. 2020

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“…However, these recent observations failed to resolve whether it is possible to generate a full Bub1 deletion in human cell lines. Using two gRNAs to remove almost the entire Bub1 gene in HAP1 cells, Raaijmakers and Medema () have now successfully achieved this. Consistent with this notion, we find here that these new cell lines are no longer sensitive to additional Bub1 depletion by RNAi under our experimental conditions (Fig A), in contrast to the original reported HAP1 “Bub1 null” cell line.…”

Section: Analysis Of New Full Bub1 Knock‐out Hap1 Cellsmentioning

confidence: 99%

Response to Raaijmakers & Medema

et al. 2019

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“…31-34 A potent, specific Bub1 kinase inhibitor is of particular value since Bub1 has a complex role in both chromosome alignment and SAC signalling, 9 with studies yielding conflicting results regarding the requirement of Bub1 for the SAC. 20 In particular, the role of Bub1 kinase activity in SAC signalling remains controversial, with some reports showing importance, 16,25,26 and others indicating that Bub1 kinase activity is redundant for SAC activity. 11,13,14,17,22-24 Therefore Bub1-specific inhibitors represent a useful tool for clarification of its role.…”

Section: Discussionmentioning

confidence: 99%

“…12 However, more recently, HAP1 cells with several BUB1 exons absent from genomic DNA were created following use of two guide RNAs for CRISPR-CAS9. 20 Surprisingly, the SAC remained functional in these cells, even when the more extensive approach was combined with Bub1 siRNA knockdown. However, generation of a complete BUB1 deletion was only possible in haploid HAP1 cells, but not in in several other cell lines.…”

Section: Introductionmentioning

confidence: 96%

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Inhibitors of the Bub1 spindle assembly checkpoint kinase: Synthesis of BAY-320 and comparison with 2OH-BNPP1

Amalina

Whalley

Perera

et al. 2020

Preprint

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SummaryBub1 is a serine/threonine kinase proposed to function centrally in both mitotic chromosome alignment and the spindle assembly checkpoint (SAC), however its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors can be invaluable tools for investigation of kinase function, we decided to evaluate the relative potential of two agents (2OH-BNPPI and BAY-320) as Bub1 inhibitors. After confirming that both agents inhibit Bub1 in vitro, we developed a cell based-assay to specifically measure Bub1 inhibition in vivo. For this assay we overexpressed a fusion of Histone 2B and the Bub1 kinase region (Bub1C) tethering it in close proximity to H2A, which generated a strong ectopic H2ApT120 signal along chromosome arms. The ectopic signal generated from Bub1C activity was effectively inhibited by BAY-320, but not 2OH-BNPP1. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localisation. Preliminary experiments using BAY-320 suggested a minor role for Bub1 kinase activity in chromosome alignment and the SAC, however results suggest that BAY-320 may exhibit off-target effects at the concentration required to demonstrate these outcomes. In conclusion, 2OH-BNPP1 may not be an effective Bub1 inhibitor in vivo, and while BAY-320 is able to inhibit Bub1 in vivo, the high concentrations required and potential for off-target effects highlight the ongoing need for improved Bub1 inhibitors.

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Killing a zombie: a full deletion of the BUB 1 gene in HAP 1 cells

Cited by 16 publications

References 11 publications

Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint

Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint

Response to Raaijmakers & Medema

Inhibitors of the Bub1 spindle assembly checkpoint kinase: Synthesis of BAY-320 and comparison with 2OH-BNPP1

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