Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling

Zhang, Gang; Kruse, Thomas; López-Méndez, Blanca; Sylvestersen, Kathrine B.; Garvanska, Dimitriya H; Schopper, Simone; Nielsen, Michael L.; Nilsson, Jakob

doi:10.1038/ncomms15822

Cited by 89 publications

(156 citation statements)

References 60 publications

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“…This approach avoids issues such as adaption during selection or that residual protein sufficient to support function remains. However, we do not favor this because all the mitotic timings are tightly clustered around 110 min in nocodazole which is very similar to that obtained when dominant negative versions of Bub1 are expressed (Zhang et al, 2017). We provide evidence that reported RPE1 and HAP1 Bub1 knockout cells express residual levels of Bub1, explaining why the response to unattached kinetochores is not strongly affected in these cells (Currie et al, 2018;Raaijmakers et al, 2018).…”

Section: Discussionmentioning

confidence: 56%

“…To further investigate this, we first determined whether the RZZ complex is required for the interaction between Mad1 and Bub1 using our recently established proximity-dependent ligation assay (Zhang et al, 2017). Given that our quantitative analysis of Bub1 CR and Rod CR cells indicated an integrated function of the two checkpoint components, we hypothesized that RZZ localizes Mad1 on kinetochores to facilitate the interaction between Bub1 and Mad1.…”

Section: Increasing the Interaction Between Bub1 And Mad1 Bypasses Thmentioning

confidence: 99%

“…This was done essentially as in Zhang et al (2017). Stable HeLa cell lines expressing the Mad1 BirA fusion protein were exposed to 0.1 ng/ml doxycycline for 18 h to obtain near endogenous Mad1 expression levels.…”

Section: Purification Of Biotinylated Protein Complexesmentioning

confidence: 99%

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Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

et al. 2019

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Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod‐Zw10‐Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1‐Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1‐Bub1 complex.

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Section: Discussionmentioning

confidence: 56%

Section: Increasing the Interaction Between Bub1 And Mad1 Bypasses Thmentioning

confidence: 99%

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Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

et al. 2019

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“…Bub1 consists of a C-terminal kinase domain and a N-terminal region with which Bub1 binds its partner protein Bub3 and localizes to kinetochores ( Fig EV1F) (Taylor et al, 1998;Kawashima et al, 2010;London & Biggins, 2014;Vleugel et al, 2015a;Zhang et al, 2015Zhang et al, , 2017. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on KNL1, a scaffold component of the KNL1/Mis12 complex/Ndc80 complex network (Cheeseman et al, 2006), creating the direct docking site for Bub3 (Kiyomitsu et al, 2007;London et al, 2012;Shepperd et al, 2012;Yamagishi et al, 2012;Primorac et al, 2013;Vleugel et al, 2015b).…”

Section: Resultsmentioning

confidence: 99%

Histone H2A phosphorylation recruits topoisomerase II α to centromeres to safeguard genomic stability

et al. 2019

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Chromosome segregation in mitosis requires the removal of catenation between sister chromatids. Timely decatenation of sister DNAs at mitotic centromeres by topoisomerase IIa (TOP2A) is crucial to maintain genomic stability. The chromatin factors that recruit TOP2A to centromeres during mitosis remain unknown. Here, we show that histone H2A Thr-120 phosphorylation (H2ApT120), a modification generated by the mitotic kinase Bub1, is necessary and sufficient for the centromeric localization of TOP2A. Phosphorylation at residue-120 enhances histone H2A binding to TOP2A in vitro. The C-gate and the extreme C-terminal region are important for H2ApT120-dependent localization of TOP2A at centromeres. Preventing H2ApT120mediated accumulation of TOP2A at mitotic centromeres interferes with sister chromatid disjunction, as evidenced by increased frequency of anaphase ultra-fine bridges (UFBs) that contain catenated DNA. Tethering TOP2A to centromeres bypasses the requirement for H2ApT120 in suppressing anaphase UFBs. These results demonstrate that H2ApT120 acts as a landmark that recruits TOP2A to mitotic centromeres to decatenate sister DNAs. Our study reveals a fundamental role for histone phosphorylation in resolving centromere DNA entanglements and safeguarding genomic stability during mitosis.MZ designed and performed the majority of the experiments and analysis. CL designed and carried out the GST pulldown assays (Figs 3E and F, and 6L), the localization of TOP2A in HeLa and Sgo1-K492A cells (Fig 4C and D), the competitive localization of EGFP-TOP2A and Sgo1 (Fig 7A-D), as well as the deconvolution microscopy (Appendix Fig S4). HZ and SL helped express the GST-H2ApS120 protein. XY performed the experiments shown in Appendix Fig S1. QC, HY, JX, and JL provided technical assistance. WL provided reagents and constructive suggestions. FW conceived and supervised the project, designed the experiments, analyzed the data, and wrote the manuscript with the input from HY.

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“…). Further MPS1 phosphorylations on BUB1 and MAD1 lead to the recruitment of the MAD1–MAD2 complex and catalyse the formation of the diffusible MCC . In mammalian cells, maintenance of MAD1–MAD2 at kinetochores additionally requires the RZZ complex previously alluded to.…”

Section: Key Components Of the Sac And Their Spatio‐temporal Localizamentioning

confidence: 99%

Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

2019

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In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule‐kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A‐B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1‐cyclin B1 and its counteracting phosphatase PP2A‐B55. Furthermore, we discuss how CDK1‐cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis.

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Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling

Cited by 89 publications

References 60 publications

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Histone H2A phosphorylation recruits topoisomerase II α to centromeres to safeguard genomic stability

Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

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