Author response: CDK-regulated dimerization of M18BP1 on a Mis18 hexamer is necessary for CENP-A loading

Pan, Dongqing; Klare, Kerstin; Petrovic, Arsen; Take, Annika; Walstein, Kai; Singh, Priyanka; Rondelet, Arnaud; Bird, Alexander W.; Musacchio, Andrea

doi:10.7554/elife.23352.022

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(4 citation statements)

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“…2B and fig. S4A) [note that MBP is a monomer, as shown previously (42)]. When expressed in interphase HeLa cells depleted of endogenous CENP-C (fig.…”

Section: Role Of Cenp-c Dimerizationsupporting

confidence: 59%

“…Two features of CENP-A nucleosomes that must be considered in this context are (i) that once incorporated into centromeric chromatin, CENP-A is usually retained over extended, sometimes yearlong periods of time, and (ii) that the levels of CENP-A at each chromosome appear to be maintained intergenerationally, implying that the amounts of newly loaded CENP-A are at least in first approximation identical to those of the centromere-residing CENP-A (66). Suppression by CDK limits loading to the early G 1 phase of the cell cycle (42,67,68). The levels of CENP-A then halve during DNA replication, when CENP-A is equally distributed to the sister chromatids.…”

Section: Discussionmentioning

confidence: 99%

“…Eight tandem repeats of CEN1-like DNA fragment 1 or CEN1-like DNA fragment 2 were cloned into a pUC18 plasmid using restriction enzyme-based cloning and Gibson assembly. The plasmid pETDuet1-mChP-CENPW-HaloT-CENPT-8His was generated by insertion of the sequence of Halotagged CENP-T between Nco I and Xho I sites of pETDuet-8His (42). The plasmids pETDuet1-mChP-CENPW-HaloT-CENPT(1-549)-8His and pETDuet1-mChP-CENPW-HaloT-CENPT(1-529)-8His were generated by exchanging the coding sequence of CENP-T with CENP-T 1-549 or CENP-T 1-529 using restriction enzyme-based cloning.…”

Section: Plasmidsmentioning

confidence: 99%

“…The plasmids containing the SpyCatcher and SpyTag sequences were ordered from Addgene. The plasmid pETDuet-MBP-8His was generated as previously described (42). The polymerase chain reaction (PCR)-amplified CDS (coding sequence) of CENP-C-601-943 or CENP-C-721-943 was inserted between the N-terminal MBP tag and the C-terminal 8His-tag using Bam HI and Xho I.…”

Section: Plasmidsmentioning

confidence: 99%

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Assembly principles and stoichiometry of a complete human kinetochore module

et al. 2021

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Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes and permits further assembly of all other kinetochore subunits in vitro with relative ratios closely matching those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module and may have important implications for transgenerational inheritance of centromeric chromatin.

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“…2B and fig. S4A) [note that MBP is a monomer, as shown previously (42)]. When expressed in interphase HeLa cells depleted of endogenous CENP-C (fig.…”

Section: Role Of Cenp-c Dimerizationsupporting

confidence: 59%