Figures and data in Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences

Figures
Tables
Additional files

2 figures, 5 tables and 1 additional file

Figures

Figure 1

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Recombinantly wrapped nucleosome core particles (NCPs) with W601 or CCEN DNA.

(A) Sequences of the W601 and CCEN DNA used to wrap histone octamers. The black text is W601 DNA, the black text highlighted in yellow is the portion of CDEII corresponding to the Mif2 footprint (Xiao et al., 2017), the cyan text is CDEIII, the sequence underlined in green is the CBF3 binding site (Guan et al., 2021), and the blue text is the pericentromeric DNA sequence just downstream of CDEIII on chromosome III. (B) Chromatogram representing elution fractions from size-exclusion chromatography column used to purify wrapped NCPs from excess free DNA. 260 nm signal is shown for both W601 and CCEN NCPs. (C) Native gel of elution fractions indicated in the chromatogram in (B). NCPs from both SEC Fraction 6 and SEC Fraction 7 were used to collect data on the optical trap. There was no statistically significant difference between rupture forces of assemblies measured with NCPs from either Fraction 6 or Fraction 7.

Figure 1—source data 1 Native gel for Figure 1C, including the relevant bands and conditions.: https://cdn.elifesciences.org/articles/105150/elife-105150-fig1-data1-v1.zip
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Figure 1—source data 2 Original file of native gel for Figure 1C.: https://cdn.elifesciences.org/articles/105150/elife-105150-fig1-data2-v1.zip
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Figure 2

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Kinetochore assemblies built on CCEN-NCPs and OA form stronger attachments to microtubules.

(A) Percentages of beads that had microtubule binding capability. Error bars indicate the standard error of the proportion. Barnard’s test was used to compare contingency tables. The p-values for the significance of the difference between fraction of beads bound if either OA or Mif2 were included compared to if neither were included are given in Table 1. Data were combined from two biological replicates (see Figure 2—source data 1). (B) Boxplot of rupture forces for each of the kinetochore assemblies tested. Dots represent individual rupture events, and boxes enclose the interquartile range, with indicated medians. Whiskers extend to the inner fences. Data were combined from four biological replicates of each condition (see Figure 2—source data 1). A Kolmogorov–Smirnov test was performed to compare the probability distributions of rupture forces across conditions. *** indicates p-value of 1.87 × 10^–6, n.s., not significant. (C) Survival probability curves for the data plotted in (B).

Figure 2—source data 1 Excel file of data for Figure 2A and B and a list of the number of technical and biological replicates for Figure 2A and B.: https://cdn.elifesciences.org/articles/105150/elife-105150-fig2-data1-v1.xlsx
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Tables

Table 1

Statistical analyses for microtubule binding assay*.

Condition	CCEN NCP neither OA nor Mif2	W601 NCP neither OA nor Mif2
W601 NCP, OA	N/A	2.61 × 10^–12
CCEN NCP, OA	3.55 × 10^–13	N/A
W601 NCP, Mif2	N/A	2.31 × 10^–12
CCEN NCP, Mif2	4.27 × 10^–7	N/A

N/A, not applicable.
*

p-Values for comparison of data in Figure 2A determined by a Barnard’s test.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Escherichia coli)	Rosetta (DE3) pLys competent cells	Novagen	Cat# 71403
Antibody	MonoRab anti-DYKDDDDK Affinity Resin	GenScript	Cat# L00766
Biological sample (Bos taurus)	Tubulin	Lab purification		Isolated from Bos taurus brains Protocol adopted from Castoldi and Popov, 2003
Antibody	Biotinylated Anti-His tag antibody	R&D Systems	Cat# BAM050 RRID:AB_356845
Chemical compound, drug	Glucose oxidase	MilliporeSigma	Cat# 345386
Chemical compound, drug	Catalase	MilliporeSigma	Cat# E3289
Chemical compound, drug	Biotinylated bovine serum albumin (BSA)	Vector laboratories	Cat# B-2007
Chemical compound, drug	Avidin DN	Vector Laboratories	Cat# A-3100
Chemical compound, drug	TCEP	Thermo Fisher	Cat# 20490
Other	Streptavidin-coated polystyrene beads	Spherotech	SVP-05-10
Software	Labview	National Instruments	RRID:SCR_014325
Software	Igor Pro	Wavemetrics	RRID:SCR_000325

Table 2

Plasmids used for expression of kinetochore proteins^*.

Protein	Plasmid	Components	References
Nucleosome	pScKl2	K. lactis His₆-H2A, K. lactis His₆-H2B, Cse4, K. lactis His₆-H4	Migl et al., 2020
OA	pGH3	Okp1, Ame1-FLAG	Hamilton et al., 2020
Mif2	pAZ144	Mif2-MBP	This study
2D-MIND	pGH62	FLAG-Nsl1, S240D & S250D Dsn1, Mtw1, Nnf1	Hamilton et al., 2020
(230-576) 2D-MIND	pEHM4	FLAG-Nsl1, (230-576) S240D & S250D Dsn1, Mtw1, Nnf1	Hamilton et al., 2020
Ndc80c	pJT48 Ndc80/Nuf2	Spc24-FLAG, Spc25 Ndc80, Nuf2	Kudalkar et al., 2015; Wei et al., 2005
Dam1c	pJT44	Spc34-FLAG, Dad1, Dad2, Dad3, Dad4 Duo1, Dam1, Hsk3, Spc19, Ask1	Umbreit et al., 2014

*

All proteins are from Saccharomyces cerevisiae except as noted.

Table 3

DNA sequences tested for ability to wrap centromeric nucleosomes.

Name	Sequence^*	Wrapped?	Stable duringSEC?
W601^†	ATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGC TCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTA ACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATA TATACATCCGAT	Yes	Yes
CEN 3 ^‡	AAAGCTATTCATTGAAAAAATAGTACAAATAAGTCACATGATGATATTTGATTTTATTATATTTTTAAAAAAAGTAAAAAATAAAAAGTA GTTTATTTTTAAAAAATAAAATTTAAAATATTAGTGTATTTGATT TCCGAAAGTTAAAAAAGAAATAGTAAGAAATATATATTT	Some	No
CEN 5.1	AAAGCTATTCATTGAAAAAATAGTACAAATAAGTCACATGATCGA GAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAG CACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGC CAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATAC ATCCGATTATTTGATTTCCGAAAGTTAAAAAAGAAATAGTAAGAA ATATATATTT	Yes	Yes
CEN3-601 (Xiao et al., 2017)	ATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGC TCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTA ATATTAGTGTATTTGATTTCCGAAAGTTAAAAAAGAAATAGTAAG AAATCATCCGAT	Yes	Yes
CCEN^†	ATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGC TCTAGCACCGCTTAAACAGTTTATTTTTAAAAAATAAAATTTAAA ATATTAGTGTATTTGATTTCCGAAAGTTAAAAAAGAAATAGTAAG AAATCATCCGAT	Yes	Yes
CEN 6.3	ATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGC TCTAGCACCGCTTAAACAGTTTATTTTTAAAAAATAAAATTTAAA ATATTAGAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATA TATACATCCGAT	Yes	ND ^§
CEN 6.4	ATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGC TCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTA ACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATA TATACATCCGATGCCGCCAGTTTATTTTTAAAAAATAAAATTTAA AATATTAG	Some	ND ^§
CON3 ^¶ (Dendooven et al., 2023)	ATAAGTCACATGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCT CTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAA TATTAGTGTATTTGATTTCCGAAAGTTAAAAAAGAAATAGTAAGA AATATATATTTCATTGAA	ND ^§	ND ^§

ND, not determined.
*

W601 DNA in black font; Genomic DNA upstream or downstream of CEN3 in blue font; CDEI in purple font; CDEII in red font; CDEII Mif2 footprint in red font and underlined; CDEIII in purple font and underlined; linker DNA highlighted in bold.
†

These are the DNA sequences used to wrap the nucleosomes tested in the optical trapping assay in this paper.
‡

The construct used by Xiao et al., 2017 included only the 147 bp of CEN3 and none of the upstream or downstream sequences.
§

Not determined.
¶

Shown for comparison only.

Table 4

Kinetochore protein purification buffers.

Protein	Purification buffers
Dam1c-FLAG	Lysis: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl
Mif2-MBP	Lysis: 30 mM HEPES buffer pH 7.5, 2 M NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets Amylose resin elution: 30 mM HEPES buffer pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets, 10 mM maltose QA: 30 mM HEPES buffer pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets QB: 30 mM HEPES buffer pH 7.5, 1 M NaCl, 10% glycerol, 1 mM TCEP
MIND-FLAG	Lysis: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 0.5% NP40, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 1 mM EDTA
Ndc80c-FLAG	Lysis: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x peptide SEC: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 1 mM EDTA
OA-FLAG	Lysis: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 0.5% NP40, 1 mM PMSF, Roche protease inhibitor tablets Low salt FLAG wash: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets High salt FLAG wash: 50 mM HEPES buffer pH 7.5, 2 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 1 mM EDTA