Structural Basis for a Safety-Belt Mechanism That Anchors Condensin to Chromosomes

. 2017 Oct 19;171(3):588–600.e24. doi: 10.1016/j.cell.2017.09.008

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse monoclonal anti V5-tag (anti PK6-tag)	AbD Serotec (MCA1360)	RRID: AB_322378
Mouse monoclonal anti tubulin (TAT1)	Woods et al., 1989	N/A
Rabbit polyclonal anti Sc Ycg1	Piazza et al., 2014	N/A

Bacterial and Virus Strains

Escherichia coli Rosetta (DE3) pLysS	Merck	Cat#70954

Chemicals, Peptides, and Recombinant Proteins

Crosslinker dibromobimane (bBBr)	Sigma-Aldrich	Cat#34025
Crosslinker bismaleimidoethane (BMOE)	Thermo-Fisher	Cat#22323
Crosslinker disthiobismaleimidoethane (DTME)	Thermo-Fisher	Cat#22335
C. thermophilum Ycs4 (3-1222, N-terminal 6 × HIS-tag)	modified purification protocol from Piazza et al., 2014	N/A
C. thermophilum Ycg1 (24-1006, N-terminal 6 × HIS-tag)	modified purification protocol from Piazza et al., 2014	N/A
C. thermophilum Brn1_336-714 (336-714, N-terminal GST-tag)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, BC1} (515-634, R539D, R541D, K542D, K544D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, BC2} (515-634, R554D, R556D, K557D, K559D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, BC1/2} (515-634, R539D, R541D, K542D, K544D, R554D, R556D, K557D, K559D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_YC1 (24-1006, K100D, K101D)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_YC2 (24-1006, K916D, K917D)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_YC1/2 (24-1006, K100D, K101D, K916D, K917D)	This work	N/A
C. thermophilum Brn1_225-512 (225-512, N-terminal 6 × HIS-tag) in complex with Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_539-634 (539-634, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_549-634 (549-634, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_572-634 (572-634, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_515-601 (515-601, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_78-1006 (78-1006)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_24-823 (24-823)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_24-883 (24-883)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_24-934 (24-934)	This work	N/A
C. thermophilum Brn1_515-634 (515-634, N-terminal 6 × HIS-tag) in complex with Ycg1_24-982 (24-982)	This work	N/A
C. thermophilum Brn1_{515-634, E514C-R629C} (515-634, E514C, R629C, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, E514C-R629C cleavable} (515-634, E514C, R629C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, L521C-S611C cleavable} (515-634, L521C, S611C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, D525C-S610C cleavable} (515-634, D525C, S610C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, E514C-Q613C cleavable} (515-634, E514C, Q613C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, A527C-R629C cleavable} (515-634, A527C, R629C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, S568C-R629C cleavable} (515-634, S568C, R629C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_{515-634, cleavable} (515-634, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006)	This work	N/A
C. thermophilum Brn1_225-634 (225-634, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_225-634 (225-634, N-terminal 6 × HIS-tag) in complex with Ycg1_YC1+YC2 (24-1006, K100D, K101D, K916D, K917D) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_{225-634 BC1/2} (225-634, R539D, R541D, K542D, K544D, R554D, R556D, K557D, K559D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_{225-634, E514C-R629C} (225-634, E514C, R629C, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_{225-634, E514C-R629C cleavable} (225-634, E514C, R629C, N-terminal 6 × HIS-tag, HRV3C cleavage site inserted after P549) in complex with Ycg1 (24-1006) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_225-634, _ΦD (225-634, L521D, F524D, W532D, W538D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006) and Ycs4 (3-1222)	This work	N/A
C. thermophilum Brn1_515-634, _ΦD (515-634, L521D, F524D, W532D, W538D, N-terminal 6 × HIS-tag) in complex with Ycg1 (24-1006)	This work	N/A
S. cerevisiae Smc2 (full length) in complex with Smc4 (full length, C-terminal 3 × StrepII-tag), Brn1 (full length, C-terminal 12 × HIS-tag 3 × HA-tag), Ycg1 (full length) and Ycs4 (full length)	Terekawa et al., 2017	N/A
S. cerevisiae Smc2 (full length) in complex with Smc4 (full length, C-terminal 3 × StrepII-tag), Brn1 (full length, C-terminal 12 × HIS-tag 3 × HA-tag) and Ycs4 (full length)	This work	N/A
S. cerevisiae Smc2 (full length) in complex with Smc4 (full length, C-terminal 3 × StrepII-tag), Brn1_BC1/2 (full length, K409D, R411D, K414D, K451D, K452D, K454D, K456D, K457D, C-terminal 12 × HIS-tag 3 × HA-tag), Ycg1 (full length) and Ycs4 (full length)	This work	N/A
S. cerevisiae Smc2 (full length) in complex with Smc4 (full length, C-terminal 3 × StrepII-tag), Brn1_ΦD (full length, M391D, F394D, W402D, W408D, C-terminal 12 × HIS-tag 3 × HA-tag), Ycg1 (full length) and Ycs4 (full length)	This work	N/A
S. cerevisiae Smc2_Q147L (full length) in complex with Smc4_Q302L (full length, C-terminal 3 × StrepII-tag), Brn1 (full length, C-terminal 12 × HIS-tag 3 × HA-tag), Ycg1 (full length) and Ycs4 (full length)	Terekawa et al., 2017	N/A
S. cerevisiae Brn1 (384-529) Ycg1 (6-498, 556-932)	This work	N/A
S. cerevisiae Brn1_{short kleisin loop} (384-417, 445-529) Ycg1 (6-498, 556-932)	This work	N/A
S. pombe Cnd2 (416-544) Cnd3 (1-438, 474-823)	This work	N/A

Deposited Data

S. pombe Cnd3–Cnd2	This work	PDB: 5OQR
S. cerevisiae Ycg1–Brn1	This work	PDB: 5OQQ
S. cerevisiae Ycg1–Brn1–DNA (I)	This work	PDB: 5OQP
S. cerevisiae Ycg1–Brn1–DNA (II)	This work	PDB: 5OQO
S. cerevisiae Ycg1–Brn1–DNA (short kleisin loop)	This work	PDB: 5OQN

Experimental Models: Cell Lines

HeLa Kyoto H2B-mCherry	Neumann et al., 2010	N/A

Experimental Models: Organisms/Strains

S. cerevisiae C4237 (MATa/α, ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::empty-vector::URA3/ura3)	This work	N/A
S. cerevisiae C4239 (MATa/α ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::BRN1-PK₆::URA3/ura3)	This work	N/A
S. cerevisiae C4257 (MATa/α ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::brn1(K409D, R411D, K414D)-PK₆::URA3/ura3)	This work	N/A
S. cerevisiae C4259 (MATa/α ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::brn1(K451D, K452D, K454D, K456D, K457D)-PK₆::URA3/ura3)	This work	N/A
S. cerevisiae C4261 (MATa/α ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::brn1(K409D, R411D, K414D, K451D, K452D, K454D, K456D, K457D)-PK₆::URA3/ura3)	This work	N/A
S. cerevisiae C4491 (MATa, lys2::pGAL1 GAL4::LYS2, pep4::HIS3, bar1::hisG, [2micron pGAL7 SMC4-StrepII₃, pGAL10 SMC2, pGAL1 BRN1-His₁₂-HA₃, TRP1], [2micron pGAL1 YCG1, pGAL10 YCS4, URA3])	Terekawa et al., 2017	N/A
S. cerevisiae C4493 (MATa, lys2::pGAL1 GAL4::LYS2, pep4::HIS3, bar1::hisG, [2micron pGAL7 SMC4-StrepII₃, pGAL10 SMC2, pGAL1 BRN1-His₁₂-HA₃, TRP1], [2micron pGAL10 YCS4, URA3])	This work	N/A
S. cerevisiae C4516 (MATa, lys2::pGAL1 GAL4::LYS2, pep4::HIS3, bar1::hisG, [2micron pGAL7 SMC4-StrepII₃, pGAL10 SMC2, pGAL1 BRN1(K409D, R411D, K414D, K451D, K452D, K456D, K457D)-His₁₂-HA₃, TRP1], [2micron pGAL1 YCG1, pGAL10 YCS4, URA3])	This work	N/A
S. cerevisiae C4724 (MATa, lys2::pGAL1 GAL4::LYS2, pep4::HIS3, bar1::hisG, [2micron pGAL7 SMC4(Q302L)- StrepII₃, pGAL10 SMC2(Q147L), pGAL1 BRN1-His₁₂-HA₃, TRP1], [2micron pGAL1 YCG1, pGAL10 YCS4, URA3])	Terekawa et al., 2017	N/A
S. cerevisiae C4895 (MATa/α ade2-1, trp1-1, can1-100, leu2-3,112, GAL, psi+, brn1::HIS3/BRN1, ura3::brn1(M391D, F394D, W402D, W408D)-PK₆::URA3/ura3)	This work	N/A
S. cerevisiae C5037 (MATa, lys2::pGAL1 GAL4::LYS2, pep4::HIS3, bar1::hisG, [2micron pGAL7 SMC4-StrepII₃, pGAL10 SMC2, pGAL1 BRN1(M391D, F394D, W402D, W408D)-His₁₂-HA₃, TRP1], [2micron pGAL1 YCG1, pGAL10 YCS4, URA3])	This work	N/A

Oligonucleotides

EMSA 35-mer template forward strand 5′-6-FAM-CCTATAGTGAGTCGTTCGATATTACAATTC ACTGG-3′	This work/modified from Piazza et al., 2014	N/A
EMSA 35-mer template reverse strand 5′-CCAGTGAATTGTAATATCGAACGACTCACTATAG G-3′	This work/modified from Piazza et al., 2014	N/A
ITC 25-mer annealed forward strand 5′-CCTATAGTGAGTCACAATTCACTGG-3′	This work/modified from Piazza et al., 2014	N/A
ITC 25-mer annealed reverse strand 5′-CCAGTGAATTGTGACTCACTATAGG-3′	This work/modified from Piazza et al., 2014	N/A
Crystallization 18-mer annealed palindromic dsDNA 5′-GATGTGTAGCTACACATC-3′	This work	N/A
22-mer annealed palindromic dsDNA 5′-GATTCGTGTAGCTACACGAATC-3′	Seifert et al., 2016	N/A
qPCR CEN4 (SC-77) forward primer 5′-TGGTGTGGAAGTCCTAATATCG-3′	Cuylen et al., 2011	N/A
qPCR CEN4 (SC-78) reverse primer 5′-TGCATGATCAAAAGGCTCAA-3′	Cuylen et al., 2011	N/A
qPCR rDNA (SC-41) forward primer 5′-TTTCTGCCTTTTTCGGTGAC-3′	Cuylen et al., 2011	N/A
qPCR CEN4 (SC-42) reverse primer 5′-TGGCATGGATTTCCCTTTAG-3′	Cuylen et al., 2011	N/A

Recombinant DNA

Plasmid pGEX6PI-Brn1_336-714 (N-terminal GST-tag-HRV3C-cleavage-site, residues 336-714 of C. thermophilum Brn1)	This work	N/A
Plasmid pETMCN-Ycs4 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 3-1222 of C. thermophilum Ycs4)	This work	N/A
Plasmid pETMCN-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 24-1006 of C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634, _BC1 -Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues R539D, R541D, K542D, K544D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634, _BC2 -Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues R554D, R556D, K557D, K559D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634, _BC1/2 -Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues R539D, R541D, K542D, K544D, R554D, R556D, K557D, K559D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634 -Ycg1_YC1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-1006 with mutated residues K100D, K101D of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634 -Ycg1_YC2 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-1006 with mutated residues K916D, K917D of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634 -Ycg1_YC1+YC2 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-1006 with mutated residues K100D, K101D, K916D, K917D of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_225-512-Ycs4 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-512 of C. thermophilum Brn1 and residues 3-1222 of untagged C. thermophilum Ycs4)	This work	N/A
Plasmid pETMCN-Brn1_539-634-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 539-634 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_549-634-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 549-634 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_572-634-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 572-634 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-601-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-601 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1_78-1006 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 78-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1_24-823 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-823 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1_24-883 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-883 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1_24-934 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-934 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634-Ycg1_24-982 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 of C. thermophilum Brn1 and residues 24-982 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, E514C-R629C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues E514C, R629C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, L521C-S611C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues L521C, S611C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, D525C-S610C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues D525C, S610C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, E514C-Q613C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues E514C, Q613C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, A527C-R629C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues A527C, R629C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, S568C-R629C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues S568C, R629C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, E514C-R629C}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues E514C, R629C of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{515-634, cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_225-634-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_225-634-Ycg1_YC1+YC2 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 of C. thermophilum Brn1 and residues 24-1006 with mutated residues K100D, K101D, K916D, K917D of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{225-634, BC1/2}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 with mutated residues R539D, R541D, K542D, K544D, R554D, R556D, K557D, K559D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{225-634, E514C-R629C}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 with mutated residues E514C, R629C of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{225-634, E514C-R629C cleavable}-Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 with mutated residues E514C, R629C and HRV3C cleavage site inserted after P549 of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_225-634, _ΦD -Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 225-634 with mutated residues L521D, F524D, W532D, W538D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_515-634, _ΦD -Ycg1 (N-terminal 6 × HIS-tag-TEV-cleavage-site, residues 515-634 with mutated residues L521D, F524D, W532D, W538D of C. thermophilum Brn1 and residues 24-1006 of untagged C. thermophilum Ycg1)	This work	N/A
Plasmid 2micron pGAL7 SMC4-StrepII₃ (C-terminal 3 × StrepII-tag of S. cerevisiae full length Smc4), pGAL10 SMC2 (S. cerevisiae full length Smc2), pGAL1 BRN1-His₁₂-HA₃ (C-terminal 12 × HIS-3 × HA-tag of S. cerevisiae full length Brn1), TRP1	St-Pierre et al., 2009	N/A
Plasmid 2micron pGAL7 SMC4-StrepII₃ (C-terminal 3 × StrepII-tag with mutated residue Q302L of S. cerevisiae full length Smc4), pGAL10 SMC2 (with mutated residue Q147L of S. cerevisiae full length Smc2), pGAL1 BRN1-His₁₂-HA₃ (C-terminal 12 × HIS-3 × HA-tag of S. cerevisiae full length Brn1), TRP1	Terekawa et al., 2017	N/A
Plasmid 2micron pGAL1 YCG1 (S. cerevisiae full length Ycg1), pGAL10 YCS4 (S. cerevisiae full length Ycs4), URA3	Terekawa et al., 2017	N/A
Plasmid 2micron pGAL7 SMC4-StrepII₃ (C-terminal 3 × StrepII-tag of S. cerevisiae full length Smc4), pGAL10 SMC2 (S. cerevisiae full length Smc2), pGAL1 BRN1-His₁₂-HA₃ (C-terminal 12 × HIS-3 × HA-tag with mutated residues K409D, R411D, K414D, K451D, K452D, K454D, K456D, K457D of S. cerevisiae full length Brn1), TRP1	This work	N/A
Plasmid 2micron pGAL7 SMC4-StrepII₃ (C-terminal 3 × StrepII-tag of S. cerevisiae full length Smc4), pGAL10 SMC2 (S. cerevisiae full length Smc2), pGAL1 BRN1-His₁₂-HA₃ (C-terminal 12 × HIS-3 × HA-tag with mutated residues M391D, F394D, W402D, W408D of S. cerevisiae full length Brn1), TRP1	This work	N/A
Plasmid 2micron pGAL10 YCS4 (S. cerevisiae full length Ycs4), URA3	This work	N/A
Plasmid pETMCN-Brn1_384-529-Ycg1_{6-932, Δ499-555} (N-terminal 6 × HIS-tag-HRV3C-cleavage-site, residues 384-529 of S. cerevisiae Brn1 and residues 6-932 with deleted residues 499-555 of untagged S. cerevisiae Ycg1)	This work	N/A
Plasmid pETMCN-Brn1_{384-529, short kleisin loop}-Ycg1_{6-932, Δ499-555} (N-terminal 6 × HIS-tag-HRV3C-cleavage-site, residues 384-529 with deleted residues 418-444 of S. cerevisiae Brn1 and residues 6-932 with deleted residues 499-555 of untagged S. cerevisiae Ycg1)	This work	N/A
Plasmid pETMCN-Cnd2_416-544-Cnd3_{1-823, Δ439-473} (N-terminal 6 × HIS-tag-HRV3C-cleavage-site, residues 416-544 of S. pombe Cnd2 and residues 1-823 with deleted residues 439-473 of untagged S. pombe Cnd3)	This work	N/A
Plasmid pC1-NCAPH (N-terminal Flag-EGFP-tag of H. sapiens NCAPH)	Piazza et al., 2014	N/A
Plasmid pC1-NCAPH2 (N-terminal Flag-EGFP-tag of H. sapiens NCAPH2)	Piazza et al., 2014	N/A
Plasmid pC1-NCAPH_BC1/2 (N-terminal Flag-EGFP-tag with mutations R446D, R448D, R450D, R451D, K452D, K462D, K463D, K464D, K467D, K468D of H. sapiens NCAPH)	This work	N/A
Plasmid pC1-NCAPH _ΦA (N-terminal Flag-EGFP-tag with mutations Y428A, F431A, W439A, W445A of H. sapiens NCAPH)	This work	N/A
Plasmid pC1-NCAPH _ΦD (N-terminal Flag-EGFP-tag with mutations Y428D, F431D, W439D, W445D of H. sapiens NCAPH)	This work	N/A
Plasmid pC1-NCAPH _S432A (N-terminal Flag-EGFP-tag with mutations S432A of H. sapiens NCAPH)	This work	N/A
Plasmid pC1-NCAPH2_BC1/2 (N-terminal Flag-EGFP-tag with mutations K329D, K332D, K333D, R335D, K350D, R351D, K352D, R353D, K354D of H. sapiens NCAPH2)	This work	N/A

Software and Algorithms

X-ray Detector Software (XDS)	Kabsch, 2010	http://xds.mpimf-heidelberg.mpg.de/
SHELX	Sheldrick, 2008	http://shelx.uni-ac.gwdg.de/SHELX/
Phenix suite	Adams et al., 2010	https://www.phenix-online.org/
CCP4 suite	Winn et al., 2011	http://www.ccp4.ac.uk/
COOT v0.8.2	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PyMOL	Schrödinger, LLC	https://www.pymol.org/
ConSurf	Ashkenazy et al., 2016	http://bental.tau.ac.il/new_ConSurfDB/
APBS	Baker et al., 2001	http://www.poissonboltzmann.org/
PISA	Krissinel and Henrick, 2007	http://www.ccp4.ac.uk/MG/ccp4mg_help/pisa.html