In on chromosomes [66] and that H4 mono-methylated on K20 binds CAP-D

In on chromosomes [66] and that H4 mono-methylated on K20 binds CAP-D3 of condensin II [68]. Our data confirm the association of CAP-D2 with histone H4, but suggest that K20 methylation may not be required for this association in mitotic chromosomes.5. PerspectivesRecent crystal structures and cross-linking analysis are providing a wealth of structural information about eukaryotic SMC protein complexes. The initial low-resolution structure presented here, together with other recently published work, should enable a new era of precise structure-based mutagenic analysis of the condensin complex.6.3. Native electrophoresis of the condensin complexFreshly purified condensin and cohesin complexes were separated in native PAGE Novex 3?2 bis ris gels according to the manufacturer’s instructions (Life Technologies).rsob.royalsocietypublishing.org6.4. Cross-linking of SMC complexes on chromosomes and scaffold preparationChicken chromosomes were purified as described previously [59]. To find the optimal ratio of protein : cross-linker, 1 mg of chromosomal protein was incubated with a 1-, 30-, 60-, buy HMPL-012 90-fold weight excess of BS3. The RRx-001 mechanism of action cross-linked proteins were analysed by immunoblotting. Anti-CAP-H antibody was used to reveal the ratio of cross-linker needed to optimally cross-link the condensin complex in situ. Purification of chromosomes from 500 ml of DT40 wildtype culture was carried out 11 times, and each time the chromosomes were cross-linked with a 30-fold weight excess of BS3 for 2 h on ice, followed by quenching with 50 mM ABC for 30 min. The cross-linked chromosome samples were supplemented with 2 mM CaCl2, treated with micrococcal nuclease (40 mg ml21; Worthington) for 30 min on ice, then diluted with an equal volume of freshly made TEE buffer (10 mM triethanolamine : HCl pH 9, 1 mM NaEDTA pH 9). Immediately, an equal volume of 2?NaCl lysis mix (20 mM Tris : HCl pH 9, 20 mM NaEDTA pH 9, 0.2 AMX, 4 M NaCl) was added. The samples were spun down at 14 000 r.p.m. for 5 min at 48C. The pellet containing the scaffold proteins was re-suspended in 1 ?SDS sample buffer, boiled for 5 min, sonicated for 15 min and boiled again for 5 min. The scaffold proteins were loaded into 20 wells of two 4 ?2 bis ris gels (Invitrogen) and separated in MOPS buffer for 2 h. The very top area of each lane containing the condensin complex was cut out and in-gel digested. The extracted peptides were analysed by SCX-HPLC as previously described [51].6. Material and methods6.1. Purification of SMC2/SMC4 subcomplex, condensin holocomplex and cohesin complexSMC2 knockout cells expressing SBP-tagged SMC2, CAP-H knockout cells expressing SBP-CAP-H and Scc1 knockout cells expressing 9Myc-tagged Scc1 [29,55,87] were grown as described previously [88] in 200 ng ml21 of doxycycline for at least 48 h. When cells reached a density of 106 per ml, nocodazole was added for a further 13 h to obtain a mitotic index of more than 80 . Cells were lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.25 M NaCl, 0.5 NP-40, 30 mg ml21 RNase A), supplemented with the protease inhibitors 1 mM PMSF (Sigma-Aldrich) and 1 mg ml21 CLAP (chymostatin, leupeptin, antipain, pepstatin A; Sigma-Aldrich) for 45 min on ice. After sonication, cellular debris was removed by centrifugation at 20 000g for 10 min at 48C. Cell lysates (4 ?108) were incubated either with 300 ml of streptavidin epharose beads (Streptavidin Plus UltraLink Resin, Pierce) for 2 h at 48C (SMC2/SMC4 and condensin) or with 200 ml of ant.In on chromosomes [66] and that H4 mono-methylated on K20 binds CAP-D3 of condensin II [68]. Our data confirm the association of CAP-D2 with histone H4, but suggest that K20 methylation may not be required for this association in mitotic chromosomes.5. PerspectivesRecent crystal structures and cross-linking analysis are providing a wealth of structural information about eukaryotic SMC protein complexes. The initial low-resolution structure presented here, together with other recently published work, should enable a new era of precise structure-based mutagenic analysis of the condensin complex.6.3. Native electrophoresis of the condensin complexFreshly purified condensin and cohesin complexes were separated in native PAGE Novex 3?2 bis ris gels according to the manufacturer’s instructions (Life Technologies).rsob.royalsocietypublishing.org6.4. Cross-linking of SMC complexes on chromosomes and scaffold preparationChicken chromosomes were purified as described previously [59]. To find the optimal ratio of protein : cross-linker, 1 mg of chromosomal protein was incubated with a 1-, 30-, 60-, 90-fold weight excess of BS3. The cross-linked proteins were analysed by immunoblotting. Anti-CAP-H antibody was used to reveal the ratio of cross-linker needed to optimally cross-link the condensin complex in situ. Purification of chromosomes from 500 ml of DT40 wildtype culture was carried out 11 times, and each time the chromosomes were cross-linked with a 30-fold weight excess of BS3 for 2 h on ice, followed by quenching with 50 mM ABC for 30 min. The cross-linked chromosome samples were supplemented with 2 mM CaCl2, treated with micrococcal nuclease (40 mg ml21; Worthington) for 30 min on ice, then diluted with an equal volume of freshly made TEE buffer (10 mM triethanolamine : HCl pH 9, 1 mM NaEDTA pH 9). Immediately, an equal volume of 2?NaCl lysis mix (20 mM Tris : HCl pH 9, 20 mM NaEDTA pH 9, 0.2 AMX, 4 M NaCl) was added. The samples were spun down at 14 000 r.p.m. for 5 min at 48C. The pellet containing the scaffold proteins was re-suspended in 1 ?SDS sample buffer, boiled for 5 min, sonicated for 15 min and boiled again for 5 min. The scaffold proteins were loaded into 20 wells of two 4 ?2 bis ris gels (Invitrogen) and separated in MOPS buffer for 2 h. The very top area of each lane containing the condensin complex was cut out and in-gel digested. The extracted peptides were analysed by SCX-HPLC as previously described [51].6. Material and methods6.1. Purification of SMC2/SMC4 subcomplex, condensin holocomplex and cohesin complexSMC2 knockout cells expressing SBP-tagged SMC2, CAP-H knockout cells expressing SBP-CAP-H and Scc1 knockout cells expressing 9Myc-tagged Scc1 [29,55,87] were grown as described previously [88] in 200 ng ml21 of doxycycline for at least 48 h. When cells reached a density of 106 per ml, nocodazole was added for a further 13 h to obtain a mitotic index of more than 80 . Cells were lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.25 M NaCl, 0.5 NP-40, 30 mg ml21 RNase A), supplemented with the protease inhibitors 1 mM PMSF (Sigma-Aldrich) and 1 mg ml21 CLAP (chymostatin, leupeptin, antipain, pepstatin A; Sigma-Aldrich) for 45 min on ice. After sonication, cellular debris was removed by centrifugation at 20 000g for 10 min at 48C. Cell lysates (4 ?108) were incubated either with 300 ml of streptavidin epharose beads (Streptavidin Plus UltraLink Resin, Pierce) for 2 h at 48C (SMC2/SMC4 and condensin) or with 200 ml of ant.