The supernatant was mixed with 20 μl of anti-Flag M2 Agarose Affinity. Gel (Sigma-Aldrich) and incubated at 4°C for 1 h. The M2-agarose beads were washed.
Supplementary document Experimental procedure Immunoprecipitation YNIG20 cells grown in 1L YPDA to 1 x 107 cells/ml were added with 0.2 M HU and incubated for 2 h. The cells were harvested, washed two times with cold water and suspended in 24 ml of the buffer containing 50 mM HEPES-KOH [pH 7.5], 300 mM KCl, Tween-20 0.05%, NP-40 0.005%, glycerol 10%, protease inhibitor Complete (Roche) x1, protease inhibitor cocktail (Sigma) 1%, β-glycerophosphate 2 mM, NaF 20 mM, Na3VO4 and Na-pyrophosphate. The suspended cells were frozen in a liquid nitrogen, ground with a mortar grinder (Retch RM100), and stored at -80°C. The disrupted cell extract (0.5 ml) was thawed and used for each immuno-precipitation after centrifugation. The supernatant was mixed with 20 µl of anti-Flag M2 Agarose Affinity Gel (Sigma-Aldrich) and incubated at 4°C for 1 h. The M2-agarose beads were washed three times with 500 µl of the same buffer, suspended in 50 µl of the buffer containing 150 µg/ml 3 x Flag peptide and incubated at 4°C for 30 min. The eluted proteins were subjected to SDS-PAGE and following western blotting. Cross-linked immunoprecipitation Cells were cultivated to 1.2 x107 cells/ml and 17.5 ml of culture were withdrawn, mixed with 8% formaldehyde (final concentration, 1%) and incubated at 23°C for 20 min. One milliliter of 2.5 M glycine was added and the mixture was incubated at 23°C for 5 min. The cells were washed two times with 20 ml of ice-cold TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl), suspended in 0.4 ml of Lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1% TritonX-100, 0.1% sodium deoxycholate) and disrupted by beads in a bead shocker (Yasui Kikai: 2 min x 6 at the maximal speed). The extracts were sonicated (0.5 min x 6) to reduce the DNA size less than 500 bp and centrifuged. The supernatant was mixed with anti-Flag antibody M2 on Dynabeads proteinA (Dynal) and incubated at 4°C for 3 h. The beads were washed two times with 1 ml of Lysis buffer, two times with 1 ml of Lysis buffer containing 0.5 M NaCl, two times with 1 ml of Wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5%
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deoxycholate, 1 mM EDTA), one time with Lysis buffer and suspended in 36 µl of Elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS). The suspension was incubated at 100°C for 30 min and the supernatant was subjected to SDS-PAGE and following western blotting. Purification of GINS, Pol ε, Sld2 and Dpb11 proteins For purification of GINS, all the subunits of the GINS complex with an N-terminal 6×His tag on Psf3 were expressed in BL21-CodonPlus-RIPL E. coli cells. Cell extracts were purified on Ni-NTA Agarose (Invitrogen) and Resource Q (GE Healthcare). To purify DNA polymerase ε, the yeast strain YRT1 (Tsubota et al., 2006) (dpb3::DPB3-5FLAG with a PreScission protease recognition site between the Dpb3 C terminus and the Flag tag) was used. Cells were frozen in liquid nitrogen and ground with a mortar grinder (Retsch RM100). The cell extracts were first mixed with anti-Flag M2 Agarose Affinity Gel (Sigma-Aldrich). To remove the Flag tag, the Polε complex was separated from the Flag tag bound to beads with PreScission protease treatment. Further purification was performed using HiTrap Heparin HP (GE Healthcare) and Resource Q (GE Healthcare). To purify Sld2-10Flag, SLD2-10FLAG cloned on YEplac112 (Gietz and Sugino, 1988) was expressed in the yeast strain BJ2168. The cells were disrupted as for Pol ε, and the Sld2-10Flag was purified through three consecutive column chromatography, ANTI-FLAG M2 Agarose Affinity Gel (Sigma-Aldrich), HiTrap Heparin HP (GE Healthcare) and Resource PHE (GE Healthcare). The purified Sld2-10Flag migrated like the hypo-phosphorylated form observed in vivo in SDS-PAGE. Phosphorylation of Thr84, which is essential for binding to Dpb11, was not detected by anti-pThr84 antibody. For purify Dpb11-CBP, DPB11-TAP with the galactose-inducible promoter GAL1p was cloned on YEplac181 (Gietz and Sugino, 1988) and expressed in the yeast strain BJ2168. The cells were disrupted as for Polε and subjected to the two-step procedure for TAP protein purification (Rigaut et al., 1999), and Dpb11-CBP was obtained. Similarly, CBP-tagged Cdc45 and Mcm4 were purified from the cells bearing TAP-tagged Cdc45 or Mcm4 purchased from Open Biosystems (Ghaemmaghami et al.,
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2003) by the two-step procedure for TAP protein purification (Rigaut et al., 1999).
References Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O'Shea EK, Weissman JS. 2003. Global analysis of protein expression in yeast. Nature 425: 737-741. Gietz RD, Sugino A. 1988. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74: 527-534. Kamimura Y, Tak YS, Sugino A, Araki H. 2001. Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. EMBO J 20: 2097-2107. Kohzaki H, Murakami Y. 2007. Faster and easier chromatin immunoprecipitation assay with high sensitivity. Proteomics 7: 10-14. Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin, B. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotech 17: 1030-1032. Tsubota T, Tajima R, Ode K, Kubota H, Fukuhara N, Kawabata T, Maki S, Maki, H. 2006. Double-stranded DNA binding, an unusual property of DNA polymeras epsilon, promotes epigenetic silencing in Saccharomyces cerevisiae. J Biol Chem 281: 32898-32908.
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