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. Author manuscript; available in PMC: 2016 Feb 25.
Published in final edited form as: Mol Cell. 2010 Sep 10;39(5):689–699. doi: 10.1016/j.molcel.2010.08.008

Figure 1. Efficient Smc3 Acetylation by Eco1 at the Time of S Phase Depends on DNA Replication.

Figure 1

(A) HA6-tagged Smc3 associated with three chromosomal loci (as measured by ChIP and qPCR) in K16655 (MATa SMC3-HA6) and K16653 (MATa SMC3-HA6, dbf4::SWI5p-DBF4) cells that are released from α factor-induced G1 arrest into fresh medium at 25°C. Cell cycle progression was monitored by flow cytometry of DNA content, and the time of anaphase was microscopically determined by the occurrence of binucleated cells (upper panel). An untagged strain (K699) yielded no significant ChIP-qPCR signal.

(B) Smc3-HA6 was immunoprecipitated from wild-type (K16655) or SWI5p-DBF4 (K16653) cells that were released from α factor-induced G1 arrest into fresh medium at 25°C. The acetylation status of Smc3 was analyzed by western blotting using the α-acetyl lysine antibody (ST1027, Calbiochem).

(C). Strains K17957 (MATa SMC3-PK6::KanMX4, TRP1::PMET3CDC20, pGAL1-10SCC1(R180D, R268D)-HA3::LEU2, his3::tetR-GFP::HIS3, YEplac195) K17955 (MATa SMC3-PK6::KanMX4, TRP1:: PMET3CDC20, pGAL1-10SCC1(R180D,R268D)-HA3:: LEU2, YEplac195-Gal-Eco1) were grown to log phase in synthetic medium lacking methionine and arrested in metaphase for 2 hr in YEP medium supplemented with 2 mM methionine and 2% raffinose. After 2 hr, the sample was divided into three subsamples, one being maintained in metaphase arrest, the second being released to G1 arrest, and the third being released to the next S phase. In all cases 2% galactose was added to the media for 80 min to induce expression of Scc1-NC-HA3. Samples were taken at the indicated times for preparation of whole cell extract. Scc1-HA3 was immunoprecipitated using α-HA antibody, and the acetylation level of co-immunoprecipitated Smc3 was subsequently measured by α-Smc3-acetyl-K113 (Figure S1) anti-body via western blotting.

(D) Smc3-HA6 was immunoprecipitated from K16655 (MATa SMC3-HA6), K16842 (MATa SMC3-HA6 ctf8Δ), or K16845 (MATa SMC3-HA6 ctf4Δ) cells that were released from α factor-induced G1 arrest into fresh medium at 25°C. Western blots were performed using either HA-specific or acetyl-lysine-specific antibodies (ST1027, Calbiochem). Cell cycle progression was monitored by flow cytometry of DNA content, and the time of anaphase was microscopically determined by the occurrence of binucleated cells.