1. Volume 31, Issue 1, Pages 143-151 (July 2008)
Acetylation of Smc3 by Eco1 Is Required for S Phase Sister Chromatid Cohesion in Both Human and Yeast 
Jinglan Zhang, Xiaomin Shi, Yehua Li, Beom-Jun Kim, Junling Jia, Zhiwei Huang, Tao Yang, Xiaoyong Fu, Sung Yun Jung, Yi Wang, Pumin Zhang, Seong-Tae Kim, Xuewen Pan, Jun Qin 
Molecular Cell 
Volume 31, Issue 1, Pages (July 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Human SMC3 Is Acetylated at K105 and K106 by ESCO1
(A) A MS/MS spectrum that identifies K105 and K106 in a SMC3 peptide of 101VIGAK(Ac)K(Ac)DQYFLDK113 as doubly acetylated. SMC3 was purified from HeLa cell nuclear extracts, resolved on SDS-PAGE, and analyzed with capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-gel trypsin digestion.
(B) HeLa cells were transfected with siRNA SMARTpools as indicated. SILAC coupled with Selective Ion Monitoring (SIM) was used to quantify doubly acetylated SMC3 peptide. SMC3 acetylation level in cells with each individual acetyltransferase-depletion was normalized to that in siGFP-transfected cells. Error bars in this and the next two panels represent standard deviations.
(C) SMC3 double acetylation in cells transfected with 4 different siESCO1 duplexes was quantified as in (B).
(D) Effects of siRNA-resistant ESCO1 expression on SMC3 acetylation in siESCO1-transfected cells.
(E) Acetylation of an SMC3 fragment by recombinant ESCO1 (GST-ESCO1-C). A synthesized peptide containing SMC3 acetylation sites was used as the substrate, and the acetylation levels were measured with mass spectrometry as described in Figures S1D–S1F. A GST epitope tag was used as a control.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
SMC3 Acetylation at K105 and K106 Is Important for Sister Chromatid Cohesion and Genomic Stability in Human Cells
(A) Establishment of tetracycline inducible stable cell lines expressing SMC3-WT, -AA, and -QQ. Exogenous SMC3 was induced 24 hr after doxycycline addition and detected with western blotting.
(B) Acetylated SMC3 is enriched in chromatin. SMC3-WT or SMC3-AA was immunoprecipitated with an M2 antibody from soluble (S) and chromatin (or insoluble, I) fractions of the cell extracts, respectively, and western blotted using antibodies specifically against aceK105, aceK105/K106 (also see Figure S2C), or FLAG. The soluble part was NETN extract, and the insoluble part was sonicated pellet after NETN extraction. WCE, whole cell extract.
(C) Three classes of mitotic spreads made from inducible SMC3 cells as indicated.
(D) Quantification of the three classes of mitotic spreads made from cycling cells 48 hr after induction of variant SMC3 expression. Experiments in (D) and (E) were repeated three times, with more than 150 cells being counted for each experiment. Error bars represent standard deviations.
(E) Quantification of mitotic spread phenotypes after siESCO1 or siP53 transfection.
(F) Mitotic phenotypes were analyzed by immunostaining. Forty-eight hours after induction, cells were fixed and stained with α-tubulin antibody (red), INCENP antibody (green), and DAPI (blue).
(G) Quantification of cells with scattered chromosomes and multipole spindles in cells expressing the SMC3 variants. Bars represent 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Smc3 Is Acetylated in Yeast and the Enzymatic Activity of Eco1 Is Essential for Smc3 Acetylation and Cell Viability
(A) An alignment of the Smc3 acetylation sites across species was generated by ClustalW. Arrows represent the acetylated lysine identified in human and yeast.
(B) Smc3-TAP was purified from a wild-type yeast arrested in G1- (with α-factor), S- (with hydroxyurea, HU), G2/M-phase (with nocodazole), or 40 min after released from nocodazole-arrest. The relative levels of doubly acetylated Smc3 peptide were compared by mass spectrometry (also see Figure S3B). The budding index of each yeast population was shown in Figure S3D.
(C) Smc3-TAP was purified from wild-type (WT), eco1L96S/E177A/N266D (ts), and eco1R222G/K223G mutants, and the acetylation levels were measured by mass spectrometry (also see Figure S3C).
(D) Wild-type (WT) Eco1, Eco1R222G/K223G, and Eco1G211D were purified as GST fusion proteins from E. coli. The acetyltransferase activity of each recombinant protein reflected by autoacetylation was detected using an antibody against acetyl-Lysine. A 2× serial dilution titration of the wild-type recombinant protein was used to estimate the autoacetylation levels on the mutant forms. CB, Coomassie Blue.
(E) The growth of an eco1Δ yeast strain harboring an empty vector, ECO1 (WT), eco1R222G/K223G, or eco1G211D mutant allele was tested.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Smc3 Acetylation Is Essential for Cell Viability, S Phase Cohesion, and Genomic Stability
(A) A haploid-convertible SMC3/smc3Δ heterozygous diploid was transformed with an empty plasmid vector (YCplac33) or a plasmid harboring the wild-type SMC3 (pXP510), the smc3K112R (pXP519), smc3K113R (pXP520), or smc3K112R/K113R (pXP521) mutant allele. After meiosis, haploid MATa cells containing both the chromosomal smc3Δ allele and a plasmid were derived from the transformants by selecting on a synthetic haploid selection medium at 30°C for 2 days.
(B) The rate of cohesion loss was measured using a GFP dot assay in the wild-type strain (JBY385) harboring an empty vector (pXM024) or the smc3L912P ts mutant (XPY1122a) harboring an empty vector (pXM024), the wild-type SMC3 (pXM026), the smc3K112R (pXM028), smc3K113R (pXM030), or smc3K112R/K113R (pXM032) mutant allele. The experiments were carried out in duplicate at 37°C, a nonpermissive temperature for the ts mutant. Error bars in (B) and (C) represent standard deviations.
(C) The stability of a centromere-based plasmid in the wild-type strain (JBY385) or the XPY1122a ts mutant harboring an empty vector (pXM024), wild-type SMC3 (pXM026), or the smc3K113R (pXM030) allele was investigated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions