A Physical Assay for Sister Chromatid Cohesion In Vitro Dmitri Ivanov, Kim Nasmyth  Molecular Cell  Volume 27, Issue 2, Pages 300-310 (July 2007) DOI: 10.1016/j.molcel.2007.07.002 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Two Forms of Centromeric Plasmid Identified by Sucrose-Gradient Centrifugation and Agarose-Gel Electrophoresis (A) The experimental scheme. Lysates from yeast strains carrying a centromeric plasmid are centrifuged through sucrose gradients. Fractions collected from the gradient are then separated on agarose gels without any additional purification (“native” gel). Finally, specific DNAs are detected by Southern blotting. Duplicated DNAs held together by cohesin (indicated) should sediment more rapidly and electrophorese more slowly. (B) Lysates from spheroplasts of strain K11549 arrested with nocodazole were sedimented in sucrose gradients and fractions 21–77 separated on an agarose gel. Minichromosome and 2 micron DNAs were detected by Southern blotting. Fraction 1 corresponds to the top and fraction 77 to the bottom of the gradient. Electrophoresis reveals a large and a small form of the minichromosome but only a single form of 2 micron DNA. (C) Quantification of the bands in (B) using the ImageQuant 5.2 program. Three point average of adjacent fractions was plotted as the percentage of the total sum of pixels for the particular form of the plasmid in all fractions. Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Large Minichromosomes Are Absent in G1 An exponentially growing culture of a wild-type yeast strain (K699) was arrested in G1 with α factor, in a G2/M-like phase with nocodazole, or left cycling. Cellular DNA contents were determined by FACS analysis using propidium iodide to stain DNA and are shown on the right. Cell lysates were separated on sucrose gradients and agarose gels as in Figure 1A, and minichromosome DNA was detected by Southern blotting. Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Large Minichromosomes Are Dependent on Eco1 and Sediment with the Same Velocity as Intercatenated Sister DNAs Exponential cultures of strains K699 (wild type), K14727 (eco1), and K14726 (top2-4) were arrested with α factor at the permissive (25°C) temperature for 2 hr and then released from the arrest into nocodazole-containing media at restrictive temperature (35°C) for an additional 2 hr. (A) Yeast lysates were separated on sucrose gradients, and fractions were electrophoresed in agarose gels either before (Native) or after (Purified) deproteinization. In the case of the latter, agarose gels contained ethidium bromide. The different forms of plasmids are indicated by colored arrows. (B) FACS analysis of the cellular DNA content showing that all strains underwent DNA replication at the restrictive temperature. (C) Quantification of the results of the Southern blots of the top2-4 mutant strain comparing the positions of the peaks of cohesed (estimated from the “Native plasmid” gel) and concatenated plasmids (estimated from the “Purified plasmid” gel). Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Cleavage of Scc1 Destroys Cohesion and Releases Individual Minichromosomes (A) Outline of the experiment. (B) Lysates from nocodazole-arrested yeast strains (K11549 [SCC1(TEV3)-HA6] versus K13082 [SCC1-HA6]) were separated on sucrose gradients. Individual fractions containing either mostly noncohesed, or a mixture of cohesed and noncohesed, or mostly cohesed minichromosomes were incubated with TEV protease to cut Scc1 and then separated on “native” agarose gels. Minichromosome DNA was detected by Southern blotting. (C) Individual gradient fractions from the experiment shown in (B) containing different forms of 2 micron plasmid were treated with TEV protease and then separated on a “native” agarose gel. Southern blot was probed for 2 micron plasmid. Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Minichromosome Dimers/Catenates Are Resistant to Scc1 Cleavage Yeast strains (K11549 [wild type SCC1(TEV3)-HA6] and K15189 [eco1-1 top2-4 SCC1(TEV3)-HA6]) were arrested with α factor at permissive (22.5°C) temperature for 3 hr and then released from the α factor arrest into nocodazole-containing media at restrictive temperature (35°C) for 1.5 hr. Yeast lysates were separated on sucrose gradients. (A) Individual gradient fractions were incubated with TEV protease to cut Scc1 and then separated on “native” agarose gels. Minichromosome DNA was detected by Southern blotting. (B) DNA was purified from selected fractions by means of phenol/chloroform extraction and ethanol precipitation, and separated on an agarose gel with ethidium bromide. We noticed that dimers tended to be more heterogenous in top2 mutants, and there appears to exist a new species of dimer that cannot be resolved by BglII. Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Linearization of the Circular Minichromosomes Destroys Cohesion and Releases Individual Minichromosomes (A) Outline of the experiment. (B) Yeast strain (K699) was transformed with the minichromosome plasmid containing a BglII restriction endonuclease site or with an equivalent plasmid whose BglII site had been mutated. Lysates of nocodazole-arrested culture were separated on sucrose gradients. Individual fractions were incubated with BglII enzyme and then separated on a “native” agarose gel. Minichromosome DNA was detected by Southern blotting. (C) Gradient fractions were treated with BglII enzyme or mock treated, and DNA was subsequently purified by means of phenol/chloroform extraction and ethanol precipitation and separated on an agarose gel containing ethidium bromide. The Southern blot shows that most of the minichromosome DNA was linearized by BglII. (D) Minichromosome DNA from the gradient fractions of the experiment shown in Figure 3 (top2-4 mutant) was purified by phenol/chloroform extraction and ethanol precipitation and then nicked with DNase I in the presence of ethidium bromide. Early and late fractions of the gradient are shown. The Southern blot shows that fractions contain plasmids predominantly in the closed circular form that is converted to nicked form upon treatment with ethidium bromide and DNase I. Late fractions contain in addition closed circular concatemers and a minor fraction of concatemers where one of the plasmids is presumably nicked (turquoise arrowhead). Both forms are converted into nicked concatemers upon treatment with DNase I and ethidium bromide. Molecular Cell 2007 27, 300-310DOI: (10.1016/j.molcel.2007.07.002) Copyright © 2007 Elsevier Inc. Terms and Conditions