Figure 1. Tethered Sir2 mediates cohesion.

A) Evaluation of chromosomal cohesion by DNA circle formation. Site-specific recombination is initiated by galactose-induced expression of the R recombinase in cells arrested at M phase with microtubule inhibitors. Recombinase sites are depicted with half-filled boxes. Silent chromatin domains are highlighted with pink and lac-GFP is represented with green spheres. Cohesion of DNA circles yields a singe bright dot of fluorescence whereas lack of cohesion yields two dots. B) Representative image of M phase-arrested cells with circularized HMR loci. C) Protein-targeting assay for cohesion. Unexcised recombination cassettes are drawn for simplicity. The HMR silencers (orange ovals) were deleted and HMR-E is replaced with a fragment containing lexA sites (6lex^opssEB). D) Tethered Sir2 mediates cohesion. HMR circles were counted in strain CSW19 that lacked lexA, and in strains CSW36 and CSW37 that contained integrated expression cassettes for lexA alone or lexA-Sir2^78–562, respectively. N represents the number of cells examined. The strain bearing lexA alone was used as point of comparison in significance tests. E) Tethered Sir2 mediates silencing. Strain GA-2050 was transformed with plasmids expressing either lexA alone (pBTM116H) or lexA-Sir2^78–562 (pCSW22) and spotted in 10-fold serial dilutions on SC-trp,-his to measure silencing of the TRP1 reporter and SC-his to measure growth. The strain contains an array of 4 lexA operators in place of the Rap1 and Abf1 binding sites at the HMR-E silencer (Aeb-4lex^op) [76]. Parallel assays with the HMR-E silencer of the targeted cohesion assay (6lex^opssEB) yielded similar results, albeit less dramatic ones (data not shown).