ABSTRACT: Cohesin is a multisubunit complex that mediates sister-chromatid cohesion. Its Smc1 and Smc3 subunits possess ABC-like ATPases at one end of 50 nm long coiled coils. At the other ends are pseudosymmetrical hinge domains that interact to create V-shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin's kleisin subunit Scc1 bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring. It has been suggested that cohesin associates with chromosomes by trapping DNA within its ring. Opening of the ring due to cleavage of Scc1 by separase destroys sister-chromatid cohesion and triggers anaphase. We show that cohesin's hinges are not merely dimerization domains. They are essential for cohesin's association with chromosomes, which is blocked by artificially holding hinge domains together but not by preventing Scc1's dissociation from SMC ATPase heads. Our results suggest that entry of DNA into cohesin's ring requires transient dissociation of Smc1 and Smc3 hinge domains. Keywords: Cohesin loading, Scc1, Smc1, Smc3, Hinge opening, ChIP-chip â?¢ Experimental factors Distribution of the mutant and wild type form of cohesin in mitotic G2 phase. â?¢ Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with SUP (supernatant) fraction. SUP fraction is equal to WCE (Whole Cell Extract) fraction. Essentially WCE or SUP fraction of any cell cycle phase can be used for normalization, and there was no effect on protein binding profiles. Cerevisiae chromosome VI array was used. â?¢ Quality control steps taken Confirmation using different tags. Confirmation by q-PCR. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immuno-precipitated from cells containing no tag recognized by antibody used. Samples used, extract preparation and labelling: â?¢ The origin of each biological sample Saccharomyces cerevisiae (W303). â?¢ Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VIwere performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). â?¢ Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. â?¢ Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (Lengronne et al. Nature 2004). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). â?¢ Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ?¤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ?¤0.025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. â?¢ Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636
