Project description:The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex here determined reveals that the complex works specifically on the duplicated genome in differently regulated pathways. One controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length, and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures. Keywords: ChIP-chip analysis ⢠The goal of the experiment - Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. ⢠Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended). Cell cycle, Double Strand Break Induction, time course after break induction, Saccharomyces cerevisiae, Chromosome VI array, Whole Genome Tiling Array, Smc5, Smc6, Nse1, Nse4, Nse5, Scc1, Scc2, Mre11, Rad53 ⢠Experimental factors Distribution of the Smc5/6 complex in the cell cycle, distribution of Smc5/6 complex before and after induction of a DNA double strand break on chromosome VI in G2/metaphase cells, distribution of Smc6 in the absence of functional Scc2, Mre11 or Rad53, distribution of Smc6 at collapsed replication forks, and distribution of Cohesin subunit Scc1 in G2/metaphase. Distribution was analyzed on Chromosome VI and/or whole genome arrays. Cells were grown in rich yeast cell extract media. All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae W303; ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5) ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array or Whole genome arrays were used. Total number of hybridization was 69. All description about hybridized samples is attached as the separate sheet. ⢠Quality control steps taken Duplication, confirmation using different tags, different subunits of the same complex, and time course after double-strand break induction. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. ⢠Links to the publication, any supplemental websites or database accession numbers. http://tilingarray.bio.titech.ac.jp/smc56dsb Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae W303 (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5). ⢠Manipulation of biological samples and protocols used Strains containing flag-tagged versions of Smc6, Smc5, Nse1, Nse4, or Nse5 proteins were used. For synchronization in G1, alpha factor was added (Zymo research and Innovagen AB) at 3 µg/ml final concentrations to logarithmically growing cells, and G1 arrest was obtained at indicated temperatures for 2.5 h. For S-phase arrest, cells were grown to log phase and arrested in G1 with alpha factor for 2 h, and then incubated in the presence of 200 mM hydroxyurea (HU) (Sigma) for 30 min. Cells were then washed twice with rich medium containing 100µg/ml pronase (Calbiochem). Finally cells were resuspended in medium containing HU and pronase, and growth continued for indicated time periods. G2/M arrest was achieved 2.5 h after addition of benomyl (Sigma, 80µg/ml), to logarithmically growing cells. G1, S and G2/M arrests were checked microscopically and by FACS. DSBs were induced by activation of the HO endonuclease from the GAL1-10-promoter by addition of 2% galactose to cell cultures initially grown in 2% raffinose. ⢠Technical protocols for preparing the hybridization extract Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x108 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The 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. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). 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 3'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). ⢠Measurement data and specifications: S. cerevisiae 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 or from our Web sites. 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 at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available from K.S. on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp and for whole genome arrays to 100bp. 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-value<=0.01 for chromosome VI and pvalue?0.0025 for whole genome array). Secondly, reliability of binding ratio was judged by change p-value (p-value<=0.01 for chromosome VI and p-value<=0.0025 for whole genome array). Thirdly, clusters consisting of at least 500bp 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. Under these conditions both types of arrays give essentially the same results for the binding profile. Repeated sequences were masked out and not included in the calculation of protein binding profile. The correlation coefficient of Smc6 binding profiles in G2/M from two independent experiments was 0.955, showing that the whole genome ChIP on chip data was reproducible. For comparison of the localization of Smc6 in different experiments and that of Smc6 and Scc1 on whole genome maps, peaks with signal to log ratio higher than 0.6 were chosen, and peaks which were distributed within a 5kb region was recognized as one peak. In the analysis of the number of Smc6 localization sites on each chromosome a 40 kb region spanning all centromeres were excluded. This region in addition to that downstream the rDNA repeats was also excluded from the analysis when comparing Smc6 localization in wild type, scc2-4 and scc1-73 cells. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Whole Genome Array S.cerevisiae: S.cerevisiae Tiling1.0F Arrays, Part#520286 and Part#520280
2010-06-11 | E-GEOD-4808 | biostudies-arrayexpress
