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Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways

ABSTRACT: 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 â¢ The goal of the experiment - Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. â¢ 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 â¢ 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) â¢ 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. â¢ 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. â¢ Links to the publication, any supplemental websites or database accession numbers. http://tilingarray.bio.titech.ac.jp/smc56dsb Samples used, extract preparation and labelling: â¢ The origin of each biological sample Saccharomyces cerevisiae W303 (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5). â¢ 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 Âµg/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 100Âµg/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, 80Âµg/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. â¢ 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 65ÂºC 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 95ÂºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ÂºC 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 (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 100ÂºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ÂºC 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: 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. â¢ 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

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Yuki Katou

PROVIDER: E-GEOD-4808 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways.

Betts Lindroos Hanna H Ström Lena L Itoh Takehiko T Katou Yuki Y Shirahige Katsuhiko K Sjögren Camilla C

Molecular cell 20060601 6

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 determined here reveals that the complex works specifically on the duplicated genome in differently regulated pathways. The first 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 ar ...[more]

PMID: 16793545

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Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.

Project description:During chromosome duplication the parental DNA molecule becomes over-wound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replicationinduced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of budding yeast chromosomes. This opens for the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin–related Smc5/6 complex. The chromosomal association of the Smc5/6 complex is shown to increase in response to chromosome lengthening, chromosome circularization, or inactivation of Topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings 3. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of Topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings which form behind the replication machinery. Smc6、Nse4, and Scc2 profiles with or without topological tension was analyzed (in top2 mutant, cells with circular chromosome, and fragmented chromosome) . 17 ChIP samples and corresponding WCE fractions have been analyzed.

Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386

Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip â¢ The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae â¢ Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment â¢ Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. â¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.

Project description:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress. In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by M-NM-1-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24M-BM-0C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (M-NM-^Tmec1 M-NM-^Tsml1 and mec1-100 M-NM-^Tsml1), as well as control strains (M-NM-^Tsml1).

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Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways

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Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways.

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