Chromosome length influences replication-induced topological stress
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Katsuhiko Shirahige
PROVIDER: E-GEOD-26263 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Chromosome length influences replication-induced topological stress.
Kegel Andreas A Betts-Lindroos Hanna H Kanno Takaharu T Jeppsson Kristian K Ström Lena L Katou Yuki Y Itoh Takehiko T Shirahige Katsuhiko K Sjögren Camilla C
Nature 20110302 7338
During chromosome duplication the parental DNA molecule becomes overwound, 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 unknow ...[more]
Similar Datasets
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis S. cerevisiae chromosomes IIIâV, and chromosome VI highdensity oligonucleotide microarrays were provided by Affymetrix Custom Express Service (SC3456a520015F, P/N 520015; rikDACF, P/N 510636, respectively). Sequence and position of oligonucleotides on the microarrays are available from Affymetrix. ChIP was carried out as previously described (Katou et al. 2003; Katou et al. 2006): we disrupted 1.5 x 108 cells byMulti-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc.) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich) were used for chromatin immunoprecipitation. ChIPed DNA was purified and amplified by random priming as described (Katou et al. 2003): a total of 10 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp, purified, and the fragments were end-labelled with biotin-N6-ddATP23. Hybridization, washing, staining and scanning were performed according to the manufacturerâs instruction (Affymetrix). Primary data analyses were carried out using the Affymetrix microarray Suite version 5.0 software to obtain hybridization intensity, fold change value, change P-value and detection P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIPed fraction with supernatant fraction by using three criteria. First, the reliability of strength of signal was judged by detection P-value of each locus (P â¥0.025). Second, reliability of binding ratio was judged by change P-value (P ⥠0.025). Third, clusters consisting of at least three contiguous loci that filled the above Bermejo et al. 26 two criteria were selected, because it was known that a single site of proteinâDNA interaction will result in immunoprecipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbours. For the analyses of BrdU incorporation, cells were fixed by ice-cold buffer containing 0.1% azide, and then total DNA from 3 x108 cells was purified. DNA was sheared to 300 bp by sonication, denatured, and mixed with 2μg anti-BrdU monoclonal antibody (2B1D5F5H4E2; MBL). Antibody-bound and unbound fractions were subsequently purified, amplified, labelled and hybridized to the DNA chip.
2010-06-05 | E-GEOD-8305 | biostudies-arrayexpress
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.
2011-02-28 | GSE26263 | GEO
Project description:ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication. INO80 distribution in WT cells was measured.
2010-06-20 | E-GEOD-18570 | biostudies-arrayexpress
Project description:The actin-related proteins (ARPs) comprise a conserved protein family. Arp4p is found in large multisubunits of the INO80 and SWR1 chromatin remodeling complexes and in the NuA4 histone acetyltransferase complex. Here we show that arp4 (arp4S23AD159A) temperature-sensitive cells are defective in G2/M phase function. arp4 mutants are sensitive to the microtubule depolymering agent benomyl and arrest at G2/M phase at restrictive temperature. Arp4p is associated with centromeric and telomeric regions throughout cell cycle. Ino80p, Esa1p, and Swr1p, components of the INO80, NuA4, and SWR1 complexes, respectively, also associate with centromeres. The association of many kinetochore components including Cse4p, a component of the centromere nucleosome, Mtw1p, and Ctf3p is partially impaired in arp4 cells, suggesting that the G2/M arrest of arp4 mutant cells is due to a defect in formation of the chromosomal segregation apparatus. Keywords: ChIP-chip ⢠The goal of the experiment Genome-wide localization of Arp4 binding sites in Saccharomyces cerevisiae ⢠Experimental factors Distribution of Arp4 in WT in G2/M phase in the presence of nocodazole (Saccharomyces cerevisiae). ⢠Experimental design ChIP analysis: Hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Chromosome III, IV,V,VI S. cerevisiae: SC3456a520015F, P/N# 520015, affymetrix tiling array were used.
2010-06-11 | E-GEOD-7276 | biostudies-arrayexpress
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
Project description:The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO-system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate phosphate (Pi) metabolism for adaptation to Pi-starvation. Pi-starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4 thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system, in that Pho85 monitors whether environmental conditions are adequate for cell growth, and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress-response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress-response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4- and Rpo21-binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi-signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates transcription of antisense and intragenic RNAs in the KCS1 locus to downregulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Since Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in the Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low Pi-signaling, i.e., forming a positive feedback loop. We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independently of the Pi conditions, and many of the latter genes are involved in stress-response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs, as well as direct regulation of structural gene transcription, function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi condition to activate or repress transcription, which challenges our current understanding of Pho4 regulation. Wild type and pho4 deletion mutant were grown in low or high phosphate medium and distribution of Rpo21 was analyzed.
2008-12-23 | E-GEOD-13350 | biostudies-arrayexpress
Project description:The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles, and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.
2015-11-19 | GSE72241 | GEO
Project description:Sister chromatid cohesion, established during replication by the protein complex Cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally cohesion formation is strictly limited to the S-phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation we show that it is essential for repair in post-replicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and controlled by the DNA damage response and Cohesin regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair. Keywords: ChIP-chip analysis Scc2, Scc1, and rH2AX distribution with or without DSB in wild type and mutant strains. DSB was induced by galactose promoter driven HO. All yeast strains were haploid and of W303 origin (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3, RAD5). DNA was purified and amplified as previously described by Katou et al. (nature, 2003), and hybridized to SC3456a 52005F or S.cerevisiae Tiling 1.0F arrays, Part#520286 (Affymetrix). Scc2 was C- terminally tagged with 6HIS-3xFLAG, while Mcd1UNCL was marked with 3HA epitopes. Anti-FLAG antibody M2 (Sigma), anti-HA antibody (Babco), or anti γ-H2A-antobodies kindly provided by A. Verreault were used for immuno-precipitations.
2010-06-10 | E-GEOD-8270 | biostudies-arrayexpress
Project description:The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles, and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders. BrdU incorporation profiles by ssDNA-BrdU IP on chip have been generated as described (Katou et al., 2003). Protein binding profiles by ChIP-chip analysis were generated as described (Bermejo et al., 2009). Labeled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
2015-11-19 | E-GEOD-72241 | biostudies-arrayexpress
Project description:The Structural Maintenance of Chromosome (SMC) protein complexes cohesin, condensin and the Smc5/6 complex (Smc5/6) are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6’s recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 controls the spatial organization of supercoiled chromosomal regions. The results show that Smc5/6 associates with transcription-induced positively supercoiled chromosomal DNA at cohesin-dependent chromosome loop boundaries. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes, and efficiently initiates loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.
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