Dataset Information

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions.

ABSTRACT: DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex DSBs, but in 40% of the isolated DSBs the recruitment was delayed approximately 5?min. Using base excision repair (BER) inhibitors we demonstrate that some simple DSBs are generated by enzymatic processing of base damage, while BER did not affect the complex DSBs. The results show that DSB processing and repair kinetics are dependent on the complexity of the breaks and can be different even for the same clastogenic agent.

SUBMITTER: Jakob B

PROVIDER: S-EPMC6989695 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions.

Jakob Burkhard B Dubiak-Szepietowska Monika M Janiel Ellen E Schmidt Alina A Durante Marco M Taucher-Scholz Gisela G

Scientific reports 20200129 1

DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex D ...[more]

PMID: 31996740

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Project description:This series is a supplementary data set for the manuscript titled "Postreplicative recruitment of Cohesin to double-strand breaks is required for DNA repair" All data shown in the paper plus duplicate experiments were registered (15 ChIP-chip data). Experimental design: 1. Type of experiment: ChIP (Chromatin immunoprecipitation) analysis hybridized to high density oligonucleotide arrays. The S. cerevisiae chromosome VI array described by Katou et al. (2003) Nature 424, 1078-83 and the newly developed S. cerevisiae chromosomes III-VIa have been used. 2. Experimental factors: Distribution of the sister chromatid cohesion protein Scc1, before and after induction of a DNA double strand break (dsb) on chromosome III, V and VI in G2/metaphase cells were analysed (Benomyl arrested cells). 3. Number of hybridizations performed: ChIP analysis: 15 4. Hybridisation design: ChIP analysis: comparison of ChIP fraction with SUP (supernatant) fraction 5. Quality control: Duplication of experiments, confirmation of recruitment of the analysed protein to a dsb by induction of breaks at three different positions in the genome. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Checking of the ChIP fraction by Western blotting. 6. URL of supplemental website: GEO Accession number: GSE1905 Web site: http://chromosomedynamics.bio.titech.ac.jp Samples used, extract preparation and labeling: 1. Origin of the biological sample Budding yeast (Saccharomyces cerevisiae). 2. Manipulation of the samples and the protocols used: Mitotic budding yeast cells were synchronised in G2/M with Benomyl (80 ug/ml) Cells were fixed 30 minutes at room temperature plus over night in 1% formaldehyde at 4C. 3. Protocols for preparing extracts: ChIP analysis: Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x10^8 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 65Â¨Â¬C 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 95Â¨Â¬C, fragments were labelled as detailed below. 4. Labeling protocol DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37C as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. 5. 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 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100C for 10 minutes, and then put on ice before being hybridized for 16 hours at 42C in an hybridization oven (GeneChip Hybri. Oven 320, Affymetrix). Washing and scanning protocol (FlexMidi-euk2v3-450) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). 6. Measurement data and specifications: S. cerevisiae arrays were scanned using the HP GeneArray Scanner (Affymetrix) at an emission wavelength of 560 nm, resolution 7.5 uM. Grids were aligned to scans following the library array description. Primary data analyses were carried out using Affymetrix Microarray Suite Ver.5.0 software to obtain signal intensity, fold change value, change p-value and detection p-value. The detailed information of this analysis is available at http://www.affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf and http://www.affymetrix.com/support/technical/technotes/statistical_reference_ guide.pdf To discriminate significant signals for binding to DNA (dark grey signals), signals from the ChIP fraction scan were compared to the SUP (supernatant) fraction scan using the 3 following criteria: first, the signal reliability was judged by the detection p-value of each locus (p-value <=0.025); secondly, reliability of binding ratio was judged by change p-value (p- value <=0.025); thirdly, only clusters of at least 3 contiguous loci responding to the 2 previous criteria were selected as significantly enriched locus. The light grey signals represent the statistically not significantly enriched signals. The software to present the result (chr6viewer) is available at http://everythingchromovi.gsc.riken.go.jp. The raw and transformed data files are available at GEO database website and at our web site http://chromosomedynamics.bio.titech.ac.jp. 6. Array Design 1. General array design: in situ synthesized arrays by Affymetrix 2. Availability of arrays: commercially available from Affymetrix 3. Location and ID of each spot on arrays: available at our web site (http://chromosomedynamics.bio.titech.ac.jp) and from Affymetrix on request 4. Probe type: oligonucleotide 5. The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Chromosome III,IV,V,VIa S.cerevisiae: SC3456a520015F, P/N# 520015 Keywords: other

Dataset Information

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions.

Publications

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions.

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