Dataset Information

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

ABSTRACT: High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. (56)iron and (28)silicon) by inhibiting Mre11 complex activity. Exposure of cells to (56)iron or (28)silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.

SUBMITTER: Xie M

PROVIDER: S-EPMC4333404 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

Xie Maohua M Park Dongkyoo D You Shuo S Li Rui R Owonikoko Taofeek K TK Wang Ya Y Doetsch Paul W PW Deng Xingming X

Nucleic acids research 20150107 2

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB re ...[more]

PMID: 25567982

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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

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 65¨¬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 95¨¬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 3’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

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

Publications

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

Similar Datasets

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