HSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex.
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ABSTRACT:
SUBMITTER: Richard DJ
PROVIDER: S-EPMC3061066 | biostudies-literature | 2011 Mar
REPOSITORIES: biostudies-literature
Publications
hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex.
Richard Derek J DJ Savage Kienan K Bolderson Emma E Cubeddu Liza L So Sairei S Ghita Mihaela M Chen David J DJ White Malcolm F MF Richard Kerry K Prise Kevin M KM Schettino Giuseppe G Khanna Kum Kum KK
Nucleic acids research 20101103 5
hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the ...[more]
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Project description:hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.
| S-EPMC3089470 | biostudies-other
Project description:In response to ionizing radiation, the MRE11/RAD50/NBN complex re-distributes to the sites of DNA double-strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for the activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine-to-alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine-to-glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared with control cells, whereas blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.
| S-EPMC3951136 | biostudies-literature
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 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
2010-05-27 | E-GEOD-1905 | biostudies-arrayexpress
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
2004-12-22 | GSE1905 | GEO
Project description:Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3? dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.
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Project description:Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.
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Project description: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.
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Project description:Abasic (AP) sites, the most common DNA lesions are frequently associated with double strand breaks (DSBs) and can pose a block to the final ligation. In many prokaryotes, nonhomologous end joining (NHEJ) repair of DSBs relies on a two-component machinery constituted by the ring-shaped DNA-binding Ku that recruits the multicatalytic protein Ligase D (LigD) to the ends. By using its polymerization and ligase activities, LigD fills the gaps that arise after realignment of the ends and seals the resulting nicks. Here, we show the presence of a robust AP lyase activity in the polymerization domain of Bacillus subtilis LigD (BsuLigD) that cleaves AP sites preferentially when they are proximal to recessive 5'-ends. Such a reaction depends on both, metal ions and the formation of a Watson-Crick base pair between the incoming nucleotide and the templating one opposite the AP site. Only after processing the AP site, and in the presence of the Ku protein, BsuLigD catalyzes both, the in-trans addition of the nucleotide to the 3'-end of an incoming primer and the ligation of both ends. These results imply that formation of a preternary-precatalytic complex ensures the coupling of AP sites cleavage to the end-joining reaction by the bacterial LigD.
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Project description:Resection is an early step in homology-directed recombinational repair (HDRR) of DNA double-strand breaks (DSBs). Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced "dirty" DSBs or even enzyme-induced "clean" DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX) at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility ("PFGE-shift"). Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, ?rad51 and ?rad52 cells deficient in late stages of HDRR, resection occurs at both ends of ?-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN) null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP) and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at ?-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at "dirty" and clean DSB ends. These approaches apply to resection at other DSBs. Given evolutionary conservation, the observations are relevant to DNA repair in human cells.
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