AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/Rad51 in somatic hypermutation.
Ontology highlight
ABSTRACT:
SUBMITTER: Zan H
PROVIDER: S-EPMC4625537 | biostudies-other | 2003 Jun
REPOSITORIES: biostudies-other
Publications
AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/Rad51 in somatic hypermutation.
Zan Hong H Wu Xiaoping X Komori Atsumasa A Holloman William K WK Casali Paolo P
Immunity 20030601 6
Somatic hypermutation (SHM) of immunoglobulin (Ig) genes appears to involve the generation of double-strand DNA breaks (DSBs) and their error-prone repair. Here we show that DSBs occur at a high frequency in unrearranged (germline) Ig variable (V) genes, BCL6 and c-MYC. These DSBs are blunt, target the mutational RGYW/RGY hotspot, and would be resolved through nonhomologous end-joining, as indicated by the presence of Ku70/Ku86 on these DNA ends. Upon CD40-induced expression of activation-induce ...[more]
Similar Datasets
Project description:DNA double-strand break repair allows cells to survive both exogenous and endogenous insults to the genome. In yeast, the recombinases Rad51 and Rad52 are central to multiple forms of homology-dependent repair. Classically, Rad51 and Rad52 are thought to act cooperatively, with formation of the functional Rad51 nucleofilament facilitated by the mediator function of Rad52. Several studies have now identified functions for the interaction between Rad51 and Rad52 that are independent of the mediator function of Rad52 and affect a seemingly diverse array of functions in de novo telomere addition, global chromosome mobility following DNA damage, Rad51 nucleofilament stability, checkpoint adaptation, and microhomology-mediated chromosome rearrangements. Here, we review these functions with an emphasis on our recent discovery that the Rad51-Rad52 interaction influences the probability of de novo telomere addition at sites preferentially targeted by telomerase following a double-strand break (DSB). We present data addressing the prevalence of sites within the yeast genome that are capable of stimulating de novo telomere addition following a DSB and speculate about the potential role such sites may play in genome stability.
| S-EPMC7492393 | biostudies-literature
Project description:Homologous recombination among repetitive sequences is an important mode of DNA repair in eukaryotes following acute radiation exposure. We have developed an assay in Saccharomyces cerevisiae that models how multiple DNA double-strand breaks form chromosomal translocations by a nonconservative homologous recombination mechanism, single-strand annealing, and identified the Rad52 paralog, Rad59, as an important factor. We show through genetic and molecular analyses that Rad59 possesses distinct Rad52-dependent and -independent functions, and that Rad59 plays a critical role in the localization of Rad52 to double-strand breaks. Our analysis further suggests that Rad52 and Rad59 act in multiple, sequential processes that determine genome structure following acute exposure to DNA damaging agents.
| S-EPMC3496973 | biostudies-literature
Project description:Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites.
| S-EPMC2790616 | 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:RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51-DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.
| S-EPMC2926616 | biostudies-literature
Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection, and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.
2014-03-23 | E-MTAB-1241 | 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:Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.
| S-EPMC380999 | biostudies-literature
Project description:A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells.
| S-EPMC6786661 | biostudies-literature
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.
| S-EPMC4030556 | biostudies-literature