Igk locus capture-based target enrichment sequencing
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE138135 | GEO | 2020/08/24
REPOSITORIES: GEO
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Project description:Cell cycle is a major determinant of DNA double-strand break (DSB) repair pathway choice with homologous recombination (HR) that is most active in S phase cells and non-homologous end-joining (NHEJ) that dominates in G1 phase cells. A third less well-defined mechanism, 'alternative end-joining', has been shown to promote error-prone repair in NHEJ- or HR-deficient cells. Here, we have used a physiologic system of NHEJ-mediated genomic rearrangements induced by the site-specific RAG1/2 endonuclease in G1 cells to investigate the fate of unrepaired G1 DSBs upon entry into the cell cycle. We show that, in the absence of XRCC4, alternative end-joining rescues RAG-induced DSB repair and promotes chromosome translocations upon G1 cell cycle exit.
2020-08-24 | GSE138136 | GEO
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-08-25 | E-MTAB-12061 | biostudies-arrayexpress
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.
2016-08-19 | E-GEOD-84102 | biostudies-arrayexpress
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.
2016-08-19 | GSE84102 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186086 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186085 | GEO
Project description:The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.
2021-03-15 | GSE166631 | GEO
Project description:DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (Suppressor of Cancer Cell Invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signaling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.
2016-12-12 | PXD004912 | Pride
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