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BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

ABSTRACT: Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage responsefactor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADPribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. Brca1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. B cells were stimulated to undergo class switch recombination in vitro. Chromatin from B cells was harvested 72 hours post-stimulation and used for RPA ChIP to study the extent of resection of DNA DSBs.

ORGANISM(S): Mus musculus

SUBMITTER: Robert Faryabi

PROVIDER: E-GEOD-35698 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Project description:Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage responsefactor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADPribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. Brca1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku.

2012-09-30 | GSE35698 | GEO

DNA repair proximity landscape analysis reveals shieldin as a key regulator of NHEJ-dependent processes

Project description:By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1.

2018-04-13 | PXD009284 | Pride

53BP1 cooperation with the REV7–shieldin complex underpins DNA structure-specific NHEJ

Project description:53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin—a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)— as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

2018-07-26 | PXD009650 | Pride

53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

Project description:53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks (DSBs), thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination (HR) in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the 'leaky' DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic dsDNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer (NK) cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer (HGSOC)s and pancreatic ductal adenocarcinoma (PDAC)s, which are refractory to ICBs, revealed that lower 53BP1 levels correlated with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

2024-07-09 | GSE237615 | GEO

RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity

Project description:The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

2022-02-04 | PXD031311 | Pride

Rif1 Prevents Resection of DNA Breaks and Promotes Immunoglobulin Class Switching

Project description:DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether it does so directly is not known. Here we identify Rapl-interacting factor 1 (Rif1) as an Ataxia-Telangiectasia Mutated (ATM) phosphorylation-dependent interactor of 53BP1, and show that absence of Rif1 results in 5M-bM-^@M-^Y-3M-bM-^@M-^Y DNA end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination (CSR) in B lymphocytes, and leads to accumulation of chromosome DSBs. Study of Rif1 DNA-end protection activity against resection via analysis of single-stranded DNA binding protein RPA and Rad51 accumulation at sites of AID-induced DNA damage by ChIP-seq. All samples shown in Fig. 4 are included (controls and test samples, 7 samples in total).

2013-03-01 | E-GEOD-42298 | biostudies-arrayexpress

Role of 53BP1 in End Protection and DNA synthesis at DNA breaks [END-seq]

Project description:Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single strand DNA (ssDNA) in BRCA1-deficient cells leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the (CTC1-STN1-TEN1) CST and DNA polymerase alpha (Polα) to counteract resection. We present evidence here that 53BP1-mediated exonuclease protection plays a more significant role than CST/Polα synthesis in countering hyper-resection at DSBs in G1 phase. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at AsiSI-induced DSBs. We show that in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, Exo1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, 53BP1 inhibits resection mainly through end-protection rather than by promoting fill-in.

2021-09-10 | GSE181669 | GEO

Role of 53BP1 in End Protection and DNA synthesis at DNA breaks [ChIP-seq]

2021-09-10 | GSE181668 | GEO

Role of 53BP1 in End Protection and DNA synthesis at DNA breaks [SAR-seq]

2021-09-10 | GSE181667 | GEO

Expression data of ATM, ATR, CHK1, CHK2, and 53BP1 shRNA knockdown and control shRNA in MCF-10A cells

Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNAs that target ATM, ATR, CHK1, CHK2, and 53BP1 genes were transfected in MCF-10A non-transformed breast cell line by lentiviral particles and selected stable ATM, ATR, CHK1, CHK2, and 53BP1 knockdown MCF-10A cells. Scrambled control shRNA-transfected and wild type MCF-10A cells were applying as control. All knockdown and control MCF-10A cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in MCF-10A medium and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three biological replicates were applied.

2014-01-24 | E-GEOD-54268 | biostudies-arrayexpress

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