BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE35698 | GEO | 2012/09/30
SECONDARY ACCESSION(S): PRJNA152035
REPOSITORIES: GEO
Similar Datasets
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. 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.
2012-09-30 | E-GEOD-35698 | biostudies-arrayexpress
Project description:By covalently linking Watson and Crick strands, DNA interstrand crosslinks (ICLs) are highly toxic lesions that block DNA replication and threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on one DNA strand to unhook the ICL. ICL unhooking generates a two-ended double-strand break (DSB) on one of the daughter molecules, while leaving a gap comprising the ICL-adduct on the other. Restoration of the adducted molecule by DNA polymerase zeta-mediated translesion synthesis (TLS) creates a template required for repair of the DSB via homologous recombination (HR), but how these processes are coordinated to ensure faithful ICL repair is not known. Here, we uncover a crucial role for SCAI in promoting ICL repair downstream of ID2 activation. Using Xenopus egg extracts and human cells, we show that SCAI forms a complex with DNA polymerase zeta and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display principal hallmarks of FA gene inactivation, including broken and radial chromosome accumulation. In the absence of SCAI, DSB intermediates are preferentially re-ligated by illegitimate DNA polymerase theta-dependent microhomology-mediated end-joining (MMEJ). Consistently, the hypersensitivity of SCAI-deficient cells to ICLs can be reverted by suppressing FA pathway activation. Our work establishes SCAI as a critical mediator of ICL resolution via the FA pathway, acting at the interphase of the TLS and HR steps to prevent erroneous repair and chromosomal instability.
2022-02-18 | PXD024280 | Pride
Project description:By covalently linking Watson and Crick strands, DNA interstrand crosslinks (ICLs) are highly toxic lesions that block DNA replication and threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on one DNA strand to unhook the ICL. ICL unhooking generates a two-ended double-strand break (DSB) on one of the daughter molecules, while leaving a gap comprising the ICL-adduct on the other. Restoration of the adducted molecule by DNA polymerase zeta-mediated translesion synthesis (TLS) creates a template required for repair of the DSB via homologous recombination (HR), but how these processes are coordinated to ensure faithful ICL repair is not known. Here, we uncover a crucial role for SCAI in promoting ICL repair downstream of ID2 activation. Using Xenopus egg extracts and human cells, we show that SCAI forms a complex with DNA polymerase zeta and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display principal hallmarks of FA gene inactivation, including broken and radial chromosome accumulation. In the absence of SCAI, DSB intermediates are preferentially re-ligated by illegitimate DNA polymerase theta-dependent microhomology-mediated end-joining (MMEJ). Consistently, the hypersensitivity of SCAI-deficient cells to ICLs can be reverted by suppressing FA pathway activation. Our work establishes SCAI as a critical mediator of ICL resolution via the FA pathway, acting at the interphase of the TLS and HR steps to prevent erroneous repair and chromosomal instability.
2022-02-18 | PXD029409 | Pride
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
Project description:53BP1 regulates DNA end-joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting RIF1 and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate non-homologous end-joining (NHEJ). However, how this axis regulates DNA end-joining and HR suppression remains unresolved. We investigated shieldin and its interplay with CST, a complex recently implicated in 53BP1-dependent activities. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination co-reliant on both complexes. DNA damage-responsive signalling promotes shieldin-CST interaction, demonstrating shieldin as a DSB-specific CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient cells independently of shieldin. These findings showcase the resilience of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.
2024-08-27 | PXD045534 | Pride
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
Project description:BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair
| PRJNA152035 | ENA
Project description:The twenty-three Fanconi Anemia proteins cooperate in a DNA repair pathway, called the FA/BRCA pathway, required for the monoubiquitination of FANCD2 and the excision of interstrand DNA crosslinks (ICLs). The CCAR1 (cell division cycle and apoptosis regulator 1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression and FANCD2 monoubiquitination. Interestingly, loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the so-called EF-hand domain, binds to the spliceosome SF3B complex and is required for its mRNA splicing activity. Taken together, CCAR1 is a unique splicing factor, required for normal splicing of the FANCA mRNA and perhaps other mRNAs involved in various cellular pathways.
2024-08-21 | GSE242781 | GEO
Project description:DNA interstrand crosslinks (ICLs) are highly cytotoxic DNA lesions that are commonly induced by endogenous metabolic processes and chemotherapeutic drugs, such as cisplatin. To prevent detrimental effects associated with ICLs, such as interference with both transcription and replication, cells rely on a complex coordination of different DNA repair pathways, including Fanconi Anemia (FA) and homologous recombination (HR), for their resolution. To unveil the breadth of factors that plays a role in this response, we performed CRISPR-based genome-wide screens treated with the clinically relevant cyclophosphamide agent. This approach, along with a fluorescence-based competition assay, defined C1orf112 as a novel modulator of the response to ICLs-inducing agents. Subsequent analysis showed that depletion of C1orf112 impairs genomic stability by increasing spontaneous levels of gamma-H2AX, micronuclei, and 53BP1-nuclear bodies. Using immunofluorescence approaches, we found that C1orf112 acts downstream of the FA pathways and is required for resolving ICL-induced RAD51 foci. Consistently, our results show that C1orf112 is required for homology-directed DNA repair, including HR and single-strand annealing. Proximal mapping of C1orf112 using TurboID technology identified the AAA+ ATPase FIGNL1 as a constitutive interactor and functional characterization of this complex shows that these C1orf112 and FIGNL1 cooperate in the unloading of RAD51 at ICL-induced lesion. Altogether, our findings reveal C1orf112 as a previously unidentified regulator of the AAA+ ATPase FIGNL1 in the resolution of ICLs by homology-directed DNA repair pathways.
2022-05-25 | MSV000089544 | MassIVE
Project description:The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.
2020-01-20 | PXD016256 | Pride