Similar Datasets
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: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:The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.
| S-EPMC5576896 | biostudies-literature
Project description:DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells
| PRJNA772567 | ENA
Project description:DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells [END-seq]
| PRJNA772573 | ENA
Project description:FANCJ helicase mutations are known to cause hereditary breast and ovarian cancers as well as bone marrow failure syndrome Fanconi anemia. FANCJ plays an important role in the repair of DNA inter-strand crosslinks and DNA double-strand breaks (DSBs) by homologous recombination (HR). Nonetheless, the molecular mechanism by which FANCJ controls HR mediated DSB repair is obscure. Here, we show that FANCJ promotes DNA end resection by recruiting CtIP to the sites of DSBs. This recruitment of CtIP is dependent on FANCJ K1249 acetylation. Notably, FANCJ acetylation is dependent on FANCJ S990 phosphorylation by CDK. The CDK mediated phosphorylation of FANCJ independently facilitates its interaction with BRCA1 at damaged DNA sites and promotes DNA end resection by CtIP recruitment. Strikingly, mutational studies reveal that ATP binding competent but hydrolysis deficient FANCJ partially supports end resection, indicating that in addition to the scaffolding role of FANCJ in CtIP recruitment, its helicase activity is important for promoting end resection. Together, these data unravel a novel function of FANCJ helicase in DNA end resection and provide mechanistic insights into its role in repairing DSBs by HR and in genome maintenance.
| S-EPMC7162537 | biostudies-literature
Project description:DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells [ChIP-Seq]
| PRJNA772575 | ENA
Project description:Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. Our findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.
2023-03-11 | PXD031357 | Pride
Project description:The retinoblastoma (RB) tumor suppressor is recognized as a master regulator that controls entry into the S phase of the cell cycle. Its loss leads to uncontrolled cell proliferation and is a hallmark of cancer. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double-strand breaks (DSBs) dependent on E2F1 and ATM kinase activity and promotes DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1, and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability, and renders mice hypersensitive to IR. Importantly, depletion of RB in osteosarcoma and breast cancer cell lines results in sensitivity to DNA-damaging drugs, which is further exacerbated by poly-ADP ribose polymerase (PARP) inhibitors. We uncovered a novel, nontranscriptional function for RB in HR, which could contribute to genome instability associated with RB loss.
| S-EPMC5159665 | biostudies-literature
Project description:During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.
| S-EPMC11329772 | biostudies-literature