DNA-damage-induced degradation of EXO1 exonuclease limits DNA end resection to ensure accurate DNA repair

Abstract Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair. Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork. Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork. Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

The Journal of Cell Biology ◽

10.1083/jcb.200811079 ◽

2009 ◽

Vol 185 (4) ◽

pp. 587-600 ◽

Cited By ~ 58

Author(s):

Sophie Badie ◽

Chunyan Liao ◽

Maria Thanasoula ◽

Paul Barber ◽

Mark A. Hill ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Protein A ◽

Repair Process ◽

Ataxia Telangiectasia Mutated ◽

Checkpoint Signaling ◽

Cycle Arrest ◽

Dna End Resection ◽

End Resection ◽

Late Function

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.

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DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

Nature Communications ◽

10.1038/s41467-021-24341-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Prasun Chakraborty ◽

Kevin Hiom

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Rna Polymerase Ii ◽

Critical Role ◽

Dna Breaks ◽

Double Stranded Dna ◽

Recombination Repair ◽

Dna End Resection ◽

End Resection ◽

Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

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SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

Cell Reports ◽

10.1016/j.celrep.2017.08.008 ◽

2017 ◽

Vol 20 (8) ◽

pp. 1921-1935 ◽

Cited By ~ 65

Author(s):

Waaqo Daddacha ◽

Allyson E. Koyen ◽

Amanda J. Bastien ◽

PamelaSara E. Head ◽

Vishal R. Dhere ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Dna End Resection ◽

End Resection

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DNA damage–induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2105440118 ◽

2021 ◽

Vol 118 (38) ◽

pp. e2105440118

Author(s):

Xiaobin S. Wang ◽

Demis Menolfi ◽

Foon Wu-Baer ◽

Marco Fangazio ◽

Stefanie N. Meyer ◽

...

Keyword(s):

Dna Damage ◽

Essential Gene ◽

Physiological Role ◽

Chromosomal Translocation ◽

Cell Cycle Checkpoint ◽

End Joining ◽

M Cell ◽

Dna End Resection ◽

End Resection

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4−/−Tp53−/− mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs−/−Tp53−/− mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

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DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

Nature ◽

10.1038/nature02964 ◽

2004 ◽

Vol 431 (7011) ◽

pp. 1011-1017 ◽

Cited By ~ 486

Author(s):

Grzegorz Ira ◽

Achille Pellicioli ◽

Alitukiriza Balijja ◽

Xuan Wang ◽

Simona Fiorani ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Checkpoint ◽

Checkpoint Activation ◽

Dna End Resection ◽

End Resection

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The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice

Nature Cell Biology ◽

10.1038/ncb3259 ◽

2015 ◽

Vol 17 (11) ◽

pp. 1446-1457 ◽

Cited By ~ 69

Author(s):

Ismail Hassan Ismail ◽

Jean-Philippe Gagné ◽

Marie-Michelle Genois ◽

Hilmar Strickfaden ◽

Darin McDonald ◽

...

Keyword(s):

Dna Repair ◽

E3 Ligase ◽

Repair Pathway ◽

Pathway Choice ◽

Dna End Resection ◽

End Resection

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Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling

NAR Cancer ◽

10.1093/narcan/zcaa006 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Diego Dibitetto ◽

Jennie R Sims ◽

Carolline F R Ascenção ◽

Kevin Feng ◽

Dongsung Kim ◽

...

Keyword(s):

Dna Repair ◽

Cell Division ◽

Cancer Cells ◽

Cancer Cell ◽

Replication Stress ◽

Parp Inhibitors ◽

Multiple Cell ◽

Dna End Resection ◽

Atr Kinase ◽

End Resection

Abstract Most cancer cells experience oncogene-induced replication stress and, as a result, exhibit high intrinsic activation of the ATR kinase. Although cancer cells often become more dependent on ATR for survival, the precise mechanism by which ATR signaling ensures cancer cell fitness and viability remains incompletely understood. Here, we find that intrinsic ATR signaling is crucial for the ability of cancer cells to promote DNA end resection, the first step in homology-directed DNA repair. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents the engagement of RAD51 as well as RAD52 at nuclear foci, leading to toxic DNA-PKcs signaling and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and engagement of RAD52. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.

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ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1

Cell Death Discovery ◽

10.1038/s41420-021-00633-7 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Shuang Yan ◽

Man Song ◽

Jie Ping ◽

Shu-ting Lai ◽

Xiao-yu Cao ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Homologous Recombination ◽

Mammalian Cells ◽

Cell Cycle Checkpoint ◽

Dependent Manner ◽

Dna Breaks ◽

M Cell ◽

End Resection

AbstractTo maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

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PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

10.1101/470427 ◽

2018 ◽

Author(s):

María Teresa Villoria ◽

Pilar Gutiérrez-Escribano ◽

Facundo Ramos ◽

Esmeralda Alonso-Rodríguez ◽

Eva Merino ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Kinase Activity ◽

Strand Break ◽

Double Strand Break ◽

Dna Lesion ◽

Damage Response ◽

End Resection ◽

Recombinational Dna Repair

AbstractThe role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by the Mec1 kinase and its own auto-phosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 phosphorylation and activation has been well documented, less is known about the processes that counteract its kinase activity during the response to DNA damage. Here, we describe that PP4 phosphatase dephosphorylates Rad53 during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects DNA resection and repair by single-strand annealing, defects that are bypassed by reducing the hyper-phosphorylation state of Rad53 observed in the absence of the phosphatase. These results confirm that Rad53 is one of the main targets of PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection.

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