RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

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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Fanci-FANCD2 Promotes Genome Stability and DNA Repair By Down-Regulating BLM Helicase Activity

Blood ◽

10.1182/blood-2021-152218 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1113-1113

Author(s):

Fengshan Liang ◽

Arvindhan Nagarajan ◽

Manoj M Pillai ◽

Patrick Sung ◽

Gary M. Kupfer

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Replication Fork ◽

Genome Stability ◽

Head And Neck Tumor ◽

Amino Acid Deletion ◽

Blm Helicase ◽

Dna End Resection ◽

End Resection ◽

Mutant Cells

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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The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00202-14 ◽

2014 ◽

Vol 35 (1) ◽

pp. 277-287 ◽

Cited By ~ 28

Author(s):

Howard Donninger ◽

Jennifer Clark ◽

Francesca Rinaldo ◽

Nicholas Nelson ◽

Thibaut Barnoud ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Tumor Suppressor ◽

Human Cancer ◽

Protein A ◽

Repair Process ◽

Protein Acetylation ◽

Repair Protein ◽

Dna Repair Protein ◽

Repair Activity

RASSF1A may be the most frequently inactivated tumor suppressor identified in human cancer so far. It is a proapoptotic Ras effector and plays an important role in the apoptotic DNA damage response (DDR). We now show that in addition to DDR regulation, RASSF1A also plays a key role in the DNA repair process itself. We show that RASSF1A forms a DNA damage-regulated complex with the key DNA repair protein xeroderma pigmentosum A (XPA). XPA requires RASSF1A to exert full repair activity, and RASSF1A-deficient cells exhibit an impaired ability to repair DNA. Moreover, a cancer-associated RASSF1A single-nucleotide polymorphism (SNP) variant exhibits differential XPA binding and inhibits DNA repair. The interaction of XPA with other components of the repair complex, such as replication protein A (RPA), is controlled in part by a dynamic acetylation/deacetylation cycle. We found that RASSF1A and its SNP variant differentially regulate XPA protein acetylation, and the SNP variant hyperstabilizes the XPA-RPA70 complex. Thus, we identify two novel functions for RASSF1A in the control of DNA repair and protein acetylation. As RASSF1A modulates both apoptotic DDR and DNA repair, it may play an important and unanticipated role in coordinating the balance between repair and death after DNA damage.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m116.772475 ◽

2017 ◽

Vol 292 (26) ◽

pp. 10779-10790 ◽

Cited By ~ 29

Author(s):

Nozomi Tomimatsu ◽

Bipasha Mukherjee ◽

Janelle Louise Harris ◽

Francesca Ludovica Boffo ◽

Molly Catherine Hardebeck ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna End Resection ◽

End Resection

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

Nucleic Acids Research ◽

10.1093/nar/gkz794 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10706-10727 ◽

Cited By ~ 2

Author(s):

María Teresa Villoria ◽

Pilar Gutiérrez-Escribano ◽

Esmeralda Alonso-Rodríguez ◽

Facundo Ramos ◽

Eva Merino ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Repair Process ◽

Strand Break ◽

Double Strand Break ◽

Dna Lesion ◽

Damage Response ◽

End Resection ◽

Recombinational Dna Repair

Abstract The 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 Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation 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 resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by 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 for their success.

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CtIP-dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation

The Journal of Cell Biology ◽

10.1083/jcb.201111065 ◽

2012 ◽

Vol 197 (7) ◽

pp. 869-876 ◽

Cited By ~ 49

Author(s):

Arne Nedergaard Kousholt ◽

Kasper Fugger ◽

Saskia Hoffmann ◽

Brian D. Larsen ◽

Tobias Menzel ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Progression ◽

Dna Lesions ◽

Cell Cycle Checkpoint ◽

Dna Breaks ◽

Checkpoint Signaling ◽

Cycle Checkpoint ◽

Dna End Resection ◽

End Resection

To prevent accumulation of mutations, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homology-directed repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of ATR (ataxia telangiectasia mutated and Rad3 related) and CHK1 kinases to induce the cell cycle checkpoint. In this paper, we show that CHK1 was rapidly and robustly activated before detectable end resection. Moreover, we show that the key resection factor CtIP was dispensable for initial ATR–CHK1 activation after DNA damage by camptothecin and ionizing radiation. In contrast, we find that DNA end resection was critically required for sustained ATR–CHK1 checkpoint signaling and for maintaining both the intra–S- and G2-phase checkpoints. Consequently, resection-deficient cells entered mitosis with persistent DNA damage. In conclusion, we have uncovered a temporal program of checkpoint activation, where CtIP-dependent DNA end resection is required for sustained checkpoint signaling.

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The role of the α-tubulin acetyltransferase αTAT1 in the DNA damage response

Journal of Cell Science ◽

10.1242/jcs.246702 ◽

2020 ◽

Vol 133 (17) ◽

pp. jcs246702

Author(s):

Na Mi Ryu ◽

Jung Min Kim

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Protein A ◽

Direct Repeat ◽

Microtubule Organization ◽

Cycle Arrest ◽

Recombination Repair ◽

Damage Response

ABSTRACTLysine 40 acetylation of α-tubulin (Ac-α-tubulin), catalyzed by the acetyltransferase αTAT1, marks stabilized microtubules. Recently, there is growing evidence to suggest crosstalk between the DNA damage response (DDR) and microtubule organization; we therefore investigated whether αTAT1 is involved in the DDR. Following treatment with DNA-damaging agents, increased levels of Ac-α-tubulin were detected. We also observed significant induction of Ac-α-tubulin after depletion of DNA repair proteins, suggesting that αTAT1 is positively regulated in response to DNA damage. Intriguingly, αTAT1 depletion decreased DNA damage-induced replication protein A (RPA) phosphorylation and foci formation. Moreover, DNA damage-induced cell cycle arrest was significantly delayed in αTAT1-depleted cells, indicating defective checkpoint activation. The checkpoint defects seen upon αTAT1 deficiency were restored by expression of wild-type αTAT1, but not by αTAT1-D157N (a catalytically inactive αTAT1), indicating that the role of αTAT1 in the DDR is dependent on enzymatic activity. Furthermore, αTAT1-depleted direct repeat GFP (DR-GFP) U2OS cells had a significant decrease in the frequency of homologous recombination repair. Collectively, our results suggest that αTAT1 may play an essential role in DNA damage checkpoints and DNA repair through its acetyltransferase activity.

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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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The emerging role of RNAs in DNA damage repair

Cell Death and Differentiation ◽

10.1038/cdd.2017.16 ◽

2017 ◽

Vol 24 (4) ◽

pp. 580-587 ◽

Cited By ~ 18

Author(s):

Ben R Hawley ◽

Wei-Ting Lu ◽

Ania Wilczynska ◽

Martin Bushell

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Critical Role ◽

Repair Process ◽

Rna Molecules ◽

Processing Enzymes ◽

Damage Repair ◽

New Findings ◽

Integral Role ◽

Repair Mechanisms

Abstract Many surveillance and repair mechanisms exist to maintain the integrity of our genome. All of the pathways described to date are controlled exclusively by proteins, which through their enzymatic activities identify breaks, propagate the damage signal, recruit further protein factors and ultimately resolve the break with little to no loss of genetic information. RNA is known to have an integral role in many cellular pathways, but, until very recently, was not considered to take part in the DNA repair process. Several reports demonstrated a conserved critical role for RNA-processing enzymes and RNA molecules in DNA repair, but the biogenesis of these damage-related RNAs and their mechanisms of action remain unknown. We will explore how these new findings challenge the idea of proteins being the sole participants in the response to DNA damage and reveal a new and exciting aspect of both DNA repair and RNA biology.

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Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816539115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11961-E11969 ◽

Cited By ~ 14

Author(s):

Tai-Yuan Yu ◽

Michael T. Kimble ◽

Lorraine S. Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Inhibitory Effects ◽

Checkpoint Signaling ◽

Synthetic Lethal ◽

Strand Breaks ◽

Damage Response ◽

End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

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