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1.
Nat Cell Biol ; 25(4): 550-564, 2023 04.
Article in English | MEDLINE | ID: mdl-36894671

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.


Subject(s)
COVID-19 , Animals , Mice , SARS-CoV-2 , Cellular Senescence , DNA Damage
2.
EMBO Rep ; 23(2): e53658, 2022 02 03.
Article in English | MEDLINE | ID: mdl-34854526

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), known to be more common in the elderly, who also show more severe symptoms and are at higher risk of hospitalization and death. Here, we show that the expression of the angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, increases during aging in mouse and human lungs. ACE2 expression increases upon telomere shortening or dysfunction in both cultured mammalian cells and in vivo in mice. This increase is controlled at the transcriptional level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Both pharmacological global DDR inhibition of ATM kinase activity and selective telomeric DDR inhibition by the use of antisense oligonucleotides prevent Ace2 upregulation following telomere damage in cultured cells and in mice. We propose that during aging telomere dysfunction due to telomeric shortening or damage triggers DDR activation and this causes the upregulation of ACE2, the SARS-CoV-2 cell receptor, thus contributing to make the elderly more susceptible to the infection.


Subject(s)
Aging , Angiotensin-Converting Enzyme 2/genetics , COVID-19 , DNA Damage , Telomere , Aged , Aging/genetics , Animals , Humans , Mice , SARS-CoV-2 , Telomere/genetics
3.
Nat Commun ; 9(1): 5376, 2018 12 18.
Article in English | MEDLINE | ID: mdl-30560944

ABSTRACT

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.


Subject(s)
BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , RNA, Long Noncoding/metabolism , Recombinational DNA Repair , Ribonuclease H/metabolism , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , G2 Phase/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , RNA, Long Noncoding/genetics , RNA, Small Interfering/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Ribonuclease H/genetics , S Phase/genetics
4.
Nat Cell Biol ; 19(12): 1400-1411, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29180822

ABSTRACT

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.


Subject(s)
DNA Breaks, Double-Stranded , DNA Damage , DNA Repair , RNA, Long Noncoding/metabolism , ATP-Binding Cassette Transporters/metabolism , Acid Anhydride Hydrolases , Animals , Cell Cycle Proteins/metabolism , Cell Line , Cell-Free System , DNA Damage/genetics , DNA Damage/physiology , DNA Repair/genetics , DNA Repair/physiology , DNA-Binding Proteins , MRE11 Homologue Protein/metabolism , Mice , Models, Biological , Nuclear Proteins/metabolism , Oligonucleotides, Antisense/genetics , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics , Transcription, Genetic , Tumor Suppressor p53-Binding Protein 1/metabolism
5.
J Cell Sci ; 129(7): 1468-76, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26906421

ABSTRACT

The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling.


Subject(s)
DEAD-box RNA Helicases/genetics , DNA Damage/genetics , DNA Repair/immunology , Histones/metabolism , Ribonuclease III/genetics , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins/metabolism , Cell Line , DNA Repair/genetics , Humans , Nuclear Proteins/metabolism , RNA Interference , RNA, Small Interfering/genetics , Ribonuclease, Pancreatic/metabolism , Trans-Activators/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism
6.
Nat Cell Biol ; 14(4): 355-65, 2012 Mar 18.
Article in English | MEDLINE | ID: mdl-22426077

ABSTRACT

The DNA-damage response (DDR) arrests cell-cycle progression until damage is removed. DNA-damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously induced persistent DDR markers is associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break next to a telomeric sequence resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a double-strand break induces persistent DNA damage and DDR. Linear, but not circular, telomeric DNA or scrambled DNA induces a prolonged checkpoint in normal cells. In terminally differentiated tissues of old primates, DDR markers accumulate at telomeres that are not critically short. We propose that linear genomes are not uniformly reparable and that telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.


Subject(s)
DNA Damage , Telomere/metabolism , Animals , Cell Cycle , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Humans , Mice , Neurons/cytology , Neurons/metabolism , Telomeric Repeat Binding Protein 2/metabolism
7.
Nat Cell Biol ; 13(3): 292-302, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21336312

ABSTRACT

Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.


Subject(s)
Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DNA Damage , Heterochromatin/genetics , Neoplasms/metabolism , Oncogenes , Animals , Apoptosis , Cell Line, Tumor , Chromatin/metabolism , DNA Replication , Histone Deacetylase Inhibitors/pharmacology , Humans , Mice , Microscopy, Fluorescence/methods , Neoplasm Transplantation , Plasmids/metabolism , RNA, Small Interfering/metabolism
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