DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging.

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.

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining.

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.

DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

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.

Alternative lengthening of telomeres (ALT) cells viability is dependent on C-rich telomeric RNAs.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.

SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

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.

Pharmacological boost of DNA damage response and repair by enhanced biogenesis of DNA damage response RNAs.

A novel class of small non-coding RNAs called DNA damage response RNAs (DDRNAs) generated at DNA double-strand breaks (DSBs) in a DROSHA- and DICER-dependent manner has been shown to regulate the DNA damage response (DDR). Similar molecules were also reported to guide DNA repair. Here, we show that DDR activation and DNA repair can be pharmacologically boosted by acting on such non-coding RNAs. Cells treated with enoxacin, a compound previously demonstrated to augment DICER activity, show stronger DDR signalling and faster DNA repair upon exposure to ionizing radiations compared to vehicle-only treated cells. Enoxacin stimulates DDRNA production at chromosomal DSBs and at dysfunctional telomeres, which in turn promotes 53BP1 accumulation at damaged sites, therefore in a miRNA-independent manner. Increased 53BP1 occupancy at DNA lesions induced by enoxacin ultimately suppresses homologous recombination, channelling DNA repair towards faster and more accurate non-homologous end-joining, including in post-mitotic primary neurons. Notably, augmented DNA repair stimulated by enoxacin increases the survival also of cancer cells treated with chemotherapeutic agents.

Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks.

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.

A damaged genome's transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks.

Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.