23 research outputs found
Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability
Aicardi-Gouti\ue8res syndrome (AGS) is an inflammatory encephalopathy caused by defective nucleic acids metabolism. Over 50% of AGS mutations affect RNase H2 the only enzyme able to remove single ribonucleotidemonophosphates (rNMPs) embedded in DNA. Ribonucleotide triphosphates (rNTPs) are incorporated into genomic DNA with relatively high frequency during normal replication making DNA more susceptible to strand breakage and mutations. Here we demonstrate that human cells depleted of RNase H2 show impaired cell cycle progression associated with chronic activation of post-replication repair (PRR) and genome instability. We identify a similar phenotype in cells derived from AGS patients, which indeed accumulate rNMPs in genomic DNA and exhibit markers of constitutive PRR and checkpoint activation. Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage. Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses. These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis
VID22 counteracts G-quadruplex-induced genome instability
Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.Associazione Italiana per la Ricerca sul Cancro (AIRC) [15631, 21806 to M.M.F.]; MIUR [PRIN 2015-2015SJLMB9; PRIN 2017-2017KSZZJW to M.M.F.]; Telethon [GGP15227 to M.M.F.]; F.L. was supported by the University of Milano: ‘‘Piano di Sviluppo dell’Ateneo per la Ricerca. Linea B: Supporto per i Giovani Ricercatori’’; M.C.B. was supported by Fondazione Veronesi; Research at the laboratory of A.A. was funded by the Spanish Ministry of Economy and Competitiveness [BFU2016-75058-P]; B.G.G. was funded by the Spanish Association Against Cancer; MIUR [PRIN2017-2017Z55KC to T.B.]; M.C., D.S.H. are supported by MIUR [PRIN 2017] and CNRbiomics [PIR01_00017]; H2020 Projects ELIXIR-EXCELERATE, EOSC-Life, EOSC-Pillar and Elixir-IIB; G.W.B. was supported by the Canadian Institutes of Health Research[FDN-159913]. Funding for open access charge: Associazione Italiana per la Ricerca sul Cancro (AIRC) [21806]
Non-Canonical CRL4A/4BCDT2 Interacts with RAD18 to Modulate Post Replication Repair and Cell Survival
The Cullin-4CDT2 E3 ubiquitin ligase plays an essential role in DNA replication origin licensing directing degradation of several licensing factors at the G1/S transition in order to prevent DNA re-replication. Recently a RAD18-independent role of Cullin-4CDT2 in PCNA monoubiquitylation has been proposed. In an effort to better understand the function of Cullin-4CDT2 E3 ubiquitin ligase in mammalian Post-Replication Repair during an unperturbed S-phase, we show that down-regulation of Cullin-4CDT2 leads to two distinguishable independent phenotypes in human cells that unveil at least two independent roles of Cullin-4CDT2 in S-phase. Apart from the re-replication preventing activity, we identified a non-canonical Cullin-4CDT2 complex, containing both CUL4A and CUL4B, associated to the COP9 signalosome, that controls a RAD18-dependent damage avoidance pathway essential during an unperturbed S-phase. Indeed, we show that the non-canonical Cullin-4A/4BCDT2 complex binds to RAD18 and it is required to modulate RAD18 protein levels onto chromatin and the consequent dynamics of PCNA monoubiquitylation during a normal S-phase. This function prevents replication stress, ATR hyper-signaling and, ultimately, apoptosis. A very similar PRR regulatory mechanism has been recently described for Spartan. Our findings uncover a finely regulated process in mammalian cells involving Post-Replication Repair factors, COP9 signalosome and a non-canonical Cullin4-based E3 ligase which is essential to tolerate spontaneous damage and for cell survival during physiological DNA replication. © 2013 Sertic et al.This work was supported by grants from AIRC (http://www.airc.it), MIUR, Regione Lombardia and Fondazione Cariplo to P.P. and M.M.-F. The financial support of Telethon-Italy (http://www.telethon.it, grant number GGP11003) is gratefully acknowledgedPeer Reviewe
Tau and DNA Damage in Neurodegeneration
Neurodegenerative disorders are a family of incurable conditions. Among them, Alzheimer’s disease and tauopathies are the most common. Pathological features of these two disorders are synaptic loss, neuronal cell death and increased DNA damage. A key pathological protein for the onset and progression of the conditions is the protein tau, a microtubule-binding protein highly expressed in neurons and encoded by the MAPT (microtubule-associated protein tau) gene. Tau is predominantly a cytosolic protein that interacts with numerous other proteins and molecules. Recent findings, however, have highlighted new and unexpected roles for tau in the nucleus of neuronal cells. This review summarizes the functions of tau in the metabolism of DNA, describing them in the context of the disorders
Intracellular trafficking of the human oxytocin receptor: evidence of receptor recycling via a Rab4/Rab5 “short cycle”
Non-canonical CRL4A/4B(CDT2) interacts with RAD18 to modulate post replication repair and cell survival.
The Cullin-4(CDT2) E3 ubiquitin ligase plays an essential role in DNA replication origin licensing directing degradation of several licensing factors at the G1/S transition in order to prevent DNA re-replication. Recently a RAD18-independent role of Cullin-4(CDT2) in PCNA monoubiquitylation has been proposed. In an effort to better understand the function of Cullin-4(CDT2) E3 ubiquitin ligase in mammalian Post-Replication Repair during an unperturbed S-phase, we show that down-regulation of Cullin-4(CDT2) leads to two distinguishable independent phenotypes in human cells that unveil at least two independent roles of Cullin-4(CDT2) in S-phase. Apart from the re-replication preventing activity, we identified a non-canonical Cullin-4(CDT2) complex, containing both CUL4A and CUL4B, associated to the COP9 signalosome, that controls a RAD18-dependent damage avoidance pathway essential during an unperturbed S-phase. Indeed, we show that the non-canonical Cullin-4A/4B(CDT2) complex binds to RAD18 and it is required to modulate RAD18 protein levels onto chromatin and the consequent dynamics of PCNA monoubiquitylation during a normal S-phase. This function prevents replication stress, ATR hyper-signaling and, ultimately, apoptosis. A very similar PRR regulatory mechanism has been recently described for Spartan. Our findings uncover a finely regulated process in mammalian cells involving Post-Replication Repair factors, COP9 signalosome and a non-canonical Cullin4-based E3 ligase which is essential to tolerate spontaneous damage and for cell survival during physiological DNA replication
One, No One, and One Hundred Thousand: The Many Forms of Ribonucleotides in DNA
In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo
Phosphorylation of H3-Thr3 by Haspin Is Required for Primary Cilia Regulation
Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell–cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3–HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia