Coordinated Activity of Y Family TLS Polymerases and EXO1 Protects Non-S Phase Cells from UV-Induced Cytotoxic Lesions.

UV-induced photoproducts are responsible for the pathological effects of sunlight. Mutations in nucleotide excision repair (NER) cause severe pathologies characterized by sunlight sensitivity, coupled to elevated predisposition to cancer and/or neurological dysfunctions. We have previously shown that in UV-irradiated non-cycling cells, only a particular subset of lesions activates the DNA damage response (DDR), and this requires NER and EXO1 activities. To define the molecular mechanism acting at these lesions, we demonstrate that Y family TLS polymerases are recruited at NER- and EXO1-positive lesion sites in non-S phase cells. The coordinated action of EXO1 and Y family TLS polymerases promotes checkpoint activation, leads to lesion repair, and is crucial to prevent cytotoxic double-strand break (DSB) formation.

Spotlight on G-Quadruplexes: From Structure and Modulation to Physiological and Pathological Roles.

G-quadruplexes or G4s are non-canonical secondary structures of nucleic acids characterized by guanines arranged in stacked tetraplex arrays. Decades of research into these peculiar assemblies of DNA and RNA, fueled by the development and optimization of a vast array of techniques and assays, has resulted in a large amount of information regarding their structure, stability, localization, and biological significance in native systems. A plethora of articles have reported the roles of G-quadruplexes in multiple pathways across several species, ranging from gene expression regulation to RNA biogenesis and trafficking, DNA replication, and genome maintenance. Crucially, a large amount of experimental evidence has highlighted the roles of G-quadruplexes in cancer biology and other pathologies, pointing at these structurally unique guanine assemblies as amenable drug targets. Given the rapid expansion of this field of research, this review aims at summarizing all the relevant aspects of G-quadruplex biology by combining and discussing results from seminal works as well as more recent and cutting-edge experimental evidence. Additionally, the most common methodologies used to study G4s are presented to aid the reader in critically interpreting and integrating experimental data.

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.

Ribonucleotides misincorporated into DNA act as strand-discrimination signals in eukaryotic mismatch repair.

To improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand.

RNase H activities counteract a toxic effect of Polymerase η in cells replicating with depleted dNTP pools.

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells.

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.

RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA.

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.

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.

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.

The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA.

Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.

Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability.

Aicardi-Goutières 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 ribonucleotide-monophosphates (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.

The ribonuclease DIS3 promotes let-7 miRNA maturation by degrading the pluripotency factor LIN28B mRNA.

Multiple myeloma, the second most frequent hematologic tumor after lymphomas, is an incurable cancer. Recent sequencing efforts have identified the ribonuclease DIS3 as one of the most frequently mutated genes in this disease. DIS3 represents the catalytic subunit of the exosome, a macromolecular complex central to the processing, maturation and surveillance of various RNAs. miRNAs are an evolutionarily conserved class of small noncoding RNAs, regulating gene expression at post-transcriptional level. Ribonucleases, including Drosha, Dicer and XRN2, are involved in the processing and stability of miRNAs. However, the role of DIS3 on the regulation of miRNAs remains largely unknown. Here we found that DIS3 regulates the levels of the tumor suppressor let-7 miRNAs without affecting other miRNA families. DIS3 facilitates the maturation of let-7 miRNAs by reducing in the cytoplasm the RNA stability of the pluripotency factor LIN28B, a inhibitor of let-7 processing. DIS3 inactivation, through the increase of LIN28B and the reduction of mature let-7, enhances the translation of let-7 targets such as MYC and RAS leading to enhanced tumorigenesis. Our study establishes that the ribonuclease DIS3, targeting LIN28B, sustains the maturation of let-7 miRNAs and suggests the increased translation of critical oncogenes as one of the biological outcomes of DIS3 inactivation.

Sensing of replication stress and Mec1 activation act through two independent pathways involving the 9-1-1 complex and DNA polymerase ε.

Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Δdpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non-essential subunit of DNA polymerase ε (Polε) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Polε.

14-3-3 Proteins regulate exonuclease 1-dependent processing of stalled replication forks.

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress.

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation.

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.

Detection of ribonucleotides embedded in DNA by Nanopore sequencing.

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes.

Elevated levels of the polo kinase Cdc5 override the Mec1/ATR checkpoint in budding yeast by acting at different steps of the signaling pathway.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity.

Saccharomyces cerevisiae Rad9 is required for an effective DNA damage response throughout the cell cycle. Assembly of Rad9 on chromatin after DNA damage is promoted by histone modifications that create docking sites for Rad9 recruitment, allowing checkpoint activation. Rad53 phosphorylation is also dependent upon BRCT-directed Rad9 oligomerization; however, the crosstalk between these molecular determinants and their functional significance are poorly understood. Here we report that, in the G1 and M phases of the cell cycle, both constitutive and DNA damage-dependent Rad9 chromatin association require its BRCT domains. In G1 cells, GST or FKBP dimerization motifs can substitute to the BRCT domains for Rad9 chromatin binding and checkpoint function. Conversely, forced Rad9 dimerization in M phase fails to promote its recruitment onto DNA, although it supports Rad9 checkpoint function. In fact, a parallel pathway, independent on histone modifications and governed by CDK1 activity, allows checkpoint activation in the absence of Rad9 chromatin binding. CDK1-dependent phosphorylation of Rad9 on Ser11 leads to specific interaction with Dpb11, allowing Rad53 activation and bypassing the requirement for the histone branch.

A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin.

Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.