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.

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.

MosChito rafts as a promising biocontrol tool against larvae of the common house mosquito, Culex pipiens.

Mosquito control is of paramount importance, in particular, in light of the major environmental alterations associated with human activities, from climate change to the altered distribution of pathogens, including those transmitted by Arthropods. Here, we used the common house mosquito, Culex pipiens to test the efficacy of MosChito raft, a novel tool for mosquito larval control. MosChito raft is a floating hydrogel matrix, composed of chitosan, genipin and yeast cells, as bio-attractants, developed for the delivery of a Bacillus thuringiensis israeliensis (Bti)-based bioinsecticide to mosquito larvae. To this aim, larvae of Cx. pipiens were collected in field in Northern Italy and a novel colony of mosquito species (hereafter: Trescore strain) was established. MosChito rafts, containing the Bti-based formulation, were tested on Cx. pipiens larvae from the Trescore strain to determine the doses to be used in successive experiments. Thus, bioassays with MosChito rafts were carried out under semi-field conditions, both on larvae from the Trescore strain and on pools of larvae collected from the field, at different developmental stages. Our results showed that MosChito raft is effective against Cx. pipiens. In particular, the observed mortality was over 50% after two days exposure of the larvae to MosChito rafts, and over 70-80% at days three to four, in both laboratory and wild larvae. In conclusion, our results point to the MosChito raft as a promising tool for the eco-friendly control of a mosquito species that is not only a nuisance insect but is also an important vector of diseases affecting humans and animals.

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.

EXO1: A tightly regulated nuclease.

Exonuclease 1 (EXO1) is an evolutionarily well conserved exonuclease. Its ability to resect DNA in the 5'-3' direction has been extensively characterized and shown to be implicated in several genomic DNA metabolic processes such as replication stress response, double strand break repair, mismatch repair, nucleotide excision repair and telomere maintenance. While the processing of DNA is critical for its repair, an excessive nucleolytic activity can lead to secondary lesions, increased genome instability and alterations in cellular functions. It is thus clear that different regulatory layers must be in effect to keep DNA degradation under control. Regulatory events that modulate EXO1 activity have been reported to act at different levels. Here we summarize the different post-translational modifications (PTMs) that affect EXO1 and discuss the implications of PTMs for EXO1 activities and how this regulation may be associated to cancer development.

Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity.

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

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.

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.

Study of UV-induced DNA Repair Factor Recruitment: Kinetics and Dynamics.

The local UV irradiation technique enables detection, kinetic measurements of recruitment, and quantification of DNA Damage Response (DDR) proteins at the site of UV-induced DNA damage.Using Isopore filters with high density pores of a broad range of sizes, it is possible to UV irradiate and damage only a very small portion of the nucleus of a cell by letting UV light pass only through the pores. Immunofluorescent analyses of modified DNA nucleotides, proteins, or fluorescently tagged versions of target factors can be used as markers to label and study UV-induced lesions and their repair.

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

Characterization of Structural and Configurational Properties of DNA by Atomic Force Microscopy.

We describe a method to extract quantitative information on DNA structural and configurational properties from high-resolution topographic maps recorded by atomic force microscopy (AFM). DNA molecules are deposited on mica surfaces from an aqueous solution, carefully dehydrated, and imaged in air in Tapping Mode. Upon extraction of the spatial coordinates of the DNA backbones from AFM images, several parameters characterizing DNA structure and configuration can be calculated. Here, we explain how to obtain the distribution of contour lengths, end-to-end distances, and gyration radii. This modular protocol can be also used to characterize other statistical parameters from AFM topographies.

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.

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice.

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering responses.

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.

Functional interplay between the 53BP1-ortholog Rad9 and the Mre11 complex regulates resection, end-tethering and repair of a double-strand break.

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5'-to-3' resection of Double-Strand DNA Breaks (DSBs). Extended 3' single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.

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.

To trim or not to trim: progression and control of DSB end resection.

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5'-3' nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.

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.

NER and DDR: classical music with new instruments.

Genomic insults by endogenous or exogenous sources activate the DNA damage response (DDR). After the recognition of damaged DNA by specific factors, repair mechanisms process the lesions, and a surveillance mechanism, known as DNA damage checkpoint, is triggered by single-stranded (ss) DNA covered by RPA. UV light induces DNA lesions, mainly 6,4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD), which are removed by nucleotide excision repair (NER). Recent reports shed light onto the mechanism connecting NER and DDR after UV irradiation. How does UV-induced DNA damage activate checkpoint kinases? How is ssDNA generated at UV lesions? In yeast, UV lesions persisting during S phase represent a block for the advancing of replication forks, which temporarily stop and then reinitiate downstream of the damage, leaving a ssDNA region containing the lesion. Nonreplicating yeast and human cells with defects in NER are not able to properly activate the checkpoint cascade, indicating that processing of UV lesions is a prerequisite for checkpoint activation. This pathway also requires the function of exonuclease 1, which acts on NER intermediates generating long tracts of ssDNA. Here, we review the connections between NER processing of UV-induced lesions and checkpoint activation, discussing the role of recently identified players in this mechanism.

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.