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Orp1+ is a gene periodically expressed by Schizosaccharomyces pombe. PCR studies showed that orp1+ is highly homologus in structure to the cdc18+ of Sch. pombe. It encodes an 80-kDa protein with amino acid sequences conserved in the Cdc18 and Cdc6 proteins. Genetic analysis showed that orp1+ is required by germinating pores to pursue further rounds of DNA replication. This suggests that opr1+ is essential for viability.

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., (© 2024. The Author(s).)

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.

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Cancer therapy is based on the selective clearance of malignant cells without severely damaging healthy tissues, and current clinical practice is constantly in need for new therapeutic targets in tumor management. The atypical protein kinase Haspin is conserved among most eukaryotes, and it has been shown to be particularly active in cycling cells. Along the years, several reports ascribed this protein the role to monitor chromosomal dynamics, primary cilia regulation and cellular polarization. Recently, an increasing amount of literature has depicted Haspin as a promising target to tackle tumors, as highlighted by its overexpression in malignant tissues and its requirement for cancer cell proliferation. In this work, we provide a detailed description on the current knowledge on Haspin, its physiological roles, the mechanisms underlying its regulation and its potential contribution to carcinogenesis., (Copyright © 2022. Published by Elsevier Inc.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1 Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae , where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Galli, Diani, Quadri, Nespoli, Galati, Panigada, Plevani and Muzi-Falconi.)

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae , mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

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.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Fibroblasts from a patient with Aicardi Goutières Syndrome (AGS) carrying a compound heterozygous mutation in TREX1, were reprogrammed into induced pluripotent stem cells (iPSCs) to establish isogenic clonal stem cell lines: UNIBSi006-A, UNIBSi006-B, and UNIBSi006-C. Cells were transduced using the episomal Sendai viral vectors, containing human OCT4, SOX2, c-MYC and KLF4 transcription factors. The transgene-free iPSC lines showed normal karyotype, expressed pluripotent markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the generation of three isogenic iPSC clones (UNIBSi007-A, UNIBSi007-B, and UNIBSi007-C) obtained from fibroblasts of a patient with Aicardi Goutières Syndrome (AGS) carrying a homozygous mutation in RNaseH2B. Cells were transduced using a Sendai virus based system, delivering the human OCT4, SOX2, c-MYC and KLF4 transcription factors. The resulting transgene-free iPSC lines retained the disease-causing DNA mutation, showed normal karyotype, expressed pluripotent markers and could differentiate in vitro toward cells of the three embryonic germ layers., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 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.

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.

Silencing RNA (siRNA) technologies emerge as a promising therapeutic tool for the treatment of multiple diseases. An ideal nanocarrier (NC) for siRNAs should be stable at physiological pH and release siRNAs in acidic endosomal pH, fulfilling siRNA delivery only inside cells. Here, we show a novel application of polyamine phosphate NCs (PANs) based on their capacity to load negatively charged nucleic acids and their pH stability. PANs are fabricated by complexation of phosphate anions from phosphate buffer solution (PB) with the amine groups of poly(allylamine) hydrochloride as carriers for siRNAs. PANs are stable in a narrow pH interval, from 7 to 9, and disassemble at pH's higher than 9 and lower than 6. siRNAs are encapsulated by complexation with poly(allylamine) hydrochloride before or after PAN formation. PANs with encapsulated siRNAs are stable in cell media. Once internalized in cells following endocytic pathways, PANs disassemble at the low endosomal pH and release the siRNAs into the cytoplasm. Confocal laser scanning microscopy (CLSM) images of Rhodamine Green labeled PANs (RG-PANs) with encapsulated Cy3-labeled siRNA in A549 cells show that siRNAs are released from the PANs. Colocalization experiments with labeled endosomes and either labeled siRNAs prove the translocation of siRNAs into the cytosol. As a proof of concept, it is shown that PANs with encapsulated green fluorescence protein (GFP) siRNAs silence GFP in A549 cells expressing this protein. Silencing efficacy was evaluated by flow cytometry, CLSM, and Western blot assays. These results open the way for the use of poly(allylamine) phosphate nanocarriers for the intracellular delivery of genetic materials.