bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒05‒16
thirty-one papers selected by
Sean Rudd
Karolinska Institutet
  1. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer.
  2. Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.
  3. Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.
  4. Making Choices: DNA Replication Fork Recovery Mechanisms.
  5. Ubiquitylation in DNA double-strand break repair.
  6. The Fanconi anemia ubiquitin E3 ligase complex as an anti-cancer target.
  7. The emerging determinants of replication fork stability.
  8. Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.
  9. DHODH and cancer: promising prospects to be explored.
  10. miRNA Dysregulation is an Emerging Modulator of Genomic Instability.
  11. The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription-replication conflicts.
  12. A novel assay for screening WIP1 phosphatase substrates in nuclear extracts.
  13. DNA Damage Repair Inhibitor for Breast Cancer Treatment.
  14. Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes.
  15. Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms.
  16. Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.
  17. The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage.
  18. Regulation of GC box activity by 8-oxoguanine.
  19. Structures of telomerase at several steps of telomere repeat synthesis.
  20. RNA transcripts stimulate homologous recombination by forming DR-loops.
  21. Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance.
  22. Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis.
  23. Mechanism of MRX inhibition by Rif2 at telomeres.
  24. Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication.
  25. Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells.
  26. A non-genetic, cell cycle dependent mechanism of platinum resistance in lung adenocarcinoma.
  27. Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.
  28. Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.
  29. Mitochondrial activities play a pivotal role in regulating cell cycle in response to doxorubicin.
  30. Targeting cell cycle regulation via the G2-M checkpoint for synthetic lethality in melanoma.
  31. SAMHD1 mutations in mantle cell lymphoma are recurrent and confer in vitro resistance to nucleoside analogues.
  1. Nature. 2021 May 12.
    DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer.
    Chao Mao, Xiaoguang Liu, Yilei Zhang, Guang Lei, Yuelong Yan, Hyemin Lee, Pranavi Koppula, Shiqi Wu, Li Zhuang, Bingliang Fang, Masha V Poyurovsky, Kellen Olszewski, Boyi Gan.
      Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism1-4. Glutathione peroxidase 4 (GPX4)5,6 and ferroptosis suppressor protein 1 (FSP1)7,8 constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N-carbamoyl-L-aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate-the substrate and product of dihydroorotate dehydrogenase (DHODH)-attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4low). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment.
    DOI:  https://doi.org/10.1038/s41586-021-03539-7
  2. Nat Metab. 2021 May 10.
    Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.
    Kai-Chun Li, Enrico Girardi, Felix Kartnig, Sarah Grosche, Tea Pemovska, Johannes W Bigenzahn, Ulrich Goldmann, Vitaly Sedlyarov, Ariel Bensimon, Sandra Schick, Jung-Ming G Lin, Bettina Gürtl, Daniela Reil, Kristaps Klavins, Stefan Kubicek, Sara Sdelci, Giulio Superti-Furga.
      Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.
    DOI:  https://doi.org/10.1038/s42255-021-00386-8
  3. Nat Commun. 2021 May 14. 12(1): 2804
    Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.
    Emily L Giddings, Devin P Champagne, Meng-Han Wu, Joshua M Laffin, Tina M Thornton, Felipe Valenca-Pereira, Rachel Culp-Hill, Karen A Fortner, Natalia Romero, James East, Phoebe Cao, Hugo Arias-Pulido, Karatatiwant S Sidhu, Brian Silverstrim, Yoonseok Kam, Shana Kelley, Mark Pereira, Susan E Bates, Janice Y Bunn, Steven N Fiering, Dwight E Matthews, Robert W Robey, Domink Stich, Angelo D'Alessandro, Mercedes Rincon.
      Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.
    DOI:  https://doi.org/10.1038/s41467-021-23071-6
  4. Semin Cell Dev Biol. 2021 May;113 27-37
    Making Choices: DNA Replication Fork Recovery Mechanisms.
    Christine M Kondratick, M Todd Washington, Maria Spies.
      DNA replication is laden with obstacles that slow, stall, collapse, and break DNA replication forks. At each obstacle, there is a decision to be made whether to bypass the lesion, repair or restart the damaged fork, or to protect stalled forks from further demise. Each "decision" draws upon multitude of proteins participating in various mechanisms that allow repair and restart of replication forks. Specific functions for many of these proteins have been described and an understanding of how they come together in supporting replication forks is starting to emerge. Many questions, however, remain regarding selection of the mechanisms that enable faithful genome duplication and how "normal" intermediates in these mechanisms are sometimes funneled into "rogue" processes that destabilize the genome and lead to cancer, cell death, and emergence of chemotherapeutic resistance. In this review we will discuss molecular mechanisms of DNA damage bypass and replication fork protection and repair. We will specifically focus on the key players that define which mechanism is employed including: PCNA and its control by posttranslational modifications, translesion synthesis DNA polymerases, molecular motors that catalyze reversal of stalled replication forks, proteins that antagonize fork reversal and protect reversed forks from nucleolytic degradation, and the machinery of homologous recombination that helps to reestablish broken forks. We will also discuss risks to genome integrity inherent in each of these mechanisms.
    Keywords:  BRCA2; DNA replication; HLTF; PCNA; RAD51; RAD52; RPA; SHPRH; SMARCAL1; ZRANB3; genome stability; replication fork protection; replication fork reversal; template switching; translesion synthesis; translesion synthesis DNA polymerases
    DOI:  https://doi.org/10.1016/j.semcdb.2020.10.001
  5. DNA Repair (Amst). 2021 May 07. pii: S1568-7864(21)00085-9. [Epub ahead of print]103 103129
    Ubiquitylation in DNA double-strand break repair.
    Mengfan Tang, Siting Li, Junjie Chen.
      Genome integrity is constantly challenged by various DNA lesions with DNA double-strand breaks (DSBs) as the most cytotoxic lesions. In order to faithfully repair DSBs, DNA damage response (DDR) signaling networks have evolved, which organize many multi-protein complexes to deal with the encountered DNA damage. Spatiotemporal dynamics of these protein complexes at DSBs are mainly modulated by post-translational modifications (PTMs). One of the most well-studied PTMs in DDR is ubiquitylation which can orchestrate cellular responses to DSBs, promote accurate DNA repair, and maintain genome integrity. Here, we summarize the recent advances of ubiquitin-dependent signaling in DDR and discuss how ubiquitylation crosstalks with other PTMs to control fundamental biological processes in DSB repair.
    Keywords:  DNA damage response; Double strand break; homologous recombination (HR); non-homologous end joining (NHEJ); ubiquitylation
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103129
  6. Mol Cell. 2021 May 04. pii: S1097-2765(21)00328-2. [Epub ahead of print]
    The Fanconi anemia ubiquitin E3 ligase complex as an anti-cancer target.
    Michael F Sharp, Rohan Bythell-Douglas, Andrew J Deans, Wayne Crismani.
      Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.
    DOI:  https://doi.org/10.1016/j.molcel.2021.04.023
  7. Nucleic Acids Res. 2021 May 12. pii: gkab344. [Epub ahead of print]
    The emerging determinants of replication fork stability.
    Tanay Thakar, George-Lucian Moldovan.
      A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.
    DOI:  https://doi.org/10.1093/nar/gkab344
  8. Biochemistry. 2021 May 14.
    Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.
    Elizabeth R Morris, Simone Kunzelmann, Sarah J Caswell, Andrew G Purkiss, Geoff Kelly, Ian A Taylor.
      SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.
    DOI:  https://doi.org/10.1021/acs.biochem.0c00944
  9. Cancer Metab. 2021 May 10. 9(1): 22
    DHODH and cancer: promising prospects to be explored.
    Yue Zhou, Lei Tao, Xia Zhou, Zeping Zuo, Jin Gong, Xiaocong Liu, Yang Zhou, Chunqi Liu, Na Sang, Huan Liu, Jiao Zou, Kun Gou, Xiaowei Yang, Yinglan Zhao.
      Human dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme catalyzing the fourth step in the de novo pyrimidine synthesis pathway. It is originally a target for the treatment of the non-neoplastic diseases involving in rheumatoid arthritis and multiple sclerosis, and is re-emerging as a validated therapeutic target for cancer therapy. In this review, we mainly unravel the biological function of DHODH in tumor progression, including its crucial role in de novo pyrimidine synthesis and mitochondrial respiratory chain in cancer cells. Moreover, various DHODH inhibitors developing in the past decades are also been displayed, and the specific mechanism between DHODH and its additional effects are illustrated. Collectively, we detailly discuss the association between DHODH and tumors in recent years here, and believe it will provide significant evidences and potential strategies for utilizing DHODH as a potential target in preclinical and clinical cancer therapies.
    Keywords:  Cancer metabolism; DHODH inhibitors; De novo pyrimidine biosynthesis; Dihydroorotate dehydrogenase; Mitochondria
    DOI:  https://doi.org/10.1186/s40170-021-00250-z
  10. Semin Cancer Biol. 2021 May 09. pii: S1044-579X(21)00132-2. [Epub ahead of print]
    miRNA Dysregulation is an Emerging Modulator of Genomic Instability.
    Ana P Ferragut Cardoso, Mayukh Banerjee, Alexandra N Nail, Angeliki Lykoudi, J Christopher States.
      Genomic instability consists of a range of genetic alterations within the genome that contributes to tumor heterogeneity and drug resistance. It is a well-established characteristic of most cancer cells. Genome instability induction results from defects in DNA damage surveillance mechanisms, mitotic checkpoints and DNA repair machinery. Accumulation of genetic alterations ultimately sets cells towards malignant transformation. Recent studies suggest that miRNAs are key players in mediating genome instability. miRNAs are a class of small RNAs expressed in most somatic tissues and are part of the epigenome. Importantly, in many cancers, miRNA expression is dysregulated. Consequently, this review examines the role of miRNA dysregulation as a causal step for induction of genome instability and subsequent carcinogenesis. We focus specifically on mechanistic studies assessing miRNA(s) and specific subtypes of genome instability or known modes of genome instability. In addition, we provide insight on the existing knowledge gaps within the field and possible ways to address them.
    Keywords:  DNA repair; chromosomal instability; genomic instability; miRNA
    DOI:  https://doi.org/10.1016/j.semcancer.2021.05.004
  11. Nat Genet. 2021 May 13.
    The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription-replication conflicts.
    Aleix Bayona-Feliu, Sonia Barroso, Sergio Muñoz, Andrés Aguilera.
      ATP-dependent chromatin remodelers are commonly mutated in human cancer. Mammalian SWI/SNF complexes comprise three conserved multisubunit chromatin remodelers (cBAF, ncBAF and PBAF) that share the BRG1 (also known as SMARCA4) subunit responsible for the main ATPase activity. BRG1 is the most frequently mutated Snf2-like ATPase in cancer. In the present study, we have investigated the role of SWI/SNF in genome instability, a hallmark of cancer cells, given its role in transcription, DNA replication and DNA-damage repair. We show that depletion of BRG1 increases R-loops and R-loop-dependent DNA breaks, as well as transcription-replication (T-R) conflicts. BRG1 colocalizes with R-loops and replication fork blocks, as determined by FANCD2 foci, with BRG1 depletion being epistatic to FANCD2 silencing. Our study, extended to other components of SWI/SNF, uncovers a key role of the SWI/SNF complex, in particular cBAF, in helping resolve R-loop-mediated T-R conflicts, thus, unveiling a new mechanism by which chromatin remodeling protects genome integrity.
    DOI:  https://doi.org/10.1038/s41588-021-00867-2
  12. FEBS J. 2021 May 13.
    A novel assay for screening WIP1 phosphatase substrates in nuclear extracts.
    Radka Storchova, Kamila Burdova, Matous Palek, René H Medema, Libor Macurek.
      Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ATM/ATR kinases that phosphorylate multiple targets including tumour suppressor protein p53. In addition, DNA damage can activate parallel stress response pathways (such as p38/MK2 kinases) contributing to establishing the cell cycle arrest. Protein phosphatase WIP1 controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 towards p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
    Keywords:  DNA damage response; acetylation; p53; protein phosphatase
    DOI:  https://doi.org/10.1111/febs.15965
  13. Adv Exp Med Biol. 2021 ;1187 159-179
    DNA Damage Repair Inhibitor for Breast Cancer Treatment.
    Ahrum Min, Kyung-Hun Lee, Seock-Ah Im.
      Cancer has been defined as a genetic disorder caused by the accumulation of genetic alterations, which result from various internal and external DNA damage that is left unrepaired. One of the main characteristics of cancer is a partial loss of DNA damage repair (DDR) pathway, resulting in increased DNA damage levels and replication stress. DDR inhibitors have been suggested as a new anticancer strategy, under the concept of synthetic lethality. The poly-(ADP-ribose) polymerase (PARP) inhibitor is the first DDR inhibitor to be used in clinical practice. PARP inhibitors have been tested in patients with BRCA1/2 germline mutations (gBRCA1/2mt) and shown robust clinical benefits in breast cancer with gBRCA1/2mt and serous ovarian cancer patients. The concept of synthetic lethality is not limited to gBRCAmt for PARP inhibitor, and discovering homologous recombination deficiency (HRD) markers beyond BRCA1/2 and identifying best candidates for DDR inhibitors are the active research areas. At the same time, various combinations of DDR inhibitors and other anticancer drugs are being tested in both preclinical and clinical studies. In addition, based on recent evidence of the immune-modulatory effect of PARP inhibitors, the combination of DDR inhibitors and immune checkpoint inhibitors is being actively investigated. Acquired resistance mechanism of DDR inhibitors, as well as defining best candidates and best combinations, would be future research topics for DDR inhibitors. Furthermore, it would also be crucial to establish a clinically relevant standardized method to detect HRD for future clinical use.
    Keywords:  BRCAness; DNA damage repair; Homologous recombination deficiency; PARP inhibitor; Synthetic lethality
    DOI:  https://doi.org/10.1007/978-981-32-9620-6_8
  14. Front Immunol. 2021 ;12 660560
    Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes.
    Clara Taffoni, Alizée Steer, Johanna Marines, Hanane Chamma, Isabelle K Vila, Nadine Laguette.
      The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches.
    Keywords:  DNA damage responses; DNA-PK; IFI16; cGAS-STING; cytosolic nucleic acids; inflammation; tumorigenesis
    DOI:  https://doi.org/10.3389/fimmu.2021.660560
  15. Front Pharmacol. 2021 ;12 658806
    Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms.
    Natale Belluardo, Giuseppa Mudò, Valentina Di Liberto, Monica Frinchi, Daniele F Condorelli, Ugo Traversa, Francisco Ciruela, Renata Ciccarelli, Patrizia Di Iorio, Patricia Giuliani.
      Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.
    Keywords:  SH-SY5Ydifferentiation; guanine; guanosine; guanylate cyclase; heme oxygenase; neuroblastoma; protein kinase C; purine nucleoside phosphorylase
    DOI:  https://doi.org/10.3389/fphar.2021.658806
  16. Drug Metab Dispos. 2021 May 12. pii: DMD-AR-2021-000423. [Epub ahead of print]
    Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.
    Siennah R Miller, Thomas R Lane, Kimberley M Zorn, Sean Ekins, Stephen H Wright, Nathan Cherrington.
      Equilibrative nucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug-interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created 3-D pharmacophores for ENT1 and ENT2 substrates and inhibitors, using Kt and IC50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, while the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor, NBMPR, decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ~70% decrease, p = 0.0046; ENT2, ~50% decrease p = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ~50% decrease p = 0.0498; ENT2: ~30% decrease p = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: p = 0.28; ENT2: p = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT interacting compounds. Significance Statement This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.
    Keywords:  Structure-activity relationships and modeling; Uptake transporters (OATP, OAT, OCT, PEPT, MCT, NTCP, ASBT, etc.); computer modeling and simulation; transporters
    DOI:  https://doi.org/10.1124/dmd.121.000423
  17. Plant J. 2021 May 14.
    The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage.
    Maherun Nisa, Clara Bergis, Jose-Antonio Pedroza-Garcia, Jeannine Drouin-Wahbi, Christelle Mazubert, Catherine Bergounioux, Moussa Benhamed, Cécile Raynaud.
      Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants' viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase eta, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants' responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.
    Keywords:  DNA replication; Pol θ; abiotic stress; genome stability; plants
    DOI:  https://doi.org/10.1111/tpj.15295
  18. Redox Biol. 2021 Apr 30. pii: S2213-2317(21)00155-5. [Epub ahead of print]43 101997
    Regulation of GC box activity by 8-oxoguanine.
    Nadine Müller, Andriy Khobta.
      The oxidation-induced DNA modification 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was recently implicated in the activation and repression of gene transcription. We aimed at a systematic characterisation of the impacts of 8-oxodG on the activity of a GC box placed upstream from the RNA polymerase II core promoter. With the help of reporters carrying single synthetic 8-oxodG residues at four conserved G:C base pairs (underlined) within the 5'-TGGGCGGAGC-3' GC box sequence, we identified two modes of interference of 8-oxodG with the promoter activity. Firstly, 8-oxodG in the purine-rich (but not in the pyrimidine-rich) strand caused direct impairment of transcriptional activation. In addition, and independently of the first mechanism, 8-oxodG initiated a decline of the gene expression, which was mediated by the specific DNA glycosylase OGG1. For the different 8-oxodG positions, the magnitude of this effect reflected the excision preferences of OGG1. Thus, 8-oxodG seeded in the pyrimidine-rich strand was excised with the highest efficiency and caused the most pronounced decrease of the promoter activity. Conversely, 8-oxodG in the symmetric position within the same CpG dinucleotide, was poorly excised and induced no decline of the gene expression. Of note, abasic lesions caused gene silencing in both positions. By contrast, an uncleavable apurinic lesion in the pyrimidine-rich strand enhanced the GC box activity, suggesting that the AP endonuclease step provides a switch between the active versus repressed promoter states during base excision repair.
    Keywords:  8-Oxoguanine; 8-Oxoguanine DNA Glycosylase (OGG1); AP lesion; Base excision repair (BER); GC box
    DOI:  https://doi.org/10.1016/j.redox.2021.101997
  19. Nature. 2021 May 12.
    Structures of telomerase at several steps of telomere repeat synthesis.
    Yao He, Yaqiang Wang, Baocheng Liu, Christina Helmling, Lukas Sušac, Ryan Cheng, Z Hong Zhou, Juli Feigon.
      Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.
    DOI:  https://doi.org/10.1038/s41586-021-03529-9
  20. Nature. 2021 May 12.
    RNA transcripts stimulate homologous recombination by forming DR-loops.
    Jian Ouyang, Tribhuwan Yadav, Jia-Min Zhang, Haibo Yang, Esther Rheinbay, Hongshan Guo, Daniel A Haber, Li Lan, Lee Zou.
      Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.
    DOI:  https://doi.org/10.1038/s41586-021-03538-8
  21. Nucleic Acids Res. 2021 May 12. pii: gkab308. [Epub ahead of print]
    Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance.
    Miyoung Lee, David Shorthouse, Robert Mahen, Benjamin A Hall, Ashok R Venkitaraman.
      Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.
    DOI:  https://doi.org/10.1093/nar/gkab308
  22. Cell Host Microbe. 2021 May 06. pii: S1931-3128(21)00182-7. [Epub ahead of print]
    Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis.
    Satoshi Imai, Takuya Ooki, Naoko Murata-Kamiya, Daisuke Komura, Kamrunnesa Tahmina, Weida Wu, Atsushi Takahashi-Kanemitsu, Christopher Takaya Knight, Akiko Kunita, Nobumi Suzuki, Adriana A Del Valle, Mayo Tsuboi, Masahiro Hata, Yoku Hayakawa, Naomi Ohnishi, Koji Ueda, Masashi Fukayama, Tetsuo Ushiku, Shumpei Ishikawa, Masanori Hatakeyama.
      Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.
    Keywords:  BRCA1; BRCAness; CagA; DNA double-strand break; Helicobacter pylori; PAR1b; gastric cancer; genome instability; homologous recombination; replication fork instability
    DOI:  https://doi.org/10.1016/j.chom.2021.04.006
  23. Nat Commun. 2021 May 12. 12(1): 2763
    Mechanism of MRX inhibition by Rif2 at telomeres.
    Florian Roisné-Hamelin, Sabrina Pobiega, Kévin Jézéquel, Simona Miron, Jordane Dépagne, Xavier Veaute, Didier Busso, Marie-Hélène Le Du, Isabelle Callebaut, Jean-Baptiste Charbonnier, Philippe Cuniasse, Sophie Zinn-Justin, Stéphane Marcand.
      Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.
    DOI:  https://doi.org/10.1038/s41467-021-23035-w
  24. Mol Cell. 2021 May 05. pii: S1097-2765(21)00326-9. [Epub ahead of print]
    Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication.
    Helena Santos-Rosa, Gonzalo Millán-Zambrano, Namshik Han, Tommaso Leonardi, Marie Klimontova, Simona Nasiscionyte, Luca Pandolfini, Kostantinos Tzelepis, Till Bartke, Tony Kouzarides.
      DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.
    Keywords:  H3K37methylation; Histone modifications; MCM; Origin licensing; Replication origins; Set1; Set2
    DOI:  https://doi.org/10.1016/j.molcel.2021.04.021
  25. Sci Rep. 2021 May 13. 11(1): 10181
    Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells.
    Makoto Isono, Kazuki Okubo, Takako Asano, Akinori Sato.
      Checkpoint kinases (CHKs) are involved in the DNA damage response in many cancer cells. CHK inhibitors have been used in clinical trials in combination with chemotherapeutics; however, their effect against bladder cancer remains unclear. Here, we investigated the efficacy of combining gemcitabine with MK-8776, a novel CHK1 inhibitor, in four bladder cancer cell lines. The effects of gemcitabine and MK-8776 on cell viability, clonogenicity, cell cycle, and apoptosis were examined alongside in vivo efficacy using murine xenograft tumor models. Combined treatment inhibited the viability and colony formation of bladder cancer cells compared to either single treatment. Although gemcitabine (10 nM) alone increased the cell number in S-phase, it increased the cell number in sub-G1 phase when combined with MK-8776 (0.5 µM). Combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin-V-positive cells, indicating the induction of apoptosis. In vivo, administration of gemcitabine and MK-8776 was well tolerated and suppressed tumor growth. Mechanistically, the combined treatment elevated γH2A.X and suppressed Rad51 expression. Our study demonstrates that MK-8776 and gemcitabine combined induces apoptosis and suppresses proliferation in bladder cancer cells by inhibiting CHKs and DNA repair. Therefore, CHK1 inhibition combined with gemcitabine may be a potential treatment for bladder cancer.
    DOI:  https://doi.org/10.1038/s41598-021-89684-5
  26. Elife. 2021 May 13. pii: e65234. [Epub ahead of print]10
    A non-genetic, cell cycle dependent mechanism of platinum resistance in lung adenocarcinoma.
    Alvaro Gonzalez Rajal, Kamila A Marzec, Rachael A McCloy, Max Nobis, Venessa Chin, Jordan F Hastings, Kaitao Lai, Marina Kennerson, William E Hughes, Vijesh Vaghjiani, Paul Timpson, Jason E Cain, D Neil Watkins, David R Croucher, Andrew Burgess.
      We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.
    Keywords:  cancer biology; cell biology; human
    DOI:  https://doi.org/10.7554/eLife.65234
  27. Science. 2021 May 14. 372(6543): 716-721
    Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.
    Marlies P Rossmann, Karen Hoi, Victoria Chan, Brian J Abraham, Song Yang, James Mullahoo, Malvina Papanastasiou, Ying Wang, Ilaria Elia, Julie R Perlin, Elliott J Hagedorn, Sara Hetzel, Raha Weigert, Sejal Vyas, Partha P Nag, Lucas B Sullivan, Curtis R Warren, Bilguujin Dorjsuren, Eugenia Custo Greig, Isaac Adatto, Chad A Cowan, Stuart L Schreiber, Richard A Young, Alexander Meissner, Marcia C Haigis, Siegfried Hekimi, Steven A Carr, Leonard I Zon.
      Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.
    DOI:  https://doi.org/10.1126/science.aaz2740
  28. EMBO J. 2021 May 12. e103311
    Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.
    Vanesa Lafarga, Oleksandra Sirozh, Irene Díaz-López, Antonio Galarreta, Misaru Hisaoka, Eduardo Zarzuela, Jasminka Boskovic, Bogdan Jovanovic, Rafael Fernandez-Leiro, Jaime Muñoz, Georg Stoecklin, Iván Ventoso, Oscar Fernandez-Capetillo.
      Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.
    Keywords:  ALS; arginine-rich peptides; chromatin; mRNA; protamine
    DOI:  https://doi.org/10.15252/embj.2019103311
  29. Cell Cycle. 2021 May 12. 1-13
    Mitochondrial activities play a pivotal role in regulating cell cycle in response to doxorubicin.
    Ken Dornfeld, James Bjork, Gavin Folkert, Andrew Skildum, Kendall B Wallace.
      Doxorubicin induces both DNA damage and metabolic interference. How these effects interact to modulate cellular toxicity is not completely understood but important given the widespread use of doxorubicin in cancer treatment. This study tests the hypothesis that cell cycle arrest and survival are affected by distinct mitochondrial activities during doxorubicin exposure.Parental and mutant S. cerevisiae strains deficient in selected genes with mitochondrial function were treated with doxorubicin and assayed for changes in proliferation rates, cell survival and cell cycle arrest kinetics. Mitochondrial DNA content was estimated using quantitative PCR. Mitochondrial function was assessed by measuring oxygen consumption with and without an uncoupler.Parental cells growing in a non-fermentable carbon source medium and mutants lacking mitochondria and grown in glucose medium both show abrupt cell cycle and proliferation arrest during doxorubicin exposure compared to parental cells grown in glucose. Mitochondrial DNA increases during doxorubicin exposure in S. cerevisiae and in human breast cancer cells. Yeast strains deficient in TCA cycle activity or electron transport both show more abrupt cell cycle arrest than parental cells when exposed to doxorubicin. Concurrent treatment with the mitochondrial uncoupler dinitrophenol facilitates cell cycle progression and proliferation during doxorubicin exposure.Doxorubicin exposure induces mitochondrial DNA synthesis with TCA cycle and oxidative phosphorylation activity having opposing effects on cell proliferation, survival and cell cycle kinetics. TCA cycle activity provides biosynthetic substrates to support cell cycle progression and cell proliferation while electron transport and oxidative phosphorylation facilitate cell cycle arrest and possibly increased cytotoxicity.
    Keywords:  Cell cycle arrest; DNA damage; doxorubicin toxicity; metabolism; mitochondria
    DOI:  https://doi.org/10.1080/15384101.2021.1919839
  30. Cell Cycle. 2021 May 09. 1-11
    Targeting cell cycle regulation via the G2-M checkpoint for synthetic lethality in melanoma.
    Nicholas Barnaba, Jeannine R LaRocque.
      Disruption of cell cycle checkpoints has been well established as a hallmark of cancer. In particular, the G1-S transition mediated by the cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) pathway is dysregulated in more than 90% of melanoma cases. Therefore, tumor cells mainly rely on the G2-M checkpoint to halt the cell cycle in order to repair DNA damage. Here, we review the promising method of cell cycle-mediated synthetic lethality for melanoma treatment, which entails exploiting somatically acquired mutations in the G1-S transition with inhibitors of the G2-M transition in order to specifically kill melanoma cells. The idea stems from the theory that melanoma cells lacking G1-S checkpoints are particularly vulnerable to mitotic catastrophe when presented with G2-M checkpoint inhibition in addition to DNA damage, whereas normal cells with intact G1-S checkpoints should theoretically be spared. This review explores the link between cell cycle dysregulation and synthetic lethality in melanoma cells and discusses potential future applications for this treatment.
    Keywords:  CHK1; G1-S transition; G2-M transition; WEE1; cell cycle checkpoint; melanoma; synthetic lethality
    DOI:  https://doi.org/10.1080/15384101.2021.1922806
  31. Leuk Res. 2021 May 03. pii: S0145-2126(21)00109-0. [Epub ahead of print]107 106608
    SAMHD1 mutations in mantle cell lymphoma are recurrent and confer in vitro resistance to nucleoside analogues.
    Marco M Bühler, Junyan Lu, Sebastian Scheinost, Ferran Nadeu, Damien Roos-Weil, Manfred Hensel, Tharshika Thavayogarajah, Holger Moch, Markus G Manz, Eugenia Haralambieva, Ewerton Marques Maggio, Sílvia Beà, Eva Giné, Elías Campo, Olivier A Bernard, Wolfgang Huber, Thorsten Zenz.
      
    DOI:  https://doi.org/10.1016/j.leukres.2021.106608
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