bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020–06–28
fifty-two papers selected by
Sean Rudd, Karolinska Institutet
  1. Pervasive lesion segregation shapes cancer genome evolution.
  2. Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability.
  3. Roles of Chk2/CHEK2 in guarding against environmentally-induced DNA damage and replication-stress.
  4. MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.
  5. Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition.
  6. Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.
  7. DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner.
  8. Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis.
  9. SAMHD1 is a key regulator of the lineage-specific response of acute lymphoblastic leukaemias to nelarabine.
  10. Exploiting replicative stress in gynecological cancers as a therapeutic strategy.
  11. Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases.
  12. DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.
  13. Nuclear body phase separation drives telomere clustering in ALT cancer cells.
  14. SIRT6 mono-ADP ribosylates KDM2A to locally increase H3K36me2 at DNA damage sites to inhibit transcription and promote repair.
  15. Inhibition of Serine Metabolism Promotes Resistance to Cisplatin in Gastric Cancer.
  16. PARP inhibitor resistance: the underlying mechanisms and clinical implications.
  17. ANKHD1 is an S phase protein required for histone synthesis and DNA repair in multiple myeloma cells.
  18. CDK12: A Potent Target and Biomarker for Human Cancer Therapy.
  19. Limited MOMP, ATM, and their roles in carcinogenesis and cancer treatment.
  20. Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication.
  21. The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe.
  22. Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6.
  23. Functional genomics identifies new synergistic therapies for retinoblastoma.
  24. Jianpi-yangwei decoction inhibits DNA damage repair in the drug resistance of gastric cancer by reducing FEN1 expression.
  25. AATF/Che-1-An RNA Binding Protein at the Nexus of DNA Damage Response and Ribosome Biogenesis.
  26. The iron-sulphur cluster in human DNA2 is required for all biochemical activities of DNA2.
  27. Effects of DNA topoisomerase IIα splice variants on acquired drug resistance.
  28. Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.
  29. CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production.
  30. HERC Ubiquitin Ligases in Cancer.
  31. SUMOylation stabilizes hSSB1 and enhances the recruitment of NBS1 to DNA damage sites.
  32. Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system.
  33. New Frontiers in Prostate Cancer Treatment: Are We Ready for Drug Combinations with Novel Agents?
  34. Small molecule inhibitors confirm ubiquitin-dependent removal of TOP2-DNA covalent complexes.
  35. Effect of Human XRCC1 Protein Oxidation on the Functional Activity of Its Complexes with the Key Enzymes of DNA Base Excision Repair.
  36. AR phosphorylation and CHK2 kinase activity regulates IR stabilized AR-CHK2 interaction and prostate cancer survival.
  37. Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.
  38. Induction of apoptosis in Ogg1-null mouse embryonic fibroblasts by GSH depletion is independent of DNA damage.
  39. Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair.
  40. Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells.
  41. Short-term treatment with imetelstat sensitizes hematopoietic malignant cells to a genotoxic agent via suppression of the telomerase-mediated DNA repair process.
  42. Nicotinamide adenine dinucleotide (NAD+): essential redox metabolite, co-substrate and an anti-cancer and anti-ageing therapeutic target.
  43. A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase.
  44. An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.
  45. The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.
  46. Structure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase.
  47. Fluoropyrimidine Modulation of the Anti-Tumor Immune Response-Prospects for Improved Colorectal Cancer Treatment.
  48. A Stapled POL κ Peptide Targets REV1 to Inhibit Mutagenic Translesion Synthesis.
  49. Dynamic human MutSα-MutLα complexes compact mismatched DNA.
  50. Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1.
  51. Identification of pathognomonic purine synthesis biomarkers by metabolomic profiling of adolescents with obesity and type 2 diabetes.
  52. Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.
  1. Nature. 2020 Jun 24.
    Pervasive lesion segregation shapes cancer genome evolution.
    Liver Cancer Evolution Consortium
      Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.
    DOI:  https://doi.org/10.1038/s41586-020-2435-1
  2. Sci Adv. 2020 Jun;6(24): eaaz7808
    Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability.
    Sumeet Nayak, Jennifer A Calvo, Ke Cong, Min Peng, Emily Berthiaume, Jessica Jackson, Angela M Zaino, Alessandro Vindigni, M Kyle Hadden, Sharon B Cantor.
      The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.
    DOI:  https://doi.org/10.1126/sciadv.aaz7808
  3. Environ Mol Mutagen. 2020 Jun 24.
    Roles of Chk2/CHEK2 in guarding against environmentally-induced DNA damage and replication-stress.
    Md Kawsar Mustofa, Yuki Tanoue, Chie Tateishi, Cyrus Vaziri, Satoshi Tateishi.
      Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA Double Strand Break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation (IR) but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently-stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the Ataxia Telangiectasia-Mutated (ATM) protein kinase. This review focuses on recently-defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents. This article is protected by copyright. All rights reserved.
    Keywords:  CHEK2; Chk2; RAD18; replication-associated DNA genotoxicity; translesion synthesis
    DOI:  https://doi.org/10.1002/em.22397
  4. Oncotarget. 2020 Jun 09. 11(23): 2141-2159
    MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.
    David King, Xiao Dun Li, Gilberto S Almeida, Colin Kwok, Polly Gravells, Daniel Harrison, Saoirse Burke, Albert Hallsworth, Yann Jamin, Sally George, Simon P Robinson, Christopher J Lord, Evon Poon, Daniel Yeomanson, Louis Chesler, Helen E Bryant.
      This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.
    Keywords:  MYCN; PARP inhibitor; neuroblastoma; replication fork stalling; replication stress
    DOI:  https://doi.org/10.18632/oncotarget.27329
  5. Cancer Discov. 2020 Jun 22. pii: CD-19-1228. [Epub ahead of print]
    Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition.
    Bryan Ngo, Eugenie Kim, Victoria Osorio-Vasquez, Sophia Doll, Sophia Bustraan, Roger J Liang, Alba Luengo, Shawn M Davidson, Ahmed Ali, Gino B Ferraro, Grant M Fischer, Roozbeh Eskandari, Diane S Kang, Jing Ni, Ariana Plasger, Vinagolu K Rajasekhar, Edward R Kastenhuber, Sarah Bacha, Roshan K Sriram, Benjamin D Stein, Samuel F Bakhoum, Matija Snuderl, Paolo Cotzia, John H Healey, Nello Mainolfi, Vipin Suri, Adam Friedman, Mark Manfredi, David M Sabatini, Drew R Jones, Min Yu, Jean J Zhao, Rakesh K Jain, Kayvan R Keshari, Michael A Davies, Matthew G Vander Heiden, Eva Hernando, Matthias Mann, Lewis C Cantley, Michael E Pacold.
      A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1228
  6. J Biol Chem. 2020 Jun 22. pii: jbc.RA120.013695. [Epub ahead of print]
    Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.
    Jana Škerlová, Judith Unterlass, Mona Göttmann, Petra Marttila, Evert Homan, Thomas Helleday, Ann-Sofie Jemth, Pål Stenmark.
      The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and L-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.
    Keywords:  N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR); cancer target; cancer therapy; carboxyaminoimidazole ribonucleotide (CAIR); de novo purine biosynthesis; drug design; nucleoside/nucleotide biosynthesis; purine; rational drug design; structural biology
    DOI:  https://doi.org/10.1074/jbc.RA120.013695
  7. Nat Commun. 2020 Jun 22. 11(1): 3158
    DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner.
    Patrick L Collins, Caitlin Purman, Sofia I Porter, Vincent Nganga, Ankita Saini, Katharina E Hayer, Greer L Gurewitz, Barry P Sleckman, Jeffrey J Bednarski, Craig H Bassing, Eugene M Oltz.
      Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for γH2Ax landscapes. DSBs that disrupt a topological border permit extension of γH2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with γH2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity.
    DOI:  https://doi.org/10.1038/s41467-020-16926-x
  8. Nat Commun. 2020 Jun 23. 11(1): 3165
    Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis.
    Elizabeth R Morris, Sarah J Caswell, Simone Kunzelmann, Laurence H Arnold, Andrew G Purkiss, Geoff Kelly, Ian A Taylor.
      SAMHD1 regulates cellular 2'-deoxynucleoside-5'-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2'-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5' bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.
    DOI:  https://doi.org/10.1038/s41467-020-16983-2
  9. Commun Biol. 2020 Jun 24. 3(1): 324
    SAMHD1 is a key regulator of the lineage-specific response of acute lymphoblastic leukaemias to nelarabine.
    Tamara Rothenburger, Katie-May McLaughlin, Tobias Herold, Constanze Schneider, Thomas Oellerich, Florian Rothweiler, Andrew Feber, Tim R Fenton, Mark N Wass, Oliver T Keppler, Martin Michaelis, Jindrich Cinatl.
      The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.
    DOI:  https://doi.org/10.1038/s42003-020-1052-8
  10. Int J Gynecol Cancer. 2020 Jun 22. pii: ijgc-2020-001277. [Epub ahead of print]
    Exploiting replicative stress in gynecological cancers as a therapeutic strategy.
    Natalie Yl Ngoi, Vignesh Sundararajan, David Sp Tan.
      Elevated levels of replicative stress in gynecological cancers arising from uncontrolled oncogenic activation, loss of key tumor suppressors, and frequent defects in the DNA repair machinery are an intrinsic vulnerability for therapeutic exploitation. The presence of replication stress activates the DNA damage response and downstream checkpoint proteins including ataxia telangiectasia and Rad3 related kinase (ATR), checkpoint kinase 1 (CHK1), and WEE1-like protein kinase (WEE1), which trigger cell cycle arrest while protecting and restoring stalled replication forks. Strategies that increase replicative stress while lowering cell cycle checkpoint thresholds may allow unrepaired DNA damage to be inappropriately carried forward in replicating cells, leading to mitotic catastrophe and cell death. Moreover, the identification of fork protection as a key mechanism of resistance to chemo- and poly (ADP-ribose) polymerase inhibitor therapy in ovarian cancer further increases the priority that should be accorded to the development of strategies targeting replicative stress. Small molecule inhibitors designed to target the DNA damage sensors, such as inhibitors of ataxia telangiectasia-mutated (ATM), ATR, CHK1 and WEE1, impair smooth cell cycle modulation and disrupt efficient DNA repair, or a combination of the above, have demonstrated interesting monotherapy and combinatorial activity, including the potential to reverse drug resistance and have entered developmental pipelines. Yet unresolved challenges lie in balancing the toxicity profile of these drugs in order to achieve a suitable therapeutic index while maintaining clinical efficacy, and selective biomarkers are urgently required. Here we describe the premise for targeting of replicative stress in gynecological cancers and discuss the clinical advancement of this strategy.
    Keywords:  cervical cancer; medical oncology; ovarian cancer; uterine cancer
    DOI:  https://doi.org/10.1136/ijgc-2020-001277
  11. Nat Commun. 2020 Jun 23. 11(1): 3181
    Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases.
    Matteo Ferrari, Chetan C Rawal, Samuele Lodovichi, Maria Y Vietri, Achille Pellicioli.
      The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.
    DOI:  https://doi.org/10.1038/s41467-020-16997-w
  12. Curr Protoc Stem Cell Biol. 2020 Sep;54(1): e115
    DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.
    Jason A Halliwell, Polly Gravells, Helen E Bryant.
      Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.
    Keywords:  CldU; DNA fiber; IdU; chlorodeoxyuridine; human pluripotent stem cells; iododeoxyuridine; replication
    DOI:  https://doi.org/10.1002/cpsc.115
  13. Mol Biol Cell. 2020 Jun 24. mbcE19100589
    Nuclear body phase separation drives telomere clustering in ALT cancer cells.
    Huaiying Zhang, Rongwei Zhao, Jason Tones, Michel Liu, Robert Dilley, David M Chenoweth, Roger A Greenberg, Michael A Lampson.
      Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically-induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere binding protein sumoylation nucleates APB condensation via SUMO-SIM (SUMO interaction motif) interactions, and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering independent of sumoylation. Our findings introduce a chemical dimerization approach to manipulate phase separation and demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E19-10-0589
  14. Aging (Albany NY). 2020 Jun 25. 12
    SIRT6 mono-ADP ribosylates KDM2A to locally increase H3K36me2 at DNA damage sites to inhibit transcription and promote repair.
    Sarallah Rezazadeh, David Yang, Seyed Ali Biashad, Denis Firsanov, Masaki Takasugi, Michael Gilbert, Gregory Tombline, Natarajan V Bhanu, Benjamin A Garcia, Andrei Seluanov, Vera Gorbunova.
      When transcribed DNA is damaged, the transcription and DNA repair machineries must interact to ensure successful DNA repair. The mechanisms of this interaction in the context of chromatin are still being elucidated. Here we show that the SIRT6 protein enhances non-homologous end joining (NHEJ) DNA repair by transiently repressing transcription. Specifically, SIRT6 mono-ADP ribosylates the lysine demethylase JHDM1A/KDM2A leading to rapid displacement of KDM2A from chromatin, resulting in increased H3K36me2 levels. Furthermore, we found that through HP1α binding, H3K36me2 promotes subsequent H3K9 tri-methylation. This results in transient suppression of transcription initiation by RNA polymerase II and recruitment of NHEJ factors to DNA double-stranded breaks (DSBs). These data reveal a mechanism where SIRT6 mediates a crosstalk between transcription and DNA repair machineries to promote DNA repair. SIRT6 functions in multiple pathways related to aging, and its novel function coordinating DNA repair and transcription is yet another way by which SIRT6 promotes genome stability and longevity.
    Keywords:  DNA repair; SIRT6; genome stability; longevity; transcription
    DOI:  https://doi.org/10.18632/aging.103567
  15. Onco Targets Ther. 2020 ;13 4833-4842
    Inhibition of Serine Metabolism Promotes Resistance to Cisplatin in Gastric Cancer.
    Xiaoya Zhao, Jianfei Fu, Wanfen Tang, Liangliang Yu, Wenxia Xu.
       Background: Serine provides important precursors of protein, lipid, and nucleotide synthesis needed for tumor cell growth. Phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in the serine de novo synthesis pathway, is highly expressed in many tumor types (including gastric cancer) and negatively correlated with overall survival. Cisplatin is a chemotherapeutic drug commonly used in the treatment of gastric cancer. In this study, we mainly investigated the relationship between serine metabolism and resistance to cisplatin in gastric cancer cells, as well as the regulatory mechanism involved in this process.
    Materials and Methods: We determined the effect of different concentrations of serine or a PHGDH inhibitor combined with cisplatin or oxaliplatin on the viability and apoptosis of SGC7901, BGC823, and MGC803 cells via the Cell Counting Kit-8 and Hoechst 33258 staining, respectively. Western blotting was utilized to detect the relative protein expression. Furthermore, we investigated DNA damage through the micrococcal nuclease sensitivity assay detected using agarose gels.
    Results: We found that reduced concentrations of serine or inhibition of PHGDH hindered the toxicity and pro-apoptotic effects of cisplatin on gastric cancer cells. Moreover, the addition of serine could reverse the sensitivity of gastric cancer cells to cisplatin. Moreover, we found that DNA damage was reduced by treatment with PHGDH inhibitor NCT-503 or CBR-5884. Inhibition of serine metabolism induced a decrease in H3K4 tri-methylation, which was reversed by JIB-04 (inhibitor of H3K4 demethylase). The tolerance of gastric cancer cells to cisplatin was relieved by JIB-04. Through micrococcal nuclease experiments, we further found that inhibiting the activity of PHGDH strengthened chromatin tightness.
    Conclusion: Inhibition of serine metabolism reduced H3K4 tri-methylation and increased the density of chromatin, which leads to decreased toxicity and pro-apoptotic effect of platinum chemotherapeutic drugs on gastric cancer cells.
    Keywords:  DNA damage; PHGDH; cisplatin resistance; gastric cancer; serine metabolism
    DOI:  https://doi.org/10.2147/OTT.S246430
  16. Mol Cancer. 2020 Jun 20. 19(1): 107
    PARP inhibitor resistance: the underlying mechanisms and clinical implications.
    He Li, Zhao-Yi Liu, Nayiyuan Wu, Yong-Chang Chen, Quan Cheng, Jing Wang.
      Due to the DNA repair defect, BRCA1/2 deficient tumor cells are more sensitive to PARP inhibitors (PARPi) through the mechanism of synthetic lethality. At present, several PAPRi targeting poly (ADP-ribose) polymerase (PARP) have been approved for ovarian cancer and breast cancer indications. However, PARPi resistance is ubiquitous in clinic. More than 40% BRCA1/2-deficient patients fail to respond to PARPi. In addition, lots of patients acquire PARPi resistance with prolonged oral administration of PARPi. Homologous recombination repair deficient (HRD), as an essential prerequisite of synthetic lethality, plays a vital role in killing tumor cells. Therefore, Homologous recombination repair restoration (HRR) becomes the predominant reason of PARPi resistance. Recently, it was reported that DNA replication fork protection also contributed to PARPi resistance in BRCA1/2-deficient cells and patients. Moreover, various factors, such as reversion mutations, epigenetic modification, restoration of ADP-ribosylation (PARylation) and pharmacological alteration lead to PARPi resistance as well. In this review, we reviewed the underlying mechanisms of PARP inhibitor resistance in detail and summarized the potential strategies to overcome PARPi resistance and increase PARPi sensitivity.
    Keywords:  Homologous recombination; PARPi; Resistance; Synthetic lethality
    DOI:  https://doi.org/10.1186/s12943-020-01227-0
  17. Blood Cells Mol Dis. 2020 Jun 09. pii: S1079-9796(20)30131-5. [Epub ahead of print]84 102460
    ANKHD1 is an S phase protein required for histone synthesis and DNA repair in multiple myeloma cells.
    Anamika Dhyani, Patricia Favaro, Sara T Olalla Saad.
      ANKHD1 is highly expressed in various cancers such as leukemia and multiple myeloma. Silencing of ANKHD1 expression leads to decreased cell proliferation and accumulation of cells at the S phase. In this study we found ANKHD1 expression to be higher at the S phase, suggesting it to be an S phase protein. We observed that ANKHD1 interacts with histone promoter regions and its inhibition downregulates expression of all core histones, implying a role in histone synthesis. Since histone synthesis occurs in parallel with DNA replication at S phase, we evaluated PCNA (Proliferating Cell Nuclear Antigen) expression, a protein involved in DNA replication and repair. PCNA expression was found to be significantly decreased in ANKHD1 silenced cells. We further observed accumulation γH2AX, a marker for DNA double stranded breaks and an early sign of DNA damage induced by replication stress, upon ANKHD1 silencing. The expressions of several genes implicated in DNA repair were also modulated in ANKHD1 silenced cells, confirming the role of ANKHD1 in DNA repair. Based on this study we speculate that ANKHD1 is an S phase protein required for histone synthesis and DNA repair. These results however, are preliminary and require thorough investigation.
    Keywords:  Ankyrin repeat and KH domain-containing 1; DNA damage; Histones; Myeloma; Proliferating cell nuclear antigen; S phase
    DOI:  https://doi.org/10.1016/j.bcmd.2020.102460
  18. Cells. 2020 Jun 18. pii: E1483. [Epub ahead of print]9(6):
    CDK12: A Potent Target and Biomarker for Human Cancer Therapy.
    Shujing Liang, Lifang Hu, Zixiang Wu, Zhihao Chen, Shuyu Liu, Xia Xu, Airong Qian.
      Cyclin-dependent kinases (CDKs) are a group of serine/threonine protein kinases and play crucial roles in various cellular processes by regulating cell cycle and gene transcription. Cyclin-dependent kinase 12 (CDK12) is an important transcription-associated CDK. It shows versatile roles in regulating gene transcription, RNA splicing, translation, DNA damage response (DDR), cell cycle progression and cell proliferation. Recently, increasing evidence demonstrates the important role of CDK12 in various human cancers, illustrating it as both a biomarker of cancer and a potential target for cancer therapy. Here, we summarize the current knowledge of CDK12, and review the research advances of CDK12's biological functions, especially its role in human cancers and as a potential target and biomarker for cancer therapy.
    Keywords:  DNA damage response; cancer therapy; cell cycle; cell proliferation; cyclin-dependent kinase 12; gene transcription
    DOI:  https://doi.org/10.3390/cells9061483
  19. Cell Biosci. 2020 ;10 81
    Limited MOMP, ATM, and their roles in carcinogenesis and cancer treatment.
    Xuhui Bao, Xinjian Liu, Fang Li, Chuan-Yuan Li.
      Limited mitochondria outer membrane permeability (MOMP) is a novel biological process where mammalian cells initiate the intrinsic apoptosis pathway with increased mitochondrial permeability but survive. One of the major consequences of limited MOMP is apoptotic endonuclease-induced DNA double strand breaks. Recent studies indicate that these DNA double stand breaks and ensuing activation of DNA damage response factors such as ATM play important but previously underappreciated roles in carcinogenesis and tumor growth. Furthermore, novel non-canonical roles of DNA repair factors such as ATM in tumor growth and treatment are also emerging. In this review, we try to summarize recent findings on this newly revealed link between DNA double strand break repair and cell death pathways.
    DOI:  https://doi.org/10.1186/s13578-020-00442-y
  20. Nucleic Acids Res. 2020 Jun 25. pii: gkaa541. [Epub ahead of print]
    Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication.
    Katy Stokes, Alicja Winczura, Boyuan Song, Giacomo De Piccoli, Daniel B Grabarczyk.
      The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.
    DOI:  https://doi.org/10.1093/nar/gkaa541
  21. Mol Oncol. 2020 Jun 24.
    The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe.
    Stanislav Drápela, Prashant Khirsariya, Wytske M van Weerden, Radek Fedr, Tereza Suchánková, Diana Búzová, Jan Červený, Aleš Hampl, Martin Puhr, William R Watson, Zoran Culig, Lumír Krejčí, Kamil Paruch, Karel Souček.
      As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality, hence CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here we report, that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to gemcitabine in a panel of chemo-naïve and matched docetaxel-resistant PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with gemcitabine than clinical candidate SCH900776. MU380 alone or in combination with gemcitabine significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in docetaxel-resistant models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a pre-stage of apoptosis. Finally, treatment by MU380 alone, or in combination with gemcitabine, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of gemcitabine in docetaxel-resistant mCRPC models. This approach might allow for dose reduction of gemcitabine and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable docetaxel-resistant mCRPC.
    Keywords:  MU380; castration-resistant prostate cancer; checkpoint kinase 1; docetaxel-resistance; gemcitabine; mitotic catastrophe
    DOI:  https://doi.org/10.1002/1878-0261.12756
  22. Cells. 2020 Jun 20. pii: E1506. [Epub ahead of print]9(6):
    Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6.
    Cecilia Aquino Perez, Matous Palek, Lenka Stolarova, Patrick von Morgen, Libor Macurek.
      Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.
    Keywords:  DNA damage; Polo-like kinase 3; protein kinase; protein phosphatase; stress
    DOI:  https://doi.org/10.3390/cells9061506
  23. Oncogene. 2020 Jun 22.
    Functional genomics identifies new synergistic therapies for retinoblastoma.
    Arthur Aubry, Joel D Pearson, Katherine Huang, Izhar Livne-Bar, Mohammad Ahmad, Madhavan Jagadeesan, Vikas Khetan, Troy Ketela, Kevin R Brown, Tao Yu, Suying Lu, Jeffrey L Wrana, Jason Moffat, Rod Bremner.
      Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1null and RB1wt;MYCNamp orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.
    DOI:  https://doi.org/10.1038/s41388-020-1372-7
  24. BMC Complement Med Ther. 2020 Jun 26. 20(1): 196
    Jianpi-yangwei decoction inhibits DNA damage repair in the drug resistance of gastric cancer by reducing FEN1 expression.
    Wenjie Huang, Huijuan Tang, Fang Wen, Xiaona Lu, Qingpei Li, Peng Shu.
       BACKGROUND: Flap Endonuclease 1(FEN1) has been considered as a new tumor marker in recent years and Jianpi Yangwei Decoction (JPYW) is a basic Traditional Chinese Medicine (TCM) for the treatment of gastric cancer. This study aimed to explore the role of FEN1-mediated DNA damage repair in the drug resistance of gastric cancer and the effect of JPYW on it by employing BGC823/5-Fu drug-resistant cell model.
    METHODS: The DNA repair efficiency of BGC823 and BGC823/5-Fu was compared intracellularly and extracellularly using an extrachromosomal assay system and the reconstituted base excision repair assay. By comparing gene and protein expression and identifying cell survival rates after knockdown or high expression of FEN1, the correlation between FEN1 high expression and 5-Fluorouracil (5-Fu) drug resistance was revealed. The effect of JPYW on DNA damage repair and FEN1 expression was observed by the degree of γ-H2AX phosphorylation in the cells, DNA repair efficiency and enzyme activity, et al. RESULTS: BGC823/5-Fu had a higher DNA repair efficiency than BGC823(P < 0.001), which proved to be both intracellular and extracellular. FEN1 was highly expressed in BGC823/5-Fu regardless of gene level(P < 0.001) or protein level. Furthermore, manipulating FEN1 altered the sensitivity of cancer cells to chemotherapeutic drug 5-Fu. Different concentrations of JPYW were used to investigate the inhibitory effect on the expression of FEN1 and DNA damage repair. JPYW inhibited DNA damage repair both intracellularly and extracellularly: the phosphorylation of γ-H2AX increased, with more DNA damage in the cells; the synthetic 8-oxo dG damage repair was reduced; and the ability of cell lysates to repair DNA damage decreased. The decrease of FEN1 expression in BGC823/5-Fu had a concentration dependent relationship with JYPW. In addition, JPYW inhibited the activity of FEN1 at the enzymatic level, as the amount of cut-off synthetic 32p labeled DNA substrates were decreased.
    CONCLUSION: FEN1 was highly expressed in drug-resistance gastric cancer cells BGC823/5-Fu, which leading to BGC823 resistant to (5-Fu) by acting on DNA damage repair. JPYW inhibited DNA damage repair and reversed 5-Fu drug resistance by reducing FEN1 expression and inhibiting FEN1 functional activity.
    Keywords:  DNA damage repair; Drug resistance; FEN1; Gastric cancer; Jianpi Yangwei decoction
    DOI:  https://doi.org/10.1186/s12906-020-02983-8
  25. Front Oncol. 2020 ;10 919
    AATF/Che-1-An RNA Binding Protein at the Nexus of DNA Damage Response and Ribosome Biogenesis.
    Rainer W J Kaiser, Johanna Erber, Katja Höpker, Francesca Fabretti, Roman-Ulrich Müller.
      The DNA damage response (DDR) is a complex signaling network that is activated upon genotoxic stress. It determines cellular fate by either activating cell cycle arrest or initiating apoptosis and thereby ensures genomic stability. The Apoptosis Antagonizing Transcription Factor (AATF/Che-1), an RNA polymerase II-interacting transcription factor and known downstream target of major DDR kinases, affects DDR signaling by inhibiting p53-mediated transcription of pro-apoptotic genes and promoting cell cycle arrest through various pathways instead. Specifically, AATF was shown to inhibit p53 expression at the transcriptional level and repress its pro-apoptotic activity by direct binding to p53 protein and transactivation of anti-apoptotic genes. Solid and hematological tumors of various organs exploit this function by overexpressing AATF. Both copy number gains and high expression levels of AATF were associated with worse prognosis or relapse of malignant tumors. Recently, a number of studies have enabled insights into the molecular mechanisms by which AATF affects both DDR and proliferation. AATF was found to directly localize to sites of DNA damage upon laser ablation and interact with DNA repair proteins. In addition, depletion of AATF resulted in increased DNA damage and decrease of both proliferative activity and genotoxic tolerance. Interestingly, considering the role of ribosomal stress in the regulation of p53, more recent work established AATF as ribosomal RNA binding protein and enabled insights into its role as an important factor for rRNA processing and ribosome biogenesis. This Mini Review summarizes recent findings on AATF and its important role in the DDR, malignancy, and ribosome biogenesis.
    Keywords:  AATF/Che-1; DNA damage response; p53; rRNA processing; ribosome biogenesis
    DOI:  https://doi.org/10.3389/fonc.2020.00919
  26. Commun Biol. 2020 Jun 23. 3(1): 322
    The iron-sulphur cluster in human DNA2 is required for all biochemical activities of DNA2.
    Laura Mariotti, Sebastian Wild, Giulia Brunoldi, Alessandra Piceni, Ilaria Ceppi, Sandra Kummer, Richard E Lutz, Petr Cejka, Kerstin Gari.
      The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2's nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.
    DOI:  https://doi.org/10.1038/s42003-020-1048-4
  27. Cancer Drug Resist. 2020 ;3(2): 161-170
    Effects of DNA topoisomerase IIα splice variants on acquired drug resistance.
    Terry S Elton, Hatice Gulcin Ozer, Jack C Yalowich.
      DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.
    Keywords:  DNA topoisomerase IIα; alternative splicing; chemoresistance; intron retention; topoisomerase IIα interfacial inhibitors/poisons
    DOI:  https://doi.org/10.20517/cdr.2019.117
  28. Annu Rev Biochem. 2020 Jun 20. 89 45-75
    Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.
    Brandon L Greene, Gyunghoon Kang, Chang Cui, Marina Bennati, Daniel G Nocera, Catherine L Drennan, JoAnne Stubbe.
      Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
    Keywords:  mechanisms; ribonucleotide reductases; structures; therapeutics
    DOI:  https://doi.org/10.1146/annurev-biochem-013118-111843
  29. Biol Open. 2020 Jun 24. pii: bio.050328. [Epub ahead of print]
    CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production.
    Jacqueline C Simonet, Maya J Foster, Eric M Lynch, Justin M Kollman, Emmanuelle Nicholas, Alana M O'Reilly, Jeffrey R Peterson.
      Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, H355A, prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous, wild type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in files fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.
    Keywords:  Agglomeration; Metabolic enzyme; Nucleotide biosynthesis
    DOI:  https://doi.org/10.1242/bio.050328
  30. Cancers (Basel). 2020 Jun 22. pii: E1653. [Epub ahead of print]12(6):
    HERC Ubiquitin Ligases in Cancer.
    Joan Sala-Gaston, Arturo Martinez-Martinez, Leonardo Pedrazza, L Francisco Lorenzo-Martín, Rubén Caloto, Xosé R Bustelo, Francesc Ventura, Jose Luis Rosa.
      HERC proteins are ubiquitin E3 ligases of the HECT family. The HERC subfamily is composed of six members classified by size into large (HERC1 and HERC2) and small (HERC3-HERC6). HERC family ubiquitin ligases regulate important cellular processes, such as neurodevelopment, DNA damage response, cell proliferation, cell migration, and immune responses. Accumulating evidence also shows that this family plays critical roles in cancer. In this review, we provide an integrated view of the role of these ligases in cancer, highlighting their bivalent functions as either oncogenes or tumor suppressors, depending on the tumor type. We include a discussion of both the molecular mechanisms involved and the potential therapeutic strategies.
    Keywords:  E3; ERK; HECT; MAPK; RAF; genome stability; oncogene; p38; p53; tumor suppressor
    DOI:  https://doi.org/10.3390/cancers12061653
  31. Signal Transduct Target Ther. 2020 Jun 24. 5(1): 80
    SUMOylation stabilizes hSSB1 and enhances the recruitment of NBS1 to DNA damage sites.
    Liwen Zhou, Lisi Zheng, Kaishun Hu, Xin Wang, Ruhua Zhang, Yezi Zou, Li Zhong, Shang Wang, Yuanzhong Wu, Tiebang Kang.
      Human single-stranded DNA-binding protein 1 (hSSB1) is required for the efficient recruitment of the MRN complex to DNA double-strand breaks and is essential for the maintenance of genome integrity. However, the mechanism by which hSSB1 recruits NBS1 remains elusive. Here, we determined that hSSB1 undergoes SUMOylation at both K79 and K94 under normal conditions and that this modification is dramatically enhanced in response to DNA damage. SUMOylation of hSSB1, which is specifically fine-tuned by PIAS2α, and SENP2, not only stabilizes the protein but also enhances the recruitment of NBS1 to DNA damage sites. Cells with defective hSSB1 SUMOylation are sensitive to ionizing radiation, and global inhibition of SUMOylation by either knocking out UBC9 or adding SUMOylation inhibitors significantly enhances the sensitivity of cancer cells to etoposide. Our findings reveal that SUMOylation, as a novel posttranslational modification of hSSB1, is critical for the functions of this protein, indicating that the use of SUMOylation inhibitors (e.g., 2-D08 and ML-792) may be a new strategy that would benefit cancer patients being treated with chemo- or radiotherapy.
    DOI:  https://doi.org/10.1038/s41392-020-0172-4
  32. Cell Cycle. 2020 Jun 23. 1-16
    Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system.
    Diletta Ciardo, Olivier Haccard, Hemalatha Narassimprakash, Virginie Chiodelli, Arach Goldar, Kathrin Marheineke.
      Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase. Further, we found that chromatin-bound Plk1 is phosphorylated on its activating site T201, which appears to be sensitive to dephosphorylation by protein phosphatase 2A. Extracts immunodepleted of Plk1 showed a decrease in DNA replication, rescued by wild type recombinant Plk1. Inversely, modest Plk1 overexpression accelerated DNA replication. Plk1 depletion led to an increase in Chk1 phosphorylation and to a decrease in Cdk2 activity, which strongly suggests that Plk1 could inhibit the ATR/Chk1-dependent intra-S phase checkpoint during normal S phase. In addition, we observed that phosphorylated Plk1 levels are high during the rapid, early cell cycles of Xenopus development but decrease after the mid-blastula transition when the cell cycle and the replication program slow down along with more active checkpoints. These data shed new light on the role of Plk1 as a positive regulating factor for DNA replication in early, rapidly dividing embryos.
    Keywords:   Xenopus ; DNA replication; Polo-like kinase 1 (Plk1); checkpoint
    DOI:  https://doi.org/10.1080/15384101.2020.1782589
  33. Cells. 2020 Jun 22. pii: E1522. [Epub ahead of print]9(6):
    New Frontiers in Prostate Cancer Treatment: Are We Ready for Drug Combinations with Novel Agents?
    Gaetano Aurilio, Alessia Cimadamore, Matteo Santoni, Franco Nolè, Marina Scarpelli, Francesco Massari, Antonio Lopez-Beltran, Liang Cheng, Rodolfo Montironi.
      Medical treatment for metastatic castration-resistant prostate cancer (mCRPC) patients has progressively been evolving from a nonspecific clinical approach to genomics-oriented therapies. The scientific community is in fact increasingly focusing on developing DNA damage repair (DDR) defect-driven novel molecules, both as single-agent therapy and in combined treatment strategies. Accordingly, research is under way into combined drug therapies targeting different pathways, e.g. androgen receptor signaling (ARS) and poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) enzymes, immune checkpoint (IC) and PARP, IC, and ARS, and prostate-specific membrane antigen (PSMA). In an attempt to formulate evolving treatment paradigms in mCRPC patients, here we selected clinical research into patients undergoing therapies with emerging molecules, with particular emphasis towards PARP-, IC-, and PSMA-inhibitors. In order to focus on those molecules and drug combinations most likely to be translated into routine clinical care in the near future, we selected only those clinical studies currently recruiting patients. A PubMed search focusing on the keywords "prostate cancer", "metastatic castration-resistant prostate cancer", "DDR pathways", "ARS inhibitors", "PARP inhibitors", "IC inhibitors", "PSMA-targeting agents", and "drug combinations" was performed.
    Keywords:  ARS inhibitors; DNA damage repair; PARP inhibitors; PSMA-inhibition; drug combinations; immune checkpoint inhibitors; metastatic castration-resistant prostate cancer; prostate cancer
    DOI:  https://doi.org/10.3390/cells9061522
  34. Mol Pharmacol. 2020 Jun 25. pii: mol.119.118893. [Epub ahead of print]
    Small molecule inhibitors confirm ubiquitin-dependent removal of TOP2-DNA covalent complexes.
    Rebecca Swan, Luke Poh, Ian G Cowell, Caroline A Austin.
      DNA Topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double strand break (DSB) in one DNA molecule, and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilise a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin activating enzymes reduces the processing of TOP2A- and TOP2B- DNA complexes. Depletion or inhibition of ubiquitin activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilised by etoposide were shown to be conjugated to ubiquitin and this was reduced by inhibition or depletion of ubiquitin activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of TOP2 poisons can be enhanced through combination therapy with UAE inhibitors, or by specific inhibition of the BMI/RING1A ubiquitin ligase which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.
    Keywords:  Cancer chemotherapy; Topoisomerases
    DOI:  https://doi.org/10.1124/mol.119.118893
  35. Biochemistry (Mosc). 2020 Mar;85(3): 288-299
    Effect of Human XRCC1 Protein Oxidation on the Functional Activity of Its Complexes with the Key Enzymes of DNA Base Excision Repair.
    I A Vasil'eva, N A Moor, O I Lavrik.
      Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time in vitro formation of the ternary XRCC1 complex with the key enzymes of SP BER - DNA polymerase β (Polβ) and DNA ligase IIIα (LigIIIα) - using the fluorescence-based technique. It was found that Polβ directly interacts with LigIIIα, but their complex is less stable than the XRCC1-Polβ and XRCC1-LigIIIα complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polβ), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.
    DOI:  https://doi.org/10.1134/S0006297920030049
  36. Elife. 2020 Jun 24. pii: e51378. [Epub ahead of print]9
    AR phosphorylation and CHK2 kinase activity regulates IR stabilized AR-CHK2 interaction and prostate cancer survival.
    Huy Q Ta, Natalia Dworak, Melissa L Ivey, Devin G Roller, Daniel Gioeli.
      We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR-CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.
    Keywords:  biochemistry; cancer biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.51378
  37. Sci Rep. 2020 Jun 25. 10(1): 10325
    Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.
    Hetty E Carraway, Sridhar A Malkaram, Yana Cen, Aymen Shatnawi, Jun Fan, Hamdy E A Ali, Zakaria Y Abd Elmageed, Thomm Buttolph, James Denvir, Donald A Primerano, Tamer E Fandy.
      The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.
    DOI:  https://doi.org/10.1038/s41598-020-67170-8
  38. Toxicol Lett. 2020 Jun 22. pii: S0378-4274(20)30292-7. [Epub ahead of print]
    Induction of apoptosis in Ogg1-null mouse embryonic fibroblasts by GSH depletion is independent of DNA damage.
    Ellen B Higgs, Roger Godschalk, Nicholas J Coltman, Frederik-Jan van Schooten, Nikolas J Hodges.
      Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in mOgg1-/- MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in mOgg1-/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1-/- MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in mOgg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into mOgg1-/- MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in mOgg1-/- MEFs is independent of DNA damage and OGG1-initiated DNA repair.
    Keywords:  8-oxoG; 8-oxoguanine DNA glycosylase; apoptosis; glutathione; oxidative stress
    DOI:  https://doi.org/10.1016/j.toxlet.2020.06.019
  39. Cancers (Basel). 2020 Jun 18. pii: E1617. [Epub ahead of print]12(6):
    Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair.
    Barbara N Borsos, Hajnalka Majoros, Tibor Pankotai.
      The proper function of DNA repair is indispensable for eukaryotic cells since accumulation of DNA damages leads to genome instability and is a major cause of oncogenesis. Ubiquitylation and deubiquitylation play a pivotal role in the precise regulation of DNA repair pathways by coordinating the recruitment and removal of repair proteins at the damaged site. Here, we summarize the most important post-translational modifications (PTMs) involved in DNA double-strand break repair. Although we highlight the most relevant PTMs, we focus principally on ubiquitylation-related processes since these are the most robust regulatory pathways among those of DNA repair.
    Keywords:  DNA repair; DSB; PTM; deubiquitylation; ubiquitylation
    DOI:  https://doi.org/10.3390/cancers12061617
  40. Life Sci Alliance. 2020 Aug;pii: e202000671. [Epub ahead of print]3(8):
    Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells.
    Somsundar V Muralidharan, Lisa M Nilsson, Mattias F Lindberg, Jonas A Nilsson.
      Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.
    DOI:  https://doi.org/10.26508/lsa.202000671
  41. Leuk Lymphoma. 2020 Jun 22. 1-11
    Short-term treatment with imetelstat sensitizes hematopoietic malignant cells to a genotoxic agent via suppression of the telomerase-mediated DNA repair process.
    Daisuke Hidaka, Masahiro Onozawa, Naohiro Miyashita, Shota Yokoyama, Masao Nakagawa, Daigo Hashimoto, Takanori Teshima.
      Imetelstat is a specific and competitive inhibitor of telomerase enzymatic activity. We demonstrated that imetelstat could interfere with the DNA repair process and enhance the effect of DNA damaging agents using hematological tumor cell lines. Short-term administration of imetelstat enhanced growth suppression by anticancer agents and radiation. It also upregulated γH2AX expression induced by irradiation. Immunofluorescence staining showed that both human telomerase reverse transcriptase (hTERT) and γH2AX were upregulated and co-localized in the nucleus of peripheral blood mononuclear cells after irradiation, suggesting that hTERT was involved in the DNA-DSB repair process. Imetelstat enhanced growth inhibitory effect of cytotoxic agents in short-term culture without shortening of telomeres, indicating that this effect was attributed by telomere length independent mechanism. Our results suggest that the combination of short-term treatment with imetelstat and cytotoxic agent is a promising strategy to treat a wide variety of hematopoietic malignancies.
    Keywords:  Polymorphic telomere insertion; interstitial telomeric sequences; telomerase inhibition
    DOI:  https://doi.org/10.1080/10428194.2020.1779256
  42. Biochem Soc Trans. 2020 Jun 23. pii: BST20190033. [Epub ahead of print]
    Nicotinamide adenine dinucleotide (NAD+): essential redox metabolite, co-substrate and an anti-cancer and anti-ageing therapeutic target.
    Hollie B S Griffiths, Courtney Williams, Sarah J King, Simon J Allison.
      Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are essential coupled redox metabolites that primarily promote cellular oxidative (catabolic) metabolic reactions. This enables energy generation through glycolysis and mitochondrial respiration to support cell growth and survival. In addition, many key enzymes that regulate diverse cell functions ranging from gene expression to proteostasis require NAD+ as a co-substrate for their catalytic activity. This includes the NAD+-dependent sirtuin family of protein deacetylases and the PARP family of DNA repair enzymes. Whilst their vital activity consumes NAD+ which is cleaved to nicotinamide, several pathways exist for re-generating NAD+ and sustaining NAD+ homeostasis. However, there is growing evidence of perturbed NAD+ homeostasis and NAD+-regulated processes contributing to multiple disease states. NAD+ levels decline in the human brain and other organs with age and this is associated with neurodegeneration and other age-related diseases. Dietary supplementation with NAD+ precursors is being investigated to counteract this. Paradoxically, many cancers have increased dependency on NAD+. Clinical efforts to exploit this have so far shown limited success. Emerging new opportunities to exploit dysregulation of NAD+ metabolism in cancers are critically discussed. An update is also provided on other key NAD+ research including perturbation of the NAD+ salvage enzyme NAMPT in the context of the tumour microenvironment (TME), methodology to study subcellular NAD+ dynamics in real-time and the regulation of differentiation by competing NAD+ pools.
    Keywords:  NAMPT; PARP; cancer therapeutics; lactate dehydrogenase A; nicotinamide adenine dinucleotide; sirtuins
    DOI:  https://doi.org/10.1042/BST20190033
  43. Nucleic Acids Res. 2020 Jun 23. pii: gkaa516. [Epub ahead of print]
    A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase.
    Janne Purhonen, Rishi Banerjee, Allison E McDonald, Vineta Fellman, Jukka Kallijärvi.
      Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.
    DOI:  https://doi.org/10.1093/nar/gkaa516
  44. Biochemistry (Mosc). 2020 Apr;85(4): 480-489
    An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.
    O A Kladova, D A Iakovlev, R Groisman, A A Ishchenko, M K Saparbaev, O S Fedorova, N A Kuznetsov.
      Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.
    DOI:  https://doi.org/10.1134/S0006297920040082
  45. Nat Commun. 2020 Jun 25. 11(1): 3214
    The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.
    Minakshi Gandhi, Matthias Groß, Jessica M Holler, Si'Ana A Coggins, Nitin Patil, Joerg H Leupold, Mathias Munschauer, Monica Schenone, Christina R Hartigan, Heike Allgayer, Baek Kim, Sven Diederichs.
      Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.
    DOI:  https://doi.org/10.1038/s41467-020-17007-9
  46. Nat Commun. 2020 Jun 26. 11(1): 3228
    Structure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase.
    Loïc Carrique, Lionel Ballut, Arpit Shukla, Neelakshi Varma, Resmi Ravi, Sébastien Violot, Bharath Srinivasan, Umesh Tippagondanahalli Ganeshappa, Sonia Kulkarni, Hemalatha Balaram, Nushin Aghajari.
      Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.
    DOI:  https://doi.org/10.1038/s41467-020-17013-x
  47. Cancers (Basel). 2020 Jun 21. pii: E1641. [Epub ahead of print]12(6):
    Fluoropyrimidine Modulation of the Anti-Tumor Immune Response-Prospects for Improved Colorectal Cancer Treatment.
    William H Gmeiner.
      Chemotherapy modulates the anti-tumor immune response and outcomes depend on the balance of favorable and unfavorable effects of drugs on anti-tumor immunity. 5-Florouracil (5-FU) is widely used in adjuvant chemotherapy regimens to treat colorectal cancer (CRC) and provides a survival benefit. However, survival remains poor for CRC patients with advanced and metastatic disease and immune checkpoint blockade therapy benefits only a sub-set of CRC patients. Here we discuss the effects of 5-FU-based chemotherapy regimens to the anti-tumor immune response. We consider how different aspects of 5-FU's multi-factorial mechanism differentially affect malignant and immune cell populations. We summarize recent studies with polymeric fluoropyrimidines (e.g., F10, CF10) that enhance DNA-directed effects and discuss how such approaches may be used to enhance the anti-tumor immune response and improve outcomes.
    Keywords:  5-Fluorouracil; MDSCs; T-cells; immunogenic cell death; immunotherapy; thymidylate synthase
    DOI:  https://doi.org/10.3390/cancers12061641
  48. Environ Mol Mutagen. 2020 Jun 23.
    A Stapled POL κ Peptide Targets REV1 to Inhibit Mutagenic Translesion Synthesis.
    Nimrat Chatterjee, Sanjay D'Souza, Mohammad Shabab, Cynthia A Harris, Gerard J Hilinski, Gregory L Verdine, Graham C Walker.
      Stapled α-helical RIR (Rev1-interacting region) peptides of DNA POL κ bind more effectively to the RIR-interface of the C-terminal recruitment domain of the translesion synthesis DNA polymerase Rev1 than unstapled peptide. The tightest-binding stapled peptide translocates into cells and enhances the cytotoxicity of DNA damaging agents while reducing mutagenesis. Drugs with these characteristics could potentially serve as adjuvants to improve chemotherapy and reduce acquired resistance by inhibiting Rev1-dependent mutagenic translesion synthesis. This article is protected by copyright. All rights reserved.
    Keywords:  Staple peptide; Translesion synthesis (TLS); chemoresistance; cytotoxicity; mutagenesis
    DOI:  https://doi.org/10.1002/em.22395
  49. Proc Natl Acad Sci U S A. 2020 Jun 25. pii: 201918519. [Epub ahead of print]
    Dynamic human MutSα-MutLα complexes compact mismatched DNA.
    Kira C Bradford, Hunter Wilkins, Pengyu Hao, Zimeng M Li, Bangchen Wang, Dan Burke, Dong Wu, Austin E Smith, Logan Spaller, Chunwei Du, Jacob W Gauer, Edward Chan, Peggy Hsieh, Keith R Weninger, Dorothy A Erie.
      DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.
    Keywords:  AFM; DNA repair; DREEM; MutL; MutS
    DOI:  https://doi.org/10.1073/pnas.1918519117
  50. Biochemistry (Mosc). 2020 May;85(5): 594-603
    Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1.
    D A Iakovlev, I V Alekseeva, N A Kuznetsov, O S Fedorova.
      Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme's tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.
    DOI:  https://doi.org/10.1134/S0006297920050089
  51. PLoS One. 2020 ;15(6): e0234970
    Identification of pathognomonic purine synthesis biomarkers by metabolomic profiling of adolescents with obesity and type 2 diabetes.
    Jennifer Concepcion, Katherine Chen, Rintaro Saito, Jon Gangoiti, Eric Mendez, Maria Eleni Nikita, Bruce A Barshop, Loki Natarajan, Kumar Sharma, Jane J Kim.
      The incidence of type 2 diabetes is increasing more rapidly in adolescents than in any other age group. We identified and compared metabolite signatures in obese children with type 2 diabetes (T2D), obese children without diabetes (OB), and healthy, age- and gender-matched normal weight controls (NW) by measuring 273 analytes in fasting plasma and 24-hour urine samples from 90 subjects by targeted LC-MS/MS. Diabetic subjects were within 2 years of diagnosis in an attempt to capture early-stage disease prior to declining renal function. We found 22 urine metabolites that were uniquely associated with T2D when compared to OB and NW groups. The metabolites most significantly elevated in T2D youth included members of the betaine pathway, nucleic acid metabolism, and branched-chain amino acids (BCAAs) and their catabolites. Notably, the metabolite pattern in OB and T2D groups differed between urine and plasma, suggesting that urinary BCAAs and their intermediates behaved as a more specific biomarker for T2D, while plasma BCAAs associated with the obese, insulin resistant state independent of diabetes status. Correlative analysis of metabolites in the T2D signature indicated that betaine metabolites, BCAAs, and aromatic amino acids were associated with hyperglycemia, but BCAA acylglycine derivatives and nucleic acid metabolites were linked to insulin resistance. Of major interest, we found that urine levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) were increased in diabetic youth, identifying urine SAICA-riboside as a potential biomarker for T2D.
    DOI:  https://doi.org/10.1371/journal.pone.0234970
  52. Int J Mol Sci. 2020 Jun 22. pii: E4425. [Epub ahead of print]21(12):
    Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.
    Kamila Puchałowicz, Maciej Tarnowski, Marta Tkacz, Dariusz Chlubek, Patrycja Kłos, Violetta Dziedziejko.
      A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPβS) in a 1-1000 μM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100-1000 μM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 μM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.
    Keywords:  SDF-1; acute myeloid leukemia; apoptosis; cell cycle; chemoresistance; cytotoxicity; extracellular nucleotides; proliferation; purinergic signaling
    DOI:  https://doi.org/10.3390/ijms21124425
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