bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒07‒04
sixty-one papers selected by
Sean Rudd
Karolinska Institutet
  1. A first-in-class Polymerase Theta Inhibitor selectively targets Homologous-Recombination-Deficient Tumors.
  2. Replication catastrophe induced by cyclic hypoxia leads to increased APOBEC3B activity.
  3. Daughter-strand gaps in DNA replication - substrates of lesion processing and initiators of distress signalling.
  4. Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase.
  5. Analyzing the Opportunities to Target DNA Double-Strand Breaks Repair and Replicative Stress Responses to Improve Therapeutic Index of Colorectal Cancer.
  6. EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.
  7. PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair.
  8. The Heritability of Replication Problems.
  9. Set1-dependent H3K4 methylation becomes critical for limiting DNA damage in response to changes in S-phase dynamics in Saccharomyces cerevisiae.
  10. New RAD51 Inhibitors to Target Homologous Recombination in Human Cells.
  11. KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance.
  12. Mechanism of mitotic recombination: insights from C. elegans.
  13. Loss of CENP-I Impairs Homologous Recombination and Sensitizes Cells to PARP1 Inhibition.
  14. The interplay between mitochondrial functionality and genome integrity in the prevention of human neurologic diseases.
  15. Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.
  16. Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?
  17. The Ultimate (Mis)match: When DNA Meets RNA.
  18. Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker.
  19. Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases.
  20. Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2.
  21. Consequences and Resolution of Transcription-Replication Conflicts.
  22. Targeting the replication stress response through synthetic lethal strategies in cancer medicine.
  23. DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.
  24. NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir.
  25. CUT Domain Proteins in DNA Repair and Cancer.
  26. Impacts of chromatin dynamics and compartmentalization on DNA repair.
  27. Chromatin mobility and relocation in DNA repair.
  28. Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β.
  29. Nucleotide Excision Repair: From Molecular Defects to Neurological Abnormalities.
  30. TRIM44 mediated p62 deubiquitination enhances DNA damage repair by increasing nuclear FLNA and 53BP1 expression.
  31. DNA glycosylases for 8-oxoguanine repair in Staphylococcus aureus.
  32. Serine-linked PARP1 auto-modification controls PARP inhibitor response.
  33. Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.
  34. TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.
  35. De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma.
  36. MYC-Induced Replicative Stress: A Double-Edged Sword for Cancer Development and Treatment.
  37. Role of Histone Methylation in Maintenance of Genome Integrity.
  38. Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance.
  39. TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.
  40. Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2.
  41. Resistance to different anthracycline chemotherapeutics elicits distinct and actionable primary metabolic dependencies in breast cancer.
  42. Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.
  43. One-Carbon Metabolism Associated Vulnerabilities in Glioblastoma: A Review.
  44. Consequences and Repair of Radiation-induced DNA Damage: Fifty Years of Fun Questions and Answers.
  45. The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.
  46. SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation.
  47. Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice.
  48. Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA.
  49. Targeting Future Pandemics, a Case for De Novo Purine Synthesis and Basic Research.
  50. Structural Chromosome Instability: Types, Origins, Consequences, and Therapeutic Opportunities.
  51. Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma.
  52. Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.
  53. Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.
  54. Structure specific DNA recognition by the SLX1-SLX4 endonuclease complex.
  55. Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.
  56. Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.
  57. Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain.
  58. Serine hydroxymethyltransferase 2: a novel target for human cancer therapy.
  59. Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis.
  60. Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.
  61. Integrated genomic-metabolic classification of acute myeloid leukemia defines a subgroup with NPM1 and cohesin/DNA damage mutations.
  1. Nat Cancer. 2021 Jun;2(6): 598-610
    A first-in-class Polymerase Theta Inhibitor selectively targets Homologous-Recombination-Deficient Tumors.
    Jia Zhou, Camille Gelot, Constantia Pantelidou, Adam Li, Hatice Yücel, Rachel E Davis, Anniina Farkkila, Bose Kochupurakkal, Aleem Syed, Geoffrey I Shapiro, John A Tainer, Brian S J Blagg, Raphael Ceccaldi, Alan D D'Andrea.
      DNA polymerase theta (POLθ) is synthetic lethal with Homologous Recombination (HR) deficiency and thus a candidate target for HR-deficient cancers. Through high-throughput small molecule screens we identified the antibiotic Novobiocin (NVB) as a specific POLθ inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POLθ ATPase domain, inhibits its ATPase activity, and phenocopies POLθ depletion. NVB kills HR-deficient breast and ovarian tumors in GEMM, xenograft and PDX models. Increased POLθ levels predict NVB sensitivity, and BRCA-deficient tumor cells with acquired resistance to PARP inhibitors (PARPi) are sensitive to NVB in vitro and in vivo. Mechanistically, NVB-mediated cell death in PARPi-resistant cells arises from increased double-strand break end resection, leading to accumulation of single-strand DNA intermediates and non-functional RAD51 foci. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. (151/150).
    Keywords:  Fanconi Anemia; HRD cancer; Homologous Recombination; MMEJ; Novobiocin; PARP inhibitor resistance; Polymerase theta (POLθ)
    DOI:  https://doi.org/10.1038/s43018-021-00203-x
  2. Nucleic Acids Res. 2021 Jul 01. pii: gkab551. [Epub ahead of print]
    Replication catastrophe induced by cyclic hypoxia leads to increased APOBEC3B activity.
    Samuel B Bader, Tiffany S Ma, Charlotte J Simpson, Jiachen Liang, Sakura Eri B Maezono, Monica M Olcina, Francesca M Buffa, Ester M Hammond.
      Tumor heterogeneity includes variable and fluctuating oxygen concentrations, which result in the accumulation of hypoxic regions in most solid tumors. Tumor hypoxia leads to increased therapy resistance and has been linked to genomic instability. Here, we tested the hypothesis that exposure to levels of hypoxia that cause replication stress could increase APOBEC activity and the accumulation of APOBEC-mediated mutations. APOBEC-dependent mutational signatures have been well-characterized, although the physiological conditions which underpin them have not been described. We demonstrate that fluctuating/cyclic hypoxic conditions which lead to replication catastrophe induce the expression and activity of APOBEC3B. In contrast, stable/chronic hypoxic conditions which induce replication stress in the absence of DNA damage are not sufficient to induce APOBEC3B. Most importantly, the number of APOBEC-mediated mutations in patient tumors correlated with a hypoxia signature. Together, our data support the conclusion that hypoxia-induced replication catastrophe drives genomic instability in tumors, specifically through increasing the activity of APOBEC3B.
    DOI:  https://doi.org/10.1093/nar/gkab551
  3. DNA Repair (Amst). 2021 Jun 23. pii: S1568-7864(21)00119-1. [Epub ahead of print]105 103163
    Daughter-strand gaps in DNA replication - substrates of lesion processing and initiators of distress signalling.
    Ronald P Wong, Kirill Petriukov, Helle D Ulrich.
      Dealing with DNA lesions during genome replication is particularly challenging because damaged replication templates interfere with the progression of the replicative DNA polymerases and thereby endanger the stability of the replisome. A variety of mechanisms for the recovery of replication forks exist, but both bacteria and eukaryotic cells also have the option of continuing replication downstream of the lesion, leaving behind a daughter-strand gap in the newly synthesized DNA. In this review, we address the significance of these single-stranded DNA structures as sites of DNA damage sensing and processing at a distance from ongoing genome replication. We describe the factors controlling the emergence of daughter-strand gaps from stalled replication intermediates, the benefits and risks of their expansion and repair via translesion synthesis or recombination-mediated template switching, and the mechanisms by which they activate local as well as global replication stress signals. Our growing understanding of daughter-strand gaps not only identifies them as targets of fundamental genome maintenance mechanisms, but also suggests that proper control over their activities has important practical implications for treatment strategies and resistance mechanisms in cancer therapy.
    Keywords:  Checkpoint; DNA damage bypass; DNA damage signalling; DNA replication stress; Daughter-strand gaps; Single-stranded DNA
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103163
  4. Nat Commun. 2021 Jun 29. 12(1): 4020
    Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase.
    Olga Rechkoblit, Robert E Johnson, Yogesh K Gupta, Louise Prakash, Satya Prakash, Aneel K Aggarwal.
      PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'-terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.
    DOI:  https://doi.org/10.1038/s41467-021-24317-z
  5. Cancers (Basel). 2021 Jun 23. pii: 3130. [Epub ahead of print]13(13):
    Analyzing the Opportunities to Target DNA Double-Strand Breaks Repair and Replicative Stress Responses to Improve Therapeutic Index of Colorectal Cancer.
    Paula Pellenz Tomasini, Temenouga Nikolova Guecheva, Natalia Motta Leguisamo, Sarah Péricart, Anne-Cécile Brunac, Jean Sébastien Hoffmann, Jenifer Saffi.
      Despite the ample improvements of CRC molecular landscape, the therapeutic options still rely on conventional chemotherapy-based regimens for early disease, and few targeted agents are recommended for clinical use in the metastatic setting. Moreover, the impact of cytotoxic, targeted agents, and immunotherapy combinations in the metastatic scenario is not fully satisfactory, especially the outcomes for patients who develop resistance to these treatments need to be improved. Here, we examine the opportunity to consider therapeutic agents targeting DNA repair and DNA replication stress response as strategies to exploit genetic or functional defects in the DNA damage response (DDR) pathways through synthetic lethal mechanisms, still not explored in CRC. These include the multiple actors involved in the repair of DNA double-strand breaks (DSBs) through homologous recombination (HR), classical non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ), inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP), as well as inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM). We also review the biomarkers that guide the use of these agents, and current clinical trials with targeted DDR therapies.
    Keywords:  DNA double strand break repair; colorectal cancer; replication stress; target therapy
    DOI:  https://doi.org/10.3390/cancers13133130
  6. Mol Cell. 2021 Jun 24. pii: S1097-2765(21)00420-2. [Epub ahead of print]
    EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.
    Shashank Hambarde, Chi-Lin Tsai, Raj K Pandita, Albino Bacolla, Anirban Maitra, Vijay Charaka, Clayton R Hunt, Rakesh Kumar, Oliver Limbo, Remy Le Meur, Walter J Chazin, Susan E Tsutakawa, Paul Russell, Katharina Schlacher, Tej K Pandita, John A Tainer.
      Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.
    Keywords:  ATR phosphorylation; Bloom; Fanconi anemia; SCE; exonuclease; fork restart; genetic instability; interstrand crosslink repair; replication stress; sister chromatid exchange; tumor proliferation
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.027
  7. Nucleic Acids Res. 2021 Jul 01. pii: gkab584. [Epub ahead of print]
    PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair.
    Bin Peng, Ruifeng Shi, Jing Bian, Yuwei Li, Peipei Wang, Hailong Wang, Ji Liao, Wei-Guo Zhu, Xingzhi Xu.
      Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1-PLK1-RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.
    DOI:  https://doi.org/10.1093/nar/gkab584
  8. Cells. 2021 Jun 11. pii: 1464. [Epub ahead of print]10(6):
    The Heritability of Replication Problems.
    Jean-Sébastien Hoffmann.
      The major challenge of DNA replication is to provide daughter cells with intact and fully duplicated genetic material. However, various endogenous or environmental factors can slow down or stall DNA replication forks; these replication problems are known to fuel genomic instability and associated pathology, including cancer progression. Whereas the mechanisms emphasizing the source and the cellular responses of replicative problems have attracted much consideration over the past decade, the propagation through mitosis of genome modification and its heritability in daughter cells when the stress is not strong enough to provoke a checkpoint response in G2/M was much less documented. Some recent studies addressing whether low replication stress could impact the DNA replication program of the next generation of cells made the remarkable discovery that DNA damage can indeed be transmitted to daughter cells and can be processed in the subsequent S-phase, and that the replication timing program at a subset of chromosomal domains can also be impacted in the next generation of cells. Such a progression of replication problems into mitosis and daughter cells may appear counter-intuitive, but it could offer considerable advantages by alerting the next generation of cells of potentially risky loci and offering the possibility of an adaptive mechanism to anticipate a reiteration of problems, notably for cancer cells in the context of resistance to therapy.
    Keywords:  DNA damage; DNA replication; replication timing; replicative stress
    DOI:  https://doi.org/10.3390/cells10061464
  9. DNA Repair (Amst). 2021 Jun 18. pii: S1568-7864(21)00115-4. [Epub ahead of print]105 103159
    Set1-dependent H3K4 methylation becomes critical for limiting DNA damage in response to changes in S-phase dynamics in Saccharomyces cerevisiae.
    Christophe de La Roche Saint-André, Vincent Géli.
      DNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S-phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. The opposite effects of the lack of RNase H activity and the reduction of histone levels on orc5-1 set1Δ viability are in agreement with their expected effects on replication fork progression. We propose that the role of H3K4 methylation during DNA replication becomes critical when the replication forks acceleration due to decreased origin firing in the orc5-1 background increases the risk for transcription replication conflicts. Furthermore, we show that an increase of reactive oxygen species levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.
    Keywords:  DNA damage; H3K4 methylation; Replication stress; Set1
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103159
  10. Genes (Basel). 2021 Jun 16. pii: 920. [Epub ahead of print]12(6):
    New RAD51 Inhibitors to Target Homologous Recombination in Human Cells.
    Irina S Shkundina, Alexander A Gall, Alexej Dick, Simon Cocklin, Alexander V Mazin.
      Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.
    Keywords:  DNA repair; homologous recombination; small-molecule inhibitors; triple-negative breast cancer
    DOI:  https://doi.org/10.3390/genes12060920
  11. Biol Open. 2021 May 15. pii: bio057729. [Epub ahead of print]10(5):
    KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance.
    Solenne Gaillard, Virginie Charasson, Cyril Ribeyre, Kader Salifou, Marie-Jeanne Pillaire, Jean-Sebastien Hoffmann, Angelos Constantinou, Didier Trouche, Marie Vandromme.
      KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of ribonucleotide reductase. Second, they are required for optimal levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.
    Keywords:  Chromatin; Drug tolerance; Replication stress
    DOI:  https://doi.org/10.1242/bio.057729
  12. Curr Opin Genet Dev. 2021 Jun 26. pii: S0959-437X(21)00075-7. [Epub ahead of print]71 10-18
    Mechanism of mitotic recombination: insights from C. elegans.
    Ondrej Belan, Roopesh Anand, Simon J Boulton.
      Homologous recombination (HR) plays a critical role in largely error-free repair of mitotic and meiotic DNA double-strand breaks (DSBs). DSBs are one of the most deleterious DNA lesions, which are repaired by non-homologous end joining (NHEJ), homologous recombination (HR) or, if compromised, micro-homology mediated end joining (MMEJ). If left unrepaired, DSBs can lead to cell death or if repaired incorrectly can result in chromosome rearrangements that drive cancer development. Here, we describe recent advances in the field of mitotic HR made using Caenorhabditis elegans roundworm, as a model system.
    DOI:  https://doi.org/10.1016/j.gde.2021.06.005
  13. Cancers (Basel). 2021 Jun 26. pii: 3202. [Epub ahead of print]13(13):
    Loss of CENP-I Impairs Homologous Recombination and Sensitizes Cells to PARP1 Inhibition.
    Tuyen T Dang, Julio C Morales.
      Centromere Protein I (CENP-I) is a member of the CENP-H/I/K complex. CENP-H/I/K is a major component of the inner kinetochore and aids in ensuring proper chromosomal segregation during mitosis. In addition to this chromosomal segregation function, CENP-I also plays a role in DNA double-strand break (DSB) repair. Loss of CENP-I leads to increased endogenous 53BP1 foci and R-loop formation, while reducing cellular survival after ionizing radiation and Niraparib, a PARP1 small molecule inhibitor, exposures. Cells lacking CENP-I display delayed 53BP1 foci regression, an indication that DSB repair is impaired. Additionally, loss of CENP-I impairs the homologous recombination DSB repair pathway, while having no effect on the non-homologous end-joining pathway. Interestingly, we find that RNaseH1 expression restores HR capacity in CENP-I deficient cells. Importantly, CENP-I expression is elevated in glioma tissue as compared to normal brain tissue. This elevated expression also correlates with poor overall patient survival. These data highlight the multi-functional role CENP-I plays in maintaining genetic, as well as chromosomal, stability and tumor survival.
    Keywords:  CENP-I; GBM; PARP1; homologous recombination
    DOI:  https://doi.org/10.3390/cancers13133202
  14. Arch Biochem Biophys. 2021 Jun 23. pii: S0003-9861(21)00226-5. [Epub ahead of print] 108977
    The interplay between mitochondrial functionality and genome integrity in the prevention of human neurologic diseases.
    Mariarosaria D'Errico, Eleonora Parlanti, Barbara Pascucci, Giuseppe Filomeni, Pier Giorgio Mastroberardino, Eugenia Dogliotti.
      As mitochondria are vulnerable to oxidative damage and represent the main source of reactive oxygen species (ROS), they are considered key tuners of ROS metabolism and buffering, whose dysfunction can progressively impact neuronal networks and disease. Defects in DNA repair and DNA damage response (DDR) may also affect neuronal health and lead to neuropathology. A number of congenital DNA repair and DDR defective syndromes, indeed, show neurological phenotypes, and a growing body of evidence indicate that defects in the mechanisms that control genome stability in neurons acts as aging-related modifiers of common neurodegenerative diseases such as Alzheimer, Parkinson's, Huntington diseases and Amyotrophic Lateral Sclerosis. In this review we elaborate on the established principles and recent concepts supporting the hypothesis that deficiencies in either DNA repair or DDR might contribute to neurodegeneration via mechanisms involving mitochondrial dysfunction/deranged metabolism.
    Keywords:  DNA damage Response; DNA repair; DNA repair Defective syndromes; Mitochondrial dysfunction; Neurodegenerative diseases; Oxidatively induced DNA damage
    DOI:  https://doi.org/10.1016/j.abb.2021.108977
  15. J Biol Chem. 2021 Jun 23. pii: S0021-9258(21)00712-2. [Epub ahead of print] 100912
    Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.
    Chloe Du Truong, Theodore A Craig, Gaofeng Cui, Maria Victoria Botuyan, Rachel A Serkasevich, Ka-Yi Chan, Georges Mer, Po-Lin Chiu, Rajiv Kumar.
      The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the S. cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1-binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared to its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
    Keywords:  DNA repair; Polζ; Rev1; Rev7; SHLD3; Shieldin 3; Shieldin complex; mutagenesis; single-particle cryo-EM; translesion synthesis
    DOI:  https://doi.org/10.1016/j.jbc.2021.100912
  16. Cancers (Basel). 2021 Jun 11. pii: 2930. [Epub ahead of print]13(12):
    Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?
    Esin Orhan, Carolina Velazquez, Imene Tabet, Claude Sardet, Charles Theillet.
      The RAD51 recombinase is a critical effector of Homologous Recombination (HR), which is an essential DNA repair mechanism for double-strand breaks. The RAD51 protein is recruited onto the DNA break by BRCA2 and forms homopolymeric filaments that invade the homologous chromatid and use it as a template for repair. RAD51 filaments are detectable by immunofluorescence as distinct foci in the cell nucleus, and their presence is a read out of HR proficiency. RAD51 is an essential gene, protecting cells from genetic instability. Its expression is low and tightly regulated in normal cells and, contrastingly, elevated in a large fraction of cancers, where its level of expression and activity have been linked with sensitivity to genotoxic treatment. In particular, BRCA-deficient tumors show reduced or obliterated RAD51 foci formation and increased sensitivity to platinum salt or PARP inhibitors. However, resistance to treatment sets in rapidly and is frequently based on a complete or partial restoration of RAD51 foci formation. Consequently, RAD51 could be a highly valuable therapeutic target. Here, we review the multiple levels of regulation that impact the transcription of the RAD51 gene, as well as the post-translational modifications that determine its expression level, recruitment on DNA damage sites and the efficient formation of homofilaments. Some of these regulation levels may be targeted and their impact on cancer cell survival discussed.
    Keywords:  DNA break; DNA repair; RAD51; cancer therapy; genomic instability; homologous recombination
    DOI:  https://doi.org/10.3390/cancers13122930
  17. Cells. 2021 Jun 08. pii: 1433. [Epub ahead of print]10(6):
    The Ultimate (Mis)match: When DNA Meets RNA.
    Benoit Palancade, Rodney Rothstein.
      RNA-containing structures, including ribonucleotide insertions, DNA:RNA hybrids and R-loops, have recently emerged as critical players in the maintenance of genome integrity. Strikingly, different enzymatic activities classically involved in genome maintenance contribute to their generation, their processing into genotoxic or repair intermediates, or their removal. Here we review how this substrate promiscuity can account for the detrimental and beneficial impacts of RNA insertions during genome metabolism. We summarize how in vivo and in vitro experiments support the contribution of DNA polymerases and homologous recombination proteins in the formation of RNA-containing structures, and we discuss the role of DNA repair enzymes in their removal. The diversity of pathways that are thus affected by RNA insertions likely reflects the ancestral function of RNA molecules in genome maintenance and transmission.
    Keywords:  DNA repair; DNA:RNA hybrid; R-loop; RNA; genetic recombination; genetic stability; ribonucleotide; transcription
    DOI:  https://doi.org/10.3390/cells10061433
  18. Comput Struct Biotechnol J. 2021 ;19 3293-3302
    Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker.
    Nicole M Hoitsma, Timothy H Click, Pratul K Agarwal, Bret D Freudenthal.
      Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5' of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3'-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.
    Keywords:  AP-Endonuclease; APE1; Abasic ribonucleotides; Apurinic/apyrimidinic sites; DNA repair; Structural biology
    DOI:  https://doi.org/10.1016/j.csbj.2021.05.035
  19. Cells. 2021 Jun 24. pii: 1591. [Epub ahead of print]10(7):
    Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases.
    Paulina Prorok, Inga R Grin, Bakhyt T Matkarimov, Alexander A Ishchenko, Jacques Laval, Dmitry O Zharkov, Murat Saparbaev.
      It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA's genome integrity. Cosmic radiation due to Earth's weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil-DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth's atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.
    Keywords:  AP endonucleases; DNA glycosylases; DNA repair; protein folds; structural homology
    DOI:  https://doi.org/10.3390/cells10071591
  20. Cells. 2021 Jun 11. pii: 1467. [Epub ahead of print]10(6):
    Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2.
    Emilie Ma, Laurent Maloisel, Léa Le Falher, Raphaël Guérois, Eric Coïc.
      Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.
    Keywords:  DNA repair; Homologous Recombination; Rad51; Rad52; Srs2; genome stability
    DOI:  https://doi.org/10.3390/cells10061467
  21. Life (Basel). 2021 Jun 30. pii: 637. [Epub ahead of print]11(7):
    Consequences and Resolution of Transcription-Replication Conflicts.
    Maxime Lalonde, Manuel Trauner, Marcel Werner, Stephan Hamperl.
      Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.
    Keywords:  G-MiDS; MIDAS; R-loops; chromatin; common fragile sites; early replicating fragile sites; fork reversal; genomic instability; replication stress; torsional stress; transcription–replication conflicts
    DOI:  https://doi.org/10.3390/life11070637
  22. Trends Cancer. 2021 Jun 29. pii: S2405-8033(21)00123-0. [Epub ahead of print]
    Targeting the replication stress response through synthetic lethal strategies in cancer medicine.
    Natalie Y L Ngoi, Melissa M Pham, David S P Tan, Timothy A Yap.
      The replication stress response (RSR) involves a downstream kinase cascade comprising ataxia telangiectasia-mutated (ATM), ATM and rad3-related (ATR), checkpoint kinases 1 and 2 (CHK1/2), and WEE1-like protein kinase (WEE1), which cooperate to arrest the cell cycle, protect stalled forks, and allow time for replication fork repair. In the presence of elevated replicative stress, cancers are increasingly dependent on RSR to maintain genomic integrity. An increasing number of drug candidates targeting key RSR nodes, as monotherapy through synthetic lethality, or through rational combinations with immune checkpoint inhibitors and targeted therapies, are demonstrating promising efficacy in early phase trials. RSR targeting is also showing potential in reversing PARP inhibitor resistance, an important area of unmet clinical need. In this review, we introduce the concept of targeting the RSR, detail the current landscape of monotherapy and combination strategies, and discuss emerging therapeutic approaches, such as targeting Polθ.
    Keywords:  PARP inhibition resistance; combination therapy; replicative stress response; synthetic lethality
    DOI:  https://doi.org/10.1016/j.trecan.2021.06.002
  23. DNA Repair (Amst). 2021 Jun 24. pii: S1568-7864(21)00108-7. [Epub ahead of print]105 103152
    DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.
    Sesha L Paluri, Matthew Burak, Alireza G Senejani, Madison Levinson, Tania Rahim, Kaylyn Clairmont, Michael Kashgarian, Isabel Alvarado-Cruz, Rithy Meas, Marina Cardó-Vila, Caroline Zeiss, Stephen Maher, Alfred L M Bothwell, Erdem Coskun, Melis Kant, Pawel Jaruga, Miral Dizdaroglu, R Stephen Lloyd, Joann B Sweasy.
      The Polb gene encodes DNA polymerase beta (Pol β), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol β-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol β-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol β during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.
    Keywords:  Base excision repair; DNA glycosylase; DNA polymerase beta; Oxidative DNA damage; Systemic lupus erythematosus
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103152
  24. Cell Chem Biol. 2021 Jun 22. pii: S2451-9456(21)00268-3. [Epub ahead of print]
    NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir.
    Si Min Zhang, Daniel Rehling, Ann-Sofie Jemth, Adam Throup, Natalia Landázuri, Ingrid Almlöf, Mona Göttmann, Nicholas C K Valerie, Sanjay R Borhade, Prasad Wakchaure, Brent D G Page, Matthieu Desroses, Evert J Homan, Martin Scobie, Sean G Rudd, Ulrika Warpman Berglund, Cecilia Söderberg-Nauclér, Pål Stenmark, Thomas Helleday.
      Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.
    Keywords:  NUDT15; Nudix hydrolase; TH8321; antiherpes; cytomegalovirus; ganciclovir; high-throughput infectivity assay; nucleoside analog drug; small-molecule inhibitor
    DOI:  https://doi.org/10.1016/j.chembiol.2021.06.001
  25. Cancers (Basel). 2021 Jun 12. pii: 2953. [Epub ahead of print]13(12):
    CUT Domain Proteins in DNA Repair and Cancer.
    Zubaidah M Ramdzan, Elise Vickridge, Camila C F Faraco, Alain Nepveu.
      Recent studies revealed that CUT domains function as accessory factors that accelerate DNA repair by stimulating the enzymatic activities of the base excision repair enzymes OGG1, APE1, and DNA pol β. Strikingly, the role of CUT domain proteins in DNA repair is exploited by cancer cells to facilitate their survival. Cancer cells in which the RAS pathway is activated produce an excess of reactive oxygen species (ROS) which, if not counterbalanced by increased production of antioxidants, causes sustained oxidative DNA damage and, ultimately, cell senescence. These cancer cells can adapt by increasing their capacity to repair oxidative DNA damage in part through elevated expression of CUT domain proteins such as CUX1, CUX2, or SATB1. In particular, CUX1 overexpression was shown to cooperate with RAS in the formation of mammary and lung tumors in mice. Conversely, knockdown of CUX1, CUX2, or SATB1 was found to be synthetic lethal in cancer cells exhibiting high ROS levels as a consequence of activating mutations in KRAS, HRAS, BRAF, or EGFR. Importantly, as a byproduct of their adaptation, cancer cells that overexpress CUT domain proteins exhibit increased resistance to genotoxic treatments such as ionizing radiation, temozolomide, and cisplatin.
    Keywords:  CUT domains; CUX1; CUX2; RAS; SATB1; base excision repair; reactive oxygen species
    DOI:  https://doi.org/10.3390/cancers13122953
  26. DNA Repair (Amst). 2021 Jun 19. pii: S1568-7864(21)00118-X. [Epub ahead of print]105 103162
    Impacts of chromatin dynamics and compartmentalization on DNA repair.
    Takaaki Yasuhara, Lee Zou.
      The proper spatial organization of DNA, RNA, and proteins is critical for a variety of cellular processes. The genome is organized into numerous functional units, such as topologically associating domains (TADs), the formation of which is regulated by both proteins and RNA. In addition, a group of chromatin-bound proteins with the ability to undergo liquid-liquid phase separation (LLPS) can affect the spatial organization and compartmentalization of chromatin, RNA, and proteins by forming condensates, conferring unique properties to specific chromosomal regions. Although the regulation of DNA repair by histone modifications and chromatin accessibility is well established, the impacts of higher-order chromatin and protein organization on the DNA damage response (DDR) have not been appreciated until recently. In this review, we will focus on the movement of chromatin during the DDR, the compartmentalization of DDR proteins via LLPS, and the roles of membraneless nuclear bodies and transcription in DNA repair. With this backdrop, we will discuss the importance of the spatial organization of chromatin and proteins for the maintenance of genome integrity.
    Keywords:  Chromatin; Compartmentalization; Foci; Nuclear bodies; Phase separation; TAD
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103162
  27. Trends Cell Biol. 2021 Jun 26. pii: S0962-8924(21)00118-5. [Epub ahead of print]
    Chromatin mobility and relocation in DNA repair.
    Noa Lamm, Samuel Rogers, Anthony J Cesare.
      The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.
    Keywords:  alternative lengthening of telomeres; chromatin mobility; homology directed repair; nuclear actin; nuclear pore complex
    DOI:  https://doi.org/10.1016/j.tcb.2021.06.002
  28. Chembiochem. 2021 Jul 02.
    Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β.
    Marc M Greenberg, Shelby C Yuhas, Ananya Majumdar.
      DNA polymerase β (Pol β) is a frequently overexpressed and/or mutated bifunctional repair enzyme. Pol β possesses polymerase and lyase active sites, that are employed in two steps of base excision repair. Pol β is an attractive therapeutic target for which there is a need for inhibitors. Two mechanistically inspired covalent inhibitors ( 1 , IC 50 = 21.0 μM; 9 , IC 50 = 18.7 μM) that modify lysine residues in different Pol β active sites are characterized. Despite modifying lysine residues in different active sites, 1 and 9 inactivate the polymerase and lyase activities of Pol β. Fluorescence anisotropy experiments indicate that they do so by preventing DNA binding. Inhibitors 1 and 9 provide the basis for a general approach to preparing domain selective inhibitors of bifunctional polymerases. Such molecules could prove to be useful tools for studying the role of wild type and mutant forms of Pol β in DNA repair.
    Keywords:  Covalent inhibitor; DNA damage; DNA polymerase β; DNA repair; Enzyme inhibition
    DOI:  https://doi.org/10.1002/cbic.202100247
  29. Int J Mol Sci. 2021 Jun 09. pii: 6220. [Epub ahead of print]22(12):
    Nucleotide Excision Repair: From Molecular Defects to Neurological Abnormalities.
    Yuliya Krasikova, Nadejda Rechkunova, Olga Lavrik.
      Nucleotide excision repair (NER) is the most versatile DNA repair pathway, which can remove diverse bulky DNA lesions destabilizing a DNA duplex. NER defects cause several autosomal recessive genetic disorders. Xeroderma pigmentosum (XP) is one of the NER-associated syndromes characterized by low efficiency of the removal of bulky DNA adducts generated by ultraviolet radiation. XP patients have extremely high ultraviolet-light sensitivity of sun-exposed tissues, often resulting in multiple skin and eye cancers. Some XP patients develop characteristic neurodegeneration that is believed to derive from their inability to repair neuronal DNA damaged by endogenous metabolites. A specific class of oxidatively induced DNA lesions, 8,5'-cyclopurine-2'-deoxynucleosides, is considered endogenous DNA lesions mainly responsible for neurological problems in XP. Growing evidence suggests that XP is accompanied by defective mitophagy, as in primary mitochondrial disorders. Moreover, NER pathway is absent in mitochondria, implying that the mitochondrial dysfunction is secondary to nuclear NER defects. In this review, we discuss the current understanding of the NER molecular mechanism and focuses on the NER linkage with the neurological degeneration in patients with XP. We also present recent research advances regarding NER involvement in oxidative DNA lesion repair. Finally, we highlight how mitochondrial dysfunction may be associated with XP.
    Keywords:  base excision repair; mitophagy; neurodegeneration; nucleotide excision repair; oxidative stress; xeroderma pigmentosum
    DOI:  https://doi.org/10.3390/ijms22126220
  30. Oncogene. 2021 Jul 01.
    TRIM44 mediated p62 deubiquitination enhances DNA damage repair by increasing nuclear FLNA and 53BP1 expression.
    Lin Lyu, Tsung-Chin Lin, Nami McCarty.
      Cancer cells show increases in protein degradation pathways, including autophagy, during progression to meet the increased protein degradation demand and support cell survival. On the other hand, reduced autophagy activity during aging is associated with a reduced DNA damage response and increased genomic instability. Therefore, it is a puzzling how DNA repair can be increased in cancer cells that are resistant to chemotherapies or during progression when autophagy activity is intact or increased. We discovered that tripartite motif containing 44 (TRIM44) is a pivotal element regulating the DNA damage response in cancer cells with intact autophagy. TRIM44 deubiquitinates p62, an autophagy substrate, which leads to its oligomerization. This prevents p62 localization to the nucleus upon irradiation. Increased cytoplasmic retention of p62 by TRIM44 prevents the degradation of FLNA and 53BP1, which increases DNA damage repair. Together, our data support TRIM44 a potential therapeutic target for therapy-resistant tumor cells with intact autophagy.
    DOI:  https://doi.org/10.1038/s41388-021-01890-7
  31. DNA Repair (Amst). 2021 Jun 18. pii: S1568-7864(21)00116-6. [Epub ahead of print]105 103160
    DNA glycosylases for 8-oxoguanine repair in Staphylococcus aureus.
    Anton V Endutkin, Elena P Panferova, Alexander E Barmatov, Dmitry O Zharkov.
      GO system is part of base excision DNA repair and is required for the correct repair of 8-oxoguanine (8-oxoG), one of the most abundant oxidative lesions. Due to the ability of 8-oxoG to mispair with A, this base is highly mutagenic, and its repair requires two enzymes: Fpg that removes 8-oxoG from 8-oxoG:C pairs, and MutY that excises the normal A from 8-oxoG:A mispairs. Here we characterize the properties of putative GO system DNA glycosylases from Staphylococcus aureus, an important human opportunistic pathogen that causes hospital infections and presents a serious health concern due to quick spread of antibiotic-resistant strains. In addition to Fpg and MutY from the reference NCTC 8325 strain (SauFpg1 and SauMutY), we have also studied an Fpg homolog from a multidrug-resistant C0673 isolate (SauFpg2), which is different from SauFpg1 in its sequence. Both SauFpg enzymes showed the highest activity at pH 7.0-9.0 and NaCl concentrations 25-75 mM (SauFpg1) or 50-100 mM (SauFpg2), whereas SauMutY was active at a broad pH range and had a salt optimum at ∼75 mM NaCl. Both SauFpg1 and SauFpg2 bound and cleaved duplexes containing 8-oxoG, 5-hydroxyuracil, 5,6-dihydrouracil or apurinic/apyrimidinic site paired with C, T, or G, but not with A. For SauFpg1 and SauFpg2, 8-oxoG was the best substrate tested, and 5,6-dihydrouracil was the worst one. SauMutY efficiently excised adenine from duplex substrates containing A:8-oxoG or A:G pairs. SauFpg enzymes were readily trapped on DNA by NaBH4 treatment, indicating formation of a Schiff base reaction intermediate. Surprisingly, SauMutY was also trapped significantly better than its E. coli homolog. All three S. aureus GO glycosylases drastically reduced spontaneous mutagenesis when expressed in an fpg mutY E. coli double mutant. Overall, we conclude that S. aureus possesses an active GO system, which could possibly be targeted for sensitization of this pathogen to oxidative stress.
    Keywords:  8-Oxoguanine; DNA damage; DNA glycosylases; DNA repair; GO system; Staphylococcus aureus
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103160
  32. Nat Commun. 2021 Jul 01. 12(1): 4055
    Serine-linked PARP1 auto-modification controls PARP inhibitor response.
    Evgeniia Prokhorova, Florian Zobel, Rebecca Smith, Siham Zentout, Ian Gibbs-Seymour, Kira Schützenhofer, Alessandra Peters, Joséphine Groslambert, Valentina Zorzini, Thomas Agnew, John Brognard, Michael L Nielsen, Dragana Ahel, Sébastien Huet, Marcin J Suskiewicz, Ivan Ahel.
      Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.
    DOI:  https://doi.org/10.1038/s41467-021-24361-9
  33. J Biol Chem. 2021 Jun 25. pii: S0021-9258(21)00721-3. [Epub ahead of print] 100921
    Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.
    Ishtiaque Rashid, Michal Hammel, Aleksandr Sverzhinsky, Miaw-Sheue Tsai, John M Pascal, John A Tainer, Alan E Tomkinson.
      Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped either by DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in non-malignant and cancer cells.
    Keywords:  DNA ligation; Phosphorylation; electron microscopy; protein structure; small angle x-ray scattering; topoisomerase
    DOI:  https://doi.org/10.1016/j.jbc.2021.100921
  34. Cancer Res. 2021 Jul 02. pii: canres.3761.2020. [Epub ahead of print]
    TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.
    Silvia Maifrede, Bac Viet Le, Margaret Nieborowska-Skorska, Konstantin Golovine, Katherine Sullivan-Reed, Wangisa M B Dunuwille, Joseph Nacson, Michael Hulse, Kelsey Keith, Jozef Madzo, Lisa Beatrice Caruso, Zachary Gazze, Zhaorui Lian, Antonella Padella, Kumaraswamy N Chitrala, Boris A Bartholdy, Ksenia Matlawska-Wasowska, Daniela Di Marcantonio, Giorgia Simonetti, Georg Greiner, Stephen M Sykes, Peter Valent, Elisabeth M Paietta, Martin S Tallman, Hugo F Fernandez, Mark R Litzow, Mark D Minden, Jian Huang, Giovanni Martinelli, George S Vassiliou, Italo Tempera, Katarzyna Piwocka, Neil Johnson, Grant A Challen, Tomasz Skorski.
      Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F, and MPLW515L, or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3761
  35. Sci Rep. 2021 Jun 29. 11(1): 13474
    De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma.
    Ami Maruyama, Yuzo Sato, Joji Nakayama, Junko Murai, Takamasa Ishikawa, Tomoyoshi Soga, Hideki Makinoshima.
      Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.
    DOI:  https://doi.org/10.1038/s41598-021-92948-9
  36. Int J Mol Sci. 2021 Jun 08. pii: 6168. [Epub ahead of print]22(12):
    MYC-Induced Replicative Stress: A Double-Edged Sword for Cancer Development and Treatment.
    Laura Curti, Stefano Campaner.
      MYC is a transcription factor that controls the expression of a large fraction of cellular genes linked to cell cycle progression, metabolism and differentiation. MYC deregulation in tumors leads to its pervasive genome-wide binding of both promoters and distal regulatory regions, associated with selective transcriptional control of a large fraction of cellular genes. This pairs with alterations of cell cycle control which drive anticipated S-phase entry and reshape the DNA-replication landscape. Under these circumstances, the fine tuning of DNA replication and transcription becomes critical and may pose an intrinsic liability in MYC-overexpressing cancer cells. Here, we will review the current understanding of how MYC controls DNA and RNA synthesis, discuss evidence of replicative and transcriptional stress induced by MYC and summarize preclinical data supporting the therapeutic potential of triggering replicative stress in MYC-driven tumors.
    Keywords:  DNA replication; MYC; cancer therapy; replicative stress; transcription; transcription stress
    DOI:  https://doi.org/10.3390/ijms22126168
  37. Genes (Basel). 2021 Jun 29. pii: 1000. [Epub ahead of print]12(7):
    Role of Histone Methylation in Maintenance of Genome Integrity.
    Arjamand Mushtaq, Ulfat Syed Mir, Clayton R Hunt, Shruti Pandita, Wajahat W Tantray, Audesh Bhat, Raj K Pandita, Mohammad Altaf, Tej K Pandita.
      Packaging of the eukaryotic genome with histone and other proteins forms a chromatin structure that regulates the outcome of all DNA mediated processes. The cellular pathways that ensure genomic stability detect and repair DNA damage through mechanisms that are critically dependent upon chromatin structures established by histones and, particularly upon transient histone post-translational modifications. Though subjected to a range of modifications, histone methylation is especially crucial for DNA damage repair, as the methylated histones often form platforms for subsequent repair protein binding at damaged sites. In this review, we highlight and discuss how histone methylation impacts the maintenance of genome integrity through effects related to DNA repair and repair pathway choice.
    Keywords:  DNA repair; histone methylation; homologous recombination; non-homologous end joining
    DOI:  https://doi.org/10.3390/genes12071000
  38. Nat Commun. 2021 Jun 29. 12(1): 4033
    Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance.
    Qinglei Hang, Liyong Zeng, Li Wang, Litong Nie, Fan Yao, Hongqi Teng, Yalan Deng, Shannon Yap, Yutong Sun, Steven J Frank, Junjie Chen, Li Ma.
      In response to DNA double-strand breaks (DSBs), repair proteins are recruited to the damaged sites. Ubiquitin signaling plays a critical role in coordinating protein recruitment during the DNA damage response. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. Upon radiation, the kinase ATM and the deubiquitinase USP51 mediate the activation and stabilization of DGCR8 through phosphorylation and deubiquitination. Specifically, radiation-induced ATM-dependent phosphorylation of DGCR8 at serine 677 facilitates USP51 to bind, deubiquitinate, and stabilize DGCR8, which leads to the recruitment of DGCR8 and DGCR8's binding partner RNF168 to MDC1 and RNF8 at DSBs. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through ATM- and USP51-mediated activation and upregulation of DGCR8.
    DOI:  https://doi.org/10.1038/s41467-021-24298-z
  39. Int J Mol Sci. 2021 Jun 28. pii: 6957. [Epub ahead of print]22(13):
    TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.
    Umakanta Swain, Gilgi Friedlander, Urmila Sehrawat, Avital Sarusi-Portuguez, Ron Rotkopf, Charlotte Ebert, Tamar Paz-Elizur, Rivka Dikstein, Thomas Carell, Nicholas E Geacintov, Zvi Livneh.
      TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.
    Keywords:  DNA repair; TLS; lesion bypass; poly(A) RNA polymerase; translesion
    DOI:  https://doi.org/10.3390/ijms22136957
  40. Nucleic Acids Res. 2021 Jul 01. pii: gkab540. [Epub ahead of print]
    Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2.
    Mengfan Tang, Guangsheng Pei, Dan Su, Chao Wang, Xu Feng, Mrinal Srivastava, Zhen Chen, Zhongming Zhao, Junjie Chen.
      Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.
    DOI:  https://doi.org/10.1093/nar/gkab540
  41. Elife. 2021 Jun 28. pii: e65150. [Epub ahead of print]10
    Resistance to different anthracycline chemotherapeutics elicits distinct and actionable primary metabolic dependencies in breast cancer.
    Shawn McGuirk, Yannick Audet-Delage, Matthew G Annis, Yibo Xue, Mathieu Vernier, Kaiqiong Zhao, Catherine St-Louis, Lucía Minarrieta, David A Patten, Geneviève Morin, Celia Mt Greenwood, Vincent Giguère, Sidong Huang, Peter M Siegel, Julie St-Pierre.
      Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.
    Keywords:  PGC-1; anthracyclines; bioenergetics; breast cancer; cancer biology; human; metabolomics; mouse; therapeutic resistance
    DOI:  https://doi.org/10.7554/eLife.65150
  42. Biochem J. 2021 Jun 29. pii: BCJ20210315. [Epub ahead of print]
    Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.
    Thanh Thi Le, Johanna Ainsworth, Cristian Polo Rivera, Thomas Macartney, Karim Labib.
      Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination.  In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase.  Here we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase.  Using purified human proteins, including a panel of E2 ubiquitin conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1.  The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1.  CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes.  Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7.  Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.
    Keywords:  CUL2-LRR1; DNA synthesis and repair; cmg helicase; cullin ligase; p97; ubiquitins
    DOI:  https://doi.org/10.1042/BCJ20210315
  43. Cancers (Basel). 2021 Jun 19. pii: 3067. [Epub ahead of print]13(12):
    One-Carbon Metabolism Associated Vulnerabilities in Glioblastoma: A Review.
    Kimia Ghannad-Zadeh, Sunit Das.
      Altered cell metabolism is a hallmark of cancer cell biology, and the adaptive metabolic strategies of cancer cells have been of recent interest to many groups. Metabolic reprogramming has been identified as a critical step in glial cell transformation, and the use of antimetabolites against glioblastoma has been investigated. One-carbon (1-C) metabolism and its associated biosynthetic pathways, particularly purine nucleotide synthesis, are critical for rapid proliferation and are altered in many cancers. Purine metabolism has also been identified as essential for glioma tumourigenesis. Additionally, alterations of 1-C-mediated purine synthesis have been identified as commonly present in brain tumour initiating cells (BTICs) and could serve as a phenotypic marker of cells responsible for tumour recurrence. Further research is required to elucidate mechanisms through which metabolic vulnerabilities may arise in BTICs and potential ways to therapeutically target these metabolic processes. This review aims to summarize the role of 1-C metabolism-associated vulnerabilities in glioblastoma tumourigenesis and progression and investigate the therapeutic potential of targeting this pathway in conjunction with other treatment strategies.
    Keywords:  de novo purine synthesis; glioblastoma; glioma; metabolic reprogramming; metabolic treatment; one-carbon metabolism
    DOI:  https://doi.org/10.3390/cancers13123067
  44. Int J Radiat Biol. 2021 Jun 30. 1-50
    Consequences and Repair of Radiation-induced DNA Damage: Fifty Years of Fun Questions and Answers.
    Susan S Wallace.
      PURPOSE: To summarize succinctly the fifty years of research undertaken in my laboratory and to provide an overview of my career in science. It is certainly a privilege to have been asked by Carmel Mothersill and Penny Jeggo to contribute to this special issue of the International Journal of Radiation Biology focusing on the work of women in the radiation sciences.CONCLUSION: My students, postdocs and I identified and characterized a number of the enzymes that recognize and remove radiation-damaged DNA bases, the DNA glycosylases, which are the first enzymes in the Base Excision Repair (BER) pathway. Although this pathway actually evolved to repair oxidative and other endogenous DNA damages, it is also responsible for removing the vast majority of radiation-induced DNA damages including base damages, alkali-labile lesions and single strand breaks. However, because of its high efficiency, attempted BER of clustered lesions produced by ionizing radiation, can have disastrous effects on cellular DNA. We also evaluated the potential biological consequences of many of the radiation-induced DNA lesions. In addition, with collaborators, we employed computational techniques, x-ray crystallography and single molecule approaches to answer many questions at the molecular level.
    Keywords:  Base excision repair; DNA base lesions; DNA damage consequences; DNA glycosylases
    DOI:  https://doi.org/10.1080/09553002.2021.1948141
  45. Cell Rep. 2021 Jun 29. pii: S2211-1247(21)00682-3. [Epub ahead of print]35(13): 109306
    The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.
    Janna Luessing, Maryam Sakhteh, Naoyuki Sarai, Louise Frizzell, Nikolay Tsanov, Kiefer Olaf Ramberg, Silvia Maretto, Peter Bernard Crowley, Noel Francis Lowndes.
      53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.
    Keywords:  53BP1; DDR; DSB repair; KIF18B; TIM; Tudor; end joining
    DOI:  https://doi.org/10.1016/j.celrep.2021.109306
  46. Mol Carcinog. 2021 Jun 30.
    SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation.
    Vidya Gopalakrishnan, Shivangi Sharma, Ujjayinee Ray, Meghana Manjunath, Divya Lakshmanan, Supriya V Vartak, Vindya K Gopinatha, Mrinal Srivastava, Mantelingu Kempegowda, Bibha Choudhary, Sathees C Raghavan.
      Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.
    Keywords:  DNA ligase IV; NHEJ; cancer therapy; chemotherapy; double-strand break; end joining; homologous recombination; radiotherapy
    DOI:  https://doi.org/10.1002/mc.23329
  47. Sci Transl Med. 2021 Jun 30. pii: eabc7275. [Epub ahead of print]13(600):
    Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice.
    Xiaoguang Fang, Zhi Huang, Kui Zhai, Qian Huang, Weiwei Tao, Leo Kim, Qiulian Wu, Alexandru Almasan, Jennifer S Yu, Xiaoxia Li, George R Stark, Jeremy N Rich, Shideng Bao.
      Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non-stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs-mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.
    DOI:  https://doi.org/10.1126/scitranslmed.abc7275
  48. J Biol Chem. 2021 Jun 24. pii: S0021-9258(21)00711-0. [Epub ahead of print] 100911
    Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA.
    Premlata Kumari, Rajivgandhi Sundaram, Kodavati Manohar, Dileep Vasudevan, Narottam Acharya.
      Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and PCNA exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA and compared its structure-function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2AA, a small molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility.
    Keywords:  DNA Replication; DNA damage; DNA polymerase; Isothermal titration calorimetry; PCNA; PIP box motif; X-ray crystal structure; complementation
    DOI:  https://doi.org/10.1016/j.jbc.2021.100911
  49. Front Immunol. 2021;12:12 694300
    Targeting Future Pandemics, a Case for De Novo Purine Synthesis and Basic Research.
    Randall C Mazzarino.
      We are currently experiencing a deadly novel viral pandemic with no efficacious, readily available anti-viral therapies to SARS-CoV-2. Viruses will hijack host cellular machinery, including metabolic processes. Here, I provide theory and evidence for targeting the host de novo purine synthetic pathway for broad spectrum anti-viral drug development as well as the pursuit of basic science to mitigate the risks of future novel viral outbreaks.
    Keywords:  immunology & infectious diseases; metabolism; nucleotide synthesis; therapeutic development; viral response
    DOI:  https://doi.org/10.3389/fimmu.2021.694300
  50. Cancers (Basel). 2021 Jun 19. pii: 3056. [Epub ahead of print]13(12):
    Structural Chromosome Instability: Types, Origins, Consequences, and Therapeutic Opportunities.
    Sebastián Omar Siri, Julieta Martino, Vanesa Gottifredi.
      Chromosomal instability (CIN) refers to an increased rate of acquisition of numerical and structural changes in chromosomes and is considered an enabling characteristic of tumors. Given its role as a facilitator of genomic changes, CIN is increasingly being considered as a possible therapeutic target, raising the question of which variables may convert CIN into an ally instead of an enemy during cancer treatment. This review discusses the origins of structural chromosome abnormalities and the cellular mechanisms that prevent and resolve them, as well as how different CIN phenotypes relate to each other. We discuss the possible fates of cells containing structural CIN, focusing on how a few cell duplication cycles suffice to induce profound CIN-mediated genome alterations. Because such alterations can promote tumor adaptation to treatment, we discuss currently proposed strategies to either avoid CIN or enhance CIN to a level that is no longer compatible with cell survival.
    Keywords:  DNA damage; DNA repair; chromosome aberrations; chromosome bridges; chromosome instability; lagging chromosomes; micronuclei; ultra-fine bridges
    DOI:  https://doi.org/10.3390/cancers13123056
  51. Cancers (Basel). 2021 Jun 26. pii: 3204. [Epub ahead of print]13(13):
    Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma.
    Jian Li, Josephine Mun-Yee Ko, Wei Dai, Valen Zhuoyou Yu, Hoi Yan Ng, Jean-Sébastien Hoffmann, Maria Li Lung.
      Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.
    Keywords:  POLQ; genomic instability; innate immune response
    DOI:  https://doi.org/10.3390/cancers13133204
  52. J Enzyme Inhib Med Chem. 2021 Dec;36(1): 1282-1289
    Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.
    Birte Arlt, Guido Mastrobuoni, Jasmin Wuenschel, Kathy Astrahantseff, Angelika Eggert, Stefan Kempa, Hedwig E Deubzer.
      The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.
    Keywords:  Cancer cell metabolism; citrate synthase; de novo serine synthesis pathway; pulsed stable isotope-resolved metabolomics; thermal shift assay
    DOI:  https://doi.org/10.1080/14756366.2021.1935917
  53. Int J Mol Sci. 2021 Jun 19. pii: 6589. [Epub ahead of print]22(12):
    Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.
    Claudia Scalera, Giulio Ticli, Ilaria Dutto, Ornella Cazzalini, Lucia A Stivala, Ennio Prosperi.
      Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.
    Keywords:  R loop; XPG endonuclease; acetylation; chromatin association; genome instability; p300/CREBBP
    DOI:  https://doi.org/10.3390/ijms22126589
  54. Nucleic Acids Res. 2021 Jun 28. pii: gkab542. [Epub ahead of print]
    Structure specific DNA recognition by the SLX1-SLX4 endonuclease complex.
    Xiang Xu, Mingzhu Wang, Jixue Sun, Zhenyu Yu, Guohong Li, Na Yang, Rui-Ming Xu.
      The SLX1-SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1-SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5'-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5'-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5'-flap DNA and specification of cleavage site by the SLX1-SLX4 complex.
    DOI:  https://doi.org/10.1093/nar/gkab542
  55. Int J Mol Sci. 2021 Jun 29. pii: 7004. [Epub ahead of print]22(13):
    Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.
    Rossana Pesi, Simone Allegrini, Mercedes Garcia-Gil, Lucia Piazza, Roberta Moschini, Lars Petter Jordheim, Marcella Camici, Maria Grazia Tozzi.
      Cytosolic 5'-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.
    Keywords:  AMPK; cN-II; lactate; metabolic regulation; p53
    DOI:  https://doi.org/10.3390/ijms22137004
  56. Stem Cell Reports. 2021 Jun 22. pii: S2213-6711(21)00309-X. [Epub ahead of print]
    Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.
    Scott Bell, Vincent McCarty, Huashan Peng, Malvin Jefri, Nuwan Hettige, Lilit Antonyan, Liam Crapper, Liam A O'Leary, Xin Zhang, Ying Zhang, Hanrong Wu, Diane Sutcliffe, Ilaria Kolobova, Thad A Rosenberger, Luc Moquin, Alain Gratton, Jelena Popic, Ilse Gantois, Patrick S Stumpf, Andreas A Schuppert, Naguib Mechawar, Nahum Sonenberg, Michel L Tremblay, Hyder A Jinnah, Carl Ernst.
      Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.
    Keywords:  Lesch-Nyhan disease; dopamine; iPSC; mTORC1; neurodevelopment; purines
    DOI:  https://doi.org/10.1016/j.stemcr.2021.06.003
  57. Int J Mol Sci. 2021 Jun 11. pii: 6308. [Epub ahead of print]22(12):
    Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain.
    David J López, José A Rodríguez, Sonia Bañuelos.
      APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein-protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.
    Keywords:  APE1; BER pathway; DNA repair; NPM1; abasic; apurinic/apyrimidinic endonuclease 1; nucleophosmin; protein regulation
    DOI:  https://doi.org/10.3390/ijms22126308
  58. Invest New Drugs. 2021 Jul 03.
    Serine hydroxymethyltransferase 2: a novel target for human cancer therapy.
    Min Xie, Dong-Sheng Pei.
      Serine and glycine are the primary sources of one-carbon units that are vital for cell proliferation. Their abnormal metabolism is known to be associated with cancer progression. As the key enzyme of serine metabolism, Serine Hydroxymethyltransferase 2 (SHMT2) has been a research hotspot in recent years. SHMT2 is a PLP-dependent tetrameric enzyme that catalyzes the reversible transition from serine to glycine, thus promoting the production of one-carbon units that are indispensable for cell growth and regulation of the redox and epigenetic states of cells. Under a hypoxic environment, SHMT2 can be upregulated and could promote the generation of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione for maintaining the redox balance. Accumulating evidence confirmed that SHMT2 facilitates cell proliferation and tumor growth and is tightly associated with poor prognosis. In this review, we present insights into the function and research development of SHMT2 and summarize the possible molecular mechanisms of SHMT2 in promoting tumor growth, in the hope that it could provide clues to more effective clinical treatment of cancer.
    Keywords:  Cancer; Hypoxic environment; SHMT2; Warburg effect
    DOI:  https://doi.org/10.1007/s10637-021-01144-z
  59. Int J Mol Sci. 2021 Jun 25. pii: 6861. [Epub ahead of print]22(13):
    Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis.
    Hong-Hsiang Guan, Yen-Hua Huang, En-Shyh Lin, Chun-Jung Chen, Cheng-Yang Huang.
      Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.
    Keywords:  4T1 cell; CAD; allantoinase; anticancer; crystal structure; dihydroorotase; dihydropyrimidinase; drug design; plumbagin; pyrimidine biosynthesis
    DOI:  https://doi.org/10.3390/ijms22136861
  60. ACS Chem Biol. 2021 Jun 30.
    Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.
    Marcela S Bucardo, You Wu, Paul T Ludford, Yao Li, Andrea Fin, Yitzhak Tor.
      Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thGN, the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzXN. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.
    DOI:  https://doi.org/10.1021/acschembio.1c00232
  61. Leukemia. 2021 Jun 30.
    Integrated genomic-metabolic classification of acute myeloid leukemia defines a subgroup with NPM1 and cohesin/DNA damage mutations.
    Giorgia Simonetti, Carlo Mengucci, Antonella Padella, Eugenio Fonzi, Gianfranco Picone, Claudio Delpino, Jacopo Nanni, Rossella De Tommaso, Eugenia Franchini, Cristina Papayannidis, Giovanni Marconi, Martina Pazzaglia, Margherita Perricone, Emanuela Scarpi, Maria Chiara Fontana, Samantha Bruno, Michela Tebaldi, Anna Ferrari, Maria Teresa Bochicchio, Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Roberta Napolitano, Annalisa Astolfi, Carmen Baldazzi, Viviana Guadagnuolo, Emanuela Ottaviani, Ilaria Iacobucci, Michele Cavo, Gastone Castellani, Torsten Haferlach, Daniel Remondini, Francesco Capozzi, Giovanni Martinelli.
      Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.
    DOI:  https://doi.org/10.1038/s41375-021-01318-x
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