bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021–04–18
57 papers selected by
Sean Rudd, Karolinska Institutet
  1. AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.
  2. Structural basis of long-range to short-range synaptic transition in NHEJ.
  3. The dystonia gene THAP1 controls DNA double-strand break repair choice.
  4. Therapeutic targeting of ATR yields durable regressions in small cell lung cancers with high replication stress.
  5. USP7 limits CDK1 activity throughout the cell cycle.
  6. De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.
  7. A Review of Small-Molecule Inhibitors of One-Carbon Enzymes: SHMT2 and MTHFD2 in the Spotlight.
  8. Genetic polymorphism in ATIC is associated with effectiveness and toxicity of pemetrexed in non-small-cell lung cancer.
  9. Aicardi-Goutières syndrome-associated gene SAMHD1 preserves genome integrity by preventing R-loop formation at transcription-replication conflict regions.
  10. Trex2 responds to damaged replication forks in diverse ways.
  11. Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks.
  12. DNA damage response and PD-1/PD-L1 pathway in ovarian cancer.
  13. N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.
  14. In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer.
  15. Primary cilia and the DNA damage response: linking a cellular antenna and nuclear signals.
  16. The Ku complex: recent advances and emerging roles outside of non-homologous end-joining.
  17. Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η.
  18. Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.
  19. Pharmacological inhibition of HSP90 radiosensitizes HNSCC xenograft by inhibition of DNA damage repair, nucleotide metabolism, and radiation-induced tumor vasculogenesis.
  20. Recruitment of KMT2C/MLL3 to DNA damage sites mediates DNA damage responses and regulates PARP inhibitor sensitivity in cancer.
  21. Proton pump inhibitors suppress DNA damage repair and sensitize treatment resistance in breast cancer by targeting fatty acid synthase.
  22. RRM2 silencing suppresses malignant phenotype and enhances radiosensitivity via activating cGAS/STING signaling pathway in lung adenocarcinoma.
  23. Single-Stranded DNA Curtains for Single-Molecule Visualization of Rad51-ssDNA Filament Dynamics.
  24. Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.
  25. Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.
  26. Control of S phase duration: a replication capacity model with E2F transcription at its heart.
  27. Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe.
  28. Pathways and signatures of mutagenesis at targeted DNA nicks.
  29. ENTPD1(CD39) expression inhibits ultraviolet radiation-induced DNA damage repair via purinergic signaling and is associated with metastasis in human cutaneous squamous cell carcinoma.
  30. SLFN11 inactivation induces proteotoxic stress and sensitizes cancer cells to ubiquitin activating enzyme inhibitor TAK-243.
  31. Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling.
  32. Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.
  33. The Enigma of Substrate Recognition and Catalytic Efficiency of APE1-Like Enzymes.
  34. UV-induced activation of ATR is mediated by UHRF2.
  35. The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.
  36. Comparative Activity and Off-Target Effects in Cells of the CHK1 Inhibitors MK-8776, SRA737, and LY2606368.
  37. Phosphoglycerate mutase 1 (PGAM1) overexpression promotes radio- and chemoresistance in gliomas by activating the DNA damage response.
  38. DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells.
  39. Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy and Enzymatic Assays.
  40. A guide to interrogating immunometabolism.
  41. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.
  42. Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Binding Protein.
  43. Metabolomic profiling of pancreatic adenocarcinoma reveals key features driving clinical outcome and drug resistance.
  44. Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.
  45. Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase.
  46. It takes three to the DNA damage response tango.
  47. Genome instability from nuclear catastrophe and DNA damage.
  48. Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.
  49. Transcription, translation, and DNA repair: new insights from emerging noncanonical substrates of RNA helicases.
  50. DNA repair pathways and their roles in drug resistance for lung adenocarcinoma.
  51. Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance.
  52. c-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation.
  53. Targeting adenosine with adenosine deaminase 2 to inhibit growth of solid tumors.
  54. Structure of Rad5 provides insights into its role in tolerance to replication stress.
  55. Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded DNA-Binding Proteins.
  56. CHK2 inhibition provides a strategy to suppress hematological toxicity from PARP inhibitors.
  57. Adenosine Kinase Expression Determines DNA Methylation in Cancer Cell Lines.
  1. Nature. 2021 Apr 14.
    AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.
    Emiliano Maiani, Giacomo Milletti, Francesca Nazio, Søs Grønbæk Holdgaard, Jirina Bartkova, Salvatore Rizza, Valentina Cianfanelli, Mar Lorente, Daniele Simoneschi, Miriam Di Marco, Pasquale D'Acunzo, Luca Di Leo, Rikke Rasmussen, Costanza Montagna, Marilena Raciti, Cristiano De Stefanis, Estibaliz Gabicagogeascoa, Gergely Rona, Nélida Salvador, Emanuela Pupo, Joanna Maria Merchut-Maya, Colin J Daniel, Marianna Carinci, Valeriana Cesarini, Alfie O'sullivan, Yeon-Tae Jeong, Matteo Bordi, Francesco Russo, Silvia Campello, Angela Gallo, Giuseppe Filomeni, Letizia Lanzetti, Rosalie C Sears, Petra Hamerlik, Armando Bartolazzi, Robert E Hynds, David R Pearce, Charles Swanton, Michele Pagano, Guillermo Velasco, Elena Papaleo, Daniela De Zio, Apolinar Maya-Mendoza, Franco Locatelli, Jiri Bartek, Francesco Cecconi.
      Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.
    DOI:  https://doi.org/10.1038/s41586-021-03422-5
  2. Nature. 2021 Apr 14.
    Structural basis of long-range to short-range synaptic transition in NHEJ.
    Siyu Chen, Linda Lee, Tasmin Naila, Susan Fishbain, Annie Wang, Alan E Tomkinson, Susan P Lees-Miller, Yuan He.
      DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.
    DOI:  https://doi.org/10.1038/s41586-021-03458-7
  3. Mol Cell. 2021 Apr 06. pii: S1097-2765(21)00229-X. [Epub ahead of print]
    The dystonia gene THAP1 controls DNA double-strand break repair choice.
    Kenta Shinoda, Dali Zong, Elsa Callen, Wei Wu, Lavinia C Dumitrache, Frida Belinky, Raj Chari, Nancy Wong, Momoko Ishikawa, Andre Stanlie, Trisha Multhaupt-Buell, Nutan Sharma, Laurie Ozelius, Michelle Ehrlich, Peter J McKinnon, André Nussenzweig.
      The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.
    Keywords:  BRCA1; SHLD1; THAP1; antibody class-switch recombination; cancer; dystonia; homologous recombination; non-homologous end joining; resection; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.034
  4. Cancer Cell. 2021 Apr 12. pii: S1535-6108(21)00116-1. [Epub ahead of print]39(4): 566-579.e7
    Therapeutic targeting of ATR yields durable regressions in small cell lung cancers with high replication stress.
    Anish Thomas, Nobuyuki Takahashi, Vinodh N Rajapakse, Xiaohu Zhang, Yilun Sun, Michele Ceribelli, Kelli M Wilson, Yang Zhang, Erin Beck, Linda Sciuto, Samantha Nichols, Brian Elenbaas, Janusz Puc, Heike Dahmen, Astrid Zimmermann, Jillian Varonin, Christopher W Schultz, Sehyun Kim, Hirity Shimellis, Parth Desai, Carleen Klumpp-Thomas, Lu Chen, Jameson Travers, Crystal McKnight, Sam Michael, Zina Itkin, Sunmin Lee, Akira Yuno, Min-Jung Lee, Christophe E Redon, Jessica D Kindrick, Cody J Peer, Jun S Wei, Mirit I Aladjem, William Douglas Figg, Seth M Steinberg, Jane B Trepel, Frank T Zenke, Yves Pommier, Javed Khan, Craig J Thomas.
      Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.
    Keywords:  DNA damage response; DNA topoisomerases; SCLC; ataxia telangiectasia mutated and rad3 related; cell-cycle checkpoints; replication stress; small cell neuroendocrine cancers; translational research
    DOI:  https://doi.org/10.1016/j.ccell.2021.02.014
  5. EMBO J. 2021 Apr 15. e99692
    USP7 limits CDK1 activity throughout the cell cycle.
    Antonio Galarreta, Pablo Valledor, Patricia Ubieto-Capella, Vanesa Lafarga, Eduardo Zarzuela, Javier Muñoz, Marcos Malumbres, Emilio Lecona, Oscar Fernandez-Capetillo.
      Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.
    Keywords:  CDK1; USP7; anticancer drugs; cell cycle; deubiquitinase
    DOI:  https://doi.org/10.15252/embj.201899692
  6. Brain. 2021 Apr 14. pii: awab020. [Epub ahead of print]
    De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.
    Jack M Shireman, Fatemeh Atashi, Gina Lee, Eunus S Ali, Miranda R Saathoff, Cheol H Park, Sol Savchuk, Shivani Baisiwala, Jason Miska, Maciej S Lesniak, C David James, Roger Stupp, Priya Kumthekar, Craig M Horbinski, Issam Ben-Sahra, Atique U Ahmed.
      Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.
    Keywords:  cellular plasticity; chemoresistance; glioblastoma; purine biosynthesis
    DOI:  https://doi.org/10.1093/brain/awab020
  7. ACS Pharmacol Transl Sci. 2021 Apr 09. 4(2): 624-646
    A Review of Small-Molecule Inhibitors of One-Carbon Enzymes: SHMT2 and MTHFD2 in the Spotlight.
    Christine R Cuthbertson, Zahra Arabzada, Armand Bankhead, Armita Kyani, Nouri Neamati.
      Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.
    DOI:  https://doi.org/10.1021/acsptsci.0c00223
  8. Thorax. 2021 Apr 15. pii: thoraxjnl-2020-216504. [Epub ahead of print]
    Genetic polymorphism in ATIC is associated with effectiveness and toxicity of pemetrexed in non-small-cell lung cancer.
    Sabine Visser, Stijn Koolen, Nadine van Donk, Nico van Walree, Cor van der Leest, Robin Cornelissen, Ron van Schaik, Ron Mathijssen, Joachim Aerts, Bruno H Stricker.
      Patients with advanced non-small-cell lung cancer who are treated with pemetrexed display a wide variation in clinical response and toxicity. In this prospective, multicentre cohort study, we investigated the association with treatment effectiveness and toxicity of 10 polymorphisms in nine candidate genes, covering the folate pathway (MTHFR), cell transport (SLC19A1/ABCC2/ABCC4), intracellular metabolism (FPGS/GGH) and target enzymes (TYMS/DHFR/ATIC) of pemetrexed. Adjusted for sex, ECOG performance score and disease stage, the association between ATIC (rs12995526) and overall survival (HR 1.59, 95% CI 1.06 to 2.39) was significant. Regarding toxicity, this ATIC polymorphism was significantly associated with severe laboratory (p=0.014) and clinical (p=0.016) chemotherapy-related adverse events, severe neutropenia (p=0.007) and all-grade diarrhoea (p=0.034) in multivariable analyses.
    Keywords:  clinical epidemiology; lung cancer; lung cancer chemotherapy; non-small cell lung cancer
    DOI:  https://doi.org/10.1136/thoraxjnl-2020-216504
  9. PLoS Genet. 2021 Apr 15. 17(4): e1009523
    Aicardi-Goutières syndrome-associated gene SAMHD1 preserves genome integrity by preventing R-loop formation at transcription-replication conflict regions.
    Kiwon Park, Jeongmin Ryoo, Heena Jeong, Minsu Kim, Sungwon Lee, Sung-Yeon Hwang, Jiyoung Ahn, Doyeon Kim, Hyungseok C Moon, Daehyun Baek, Kwangsoo Kim, Hye Yoon Park, Kwangseog Ahn.
      The comorbid association of autoimmune diseases with cancers has been a major obstacle to successful anti-cancer treatment. Cancer survival rate decreases significantly in patients with preexisting autoimmunity. However, to date, the molecular and cellular profiles of such comorbidities are poorly understood. We used Aicardi-Goutières syndrome (AGS) as a model autoimmune disease and explored the underlying mechanisms of genome instability in AGS-associated-gene-deficient patient cells. We found that R-loops are highly enriched at transcription-replication conflict regions of the genome in fibroblast of patients bearing SAMHD1 mutation, which is the AGS-associated-gene mutation most frequently reported with tumor and malignancies. In SAMHD1-depleted cells, R-loops accumulated with the concomitant activation of DNA damage responses. Removal of R-loops in SAMHD1 deficiency reduced cellular responses to genome instability. Furthermore, downregulation of SAMHD1 expression is associated with various types of cancer and poor survival rate. Our findings suggest that SAMHD1 functions as a tumor suppressor by resolving R-loops, and thus, SAMHD1 and R-loop may be novel diagnostic markers and targets for patient stratification in anti-cancer therapy.
    DOI:  https://doi.org/10.1371/journal.pgen.1009523
  10. Mol Cell Oncol. 2021 ;8(2): 1881394
    Trex2 responds to damaged replication forks in diverse ways.
    Paul Hasty.
      Three prime Repair Exonuclease 2 (Trex2) alters replication fork (RF) stability and mutation levels in cells defective for homologous recombination (HR). Trex2 has multiple functions that can either cause or supress RF instability in cells with different HR-defects. Why does Trex2 have such diverse effects on RF maintenance?
    DOI:  https://doi.org/10.1080/23723556.2021.1881394
  11. EMBO J. 2021 Apr 12. e104847
    Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks.
    Nhung Pham, Zhenxin Yan, Yang Yu, Mosammat Faria Afreen, Anna Malkova, James E Haber, Grzegorz Ira.
      DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.
    Keywords:  DSB end resection; break-induced replication; double-strand break; homologous recombination; ssDNA annealing
    DOI:  https://doi.org/10.15252/embj.2020104847
  12. DNA Repair (Amst). 2021 Jun;pii: S1568-7864(21)00068-9. [Epub ahead of print]102 103112
    DNA damage response and PD-1/PD-L1 pathway in ovarian cancer.
    Tianyu Zhang, Shuangshuang Zheng, Yang Liu, Xiao Li, Jing Wu, Yue Sun, Guoyan Liu.
      Ovarian cancer has a poor prognosis due to drug resistance, relapse and metastasis. In recent years, immunotherapy has been applied in numerous cancers clinically. However, the effect of immunotherapy monotherapy in ovarian cancer is limited. DNA damage response (DDR) is an essential factor affecting the efficacy of tumor immunotherapy. Defective DNA repair may lead to carcinogenesis and tumor genomic instability, but on the other hand, it may also portend particular vulnerability of tumors and can be used as biomarkers for immunotherapy patient selection. Programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway mediates tumor immune escape, which may be a promising target for immunotherapy. Therefore, further understanding of the mechanism of PD-L1 expression after DDR may help guide the development of immunotherapy in ovarian cancer. In this review, we present the DNA damage repair pathway and summarize how DNA damage repair affects the PD-1/PD-L1 pathway in cancer cells. And then we look for biomarkers that affect efficacy or prognosis. Finally, we review the progress of PD-1/PD-L1-based immunotherapy in combination with other therapies that may affect the DDR pathway in ovarian cancer.
    Keywords:  Combined modality therapy; DNA damage response; Immune checkpoint inhibitors; Ovarian cancer; PD-1/PD-L1
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103112
  13. Gene. 2021 Apr 09. pii: S0378-1119(21)00241-9. [Epub ahead of print] 145647
    N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.
    Takashi Tsuyama, Kumiko Fujita, Ryosuke Sasaki, Shiori Hamanaka, Yuki Sotoyama, Akira Ogawa, Kana Kusuzaki, Yutaro Azuma, Shusuke Tada.
      RecQ4, a member of the RecQ helicase family, is required for the maintenance of genome integrity. RecQ4 has been shown to promote the following two DNA double-strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). However, its molecular function has not been fully elucidated. In the present study, we aimed to investigate the role of RecQ4 in NHEJ using Xenopus egg extracts. The N-terminal 598 amino acid region of Xenopus RecQ4 (N598), which lacks a central helicase domain and a downstream C-terminal region, was added to the extracts and its effect on the joining of DNA ends was analyzed. We found that N598 inhibited the joining of linearized DNA ends in the extracts. In addition, N598 inhibited DSB-induced chromatin binding of Ku70, which is essential for NHEJ, while the DSB-induced chromatin binding of the HR-associated proteins, replication protein A (RPA) and Rad51, increased upon the addition of N598. These results suggest that RecQ4 possibly influences the choice of the DSB repair pathway by influencing the association of the Ku heterodimer with the DNA ends.
    Keywords:  DNA double-strand breaks; DNA repair; DNA-dependent protein kinase; RecQ helicase; Rothmund-Thomson syndrome
    DOI:  https://doi.org/10.1016/j.gene.2021.145647
  14. Sci Rep. 2021 Apr 13. 11(1): 8042
    In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer.
    Z Ping Lin, Nour N Al Zouabi, Mark L Xu, Nicole E Bowen, Terence L Wu, Ethan S Lavi, Pamela H Huang, Yong-Lian Zhu, Baek Kim, Elena S Ratner.
      Poly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.
    DOI:  https://doi.org/10.1038/s41598-021-87325-5
  15. Biochem Soc Trans. 2021 Apr 12. pii: BST20200751. [Epub ahead of print]
    Primary cilia and the DNA damage response: linking a cellular antenna and nuclear signals.
    Ciaran G Morrison.
      The maintenance of genome stability involves integrated biochemical activities that detect DNA damage or incomplete replication, delay the cell cycle, and direct DNA repair activities on the affected chromatin. These processes, collectively termed the DNA damage response (DDR), are crucial for cell survival and to avoid disease, particularly cancer. Recent work has highlighted links between the DDR and the primary cilium, an antenna-like, microtubule-based signalling structure that extends from a centriole docked at the cell surface. Ciliary dysfunction gives rise to a range of complex human developmental disorders termed the ciliopathies. Mutations in ciliopathy genes have been shown to impact on several functions that relate to centrosome integrity, DNA damage signalling, responses to problems in DNA replication and the control of gene expression. This review covers recent findings that link cilia and the DDR and explores the various roles played by key genes in these two contexts. It outlines how proteins encoded by ciliary genes impact checkpoint signalling, DNA replication and repair, gene expression and chromatin remodelling. It discusses how these diverse activities may integrate nuclear responses with those that affect a structure of the cell periphery. Additional directions for exploration of the interplay between these pathways are highlighted, with a focus on new ciliary gene candidates that alter genome stability.
    Keywords:  DNA synthesis and repair; cell cycle; centrosomes; cilia
    DOI:  https://doi.org/10.1042/BST20200751
  16. Cell Mol Life Sci. 2021 Apr 15.
    The Ku complex: recent advances and emerging roles outside of non-homologous end-joining.
    Sanna Abbasi, Gursimran Parmar, Rachel D Kelly, Nileeka Balasuriya, Caroline Schild-Poulter.
      Since its discovery in 1981, the Ku complex has been extensively studied under multiple cellular contexts, with most work focusing on Ku in terms of its essential role in non-homologous end-joining (NHEJ). In this process, Ku is well-known as the DNA-binding subunit for DNA-PK, which is central to the NHEJ repair process. However, in addition to the extensive study of Ku's role in DNA repair, Ku has also been implicated in various other cellular processes including transcription, the DNA damage response, DNA replication, telomere maintenance, and has since been studied in multiple contexts, growing into a multidisciplinary point of research across various fields. Some advances have been driven by clarification of Ku's structure, including the original Ku crystal structure and the more recent Ku-DNA-PKcs crystallography, cryogenic electron microscopy (cryoEM) studies, and the identification of various post-translational modifications. Here, we focus on the advances made in understanding the Ku heterodimer outside of non-homologous end-joining, and across a variety of model organisms. We explore unique structural and functional aspects, detail Ku expression, conservation, and essentiality in different species, discuss the evidence for its involvement in a diverse range of cellular functions, highlight Ku protein interactions and recent work concerning Ku-binding motifs, and finally, we summarize the clinical Ku-related research to date.
    Keywords:  Cancer; Genome stability; Ku heterodimer; Ku-binding motifs; Non-homologous end-joining; Telomere maintenance
    DOI:  https://doi.org/10.1007/s00018-021-03801-1
  17. J Biol Chem. 2021 Apr 08. pii: S0021-9258(21)00428-2. [Epub ahead of print] 100642
    Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η.
    Pratibha P Ghodke, Jyotirling R Mali, Amritraj Patra, Carmelo J Rizzo, F Peter Guengerich, Martin Egli.
      Etheno (ε)-adducts, e.g. 1,N2-ε-guanine (1,N2-ε-G) and 1,N6-ε-adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N2-ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N2-ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with non-hydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5'-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N2-ε-G and T, compared to the single H-bond formed between incoming dCMPnPP and 1,N2-ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication.
    Keywords:  DNA damage; DNA polymerase; DNA replication; DNA-protein interaction; X-ray crystallography; etheno DNA adducts; mass spectrometry; translesion synthesis
    DOI:  https://doi.org/10.1016/j.jbc.2021.100642
  18. Front Genet. 2021 ;12 644803
    Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.
    Christopher B Nelson, Taghreed M Alturki, Jared J Luxton, Lynn E Taylor, David G Maranon, Keiko Muraki, John P Murnane, Susan M Bailey.
      Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5' C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5' C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.
    Keywords:  G1 human cells; alternative lengthening of telomeres (ALT); telomere double-strand breaks(DSBs); telomere repeat-containing RNA (TERRA); telomeres; telomeric C-rich overhangs
    DOI:  https://doi.org/10.3389/fgene.2021.644803
  19. Int J Radiat Oncol Biol Phys. 2021 Apr 07. pii: S0360-3016(21)00310-2. [Epub ahead of print]
    Pharmacological inhibition of HSP90 radiosensitizes HNSCC xenograft by inhibition of DNA damage repair, nucleotide metabolism, and radiation-induced tumor vasculogenesis.
    Sarwat Naz, Andrew J Leiker, Rajani Choudhuri, Olivia Preston, Anastasia L Sowers, Sangeeta Gohain, Janet Gamson, Askale Mathias, Carter Van Waes, John A Cook, James B Mitchell.
       PURPOSE: Recent pre-clinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and HNSCC. These studies emphasized that AT13387 down-regulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence.
    METHODS AND MATERIALS: HNSCC and NSCLC cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were utilized to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using LC/MS mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis.
    RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and TCA cycle by AT13387, reduction in ATP levels, and rate-limiting metabolites in nucleotide metabolism, namely PRPP and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow derived cells and inhibition of HIF-1 and SDF-1 expression thereby inhibiting IR-induced vasculogenesis.
    CONCLUSIONS: AT13387 treatment resulted in pharmacological inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.
    Keywords:  AT13387; Cancer Metabolism; HSP90; Hypoxia; Onalespib; Radiosensitizer; Vasculogenesis
    DOI:  https://doi.org/10.1016/j.ijrobp.2021.03.048
  20. Cancer Res. 2021 Apr 14. pii: canres.CAN-21-0688-E.2021. [Epub ahead of print]
    Recruitment of KMT2C/MLL3 to DNA damage sites mediates DNA damage responses and regulates PARP inhibitor sensitivity in cancer.
    Antao Chang, Liang Liu, Justin M Ashby, Dan Wu, Yanan Chen, Stacey S O'Neill, Shan Huang, Juan Wang, Guanwen Wang, Dongmei Cheng, Xiaoming Tan, W Jeffrey Petty, Boris C Pasche, Rong Xiang, Wei Zhang, Peiqing Sun.
      When recruited to promoters, histone 3 lysine 4 (H3K4) methyltransferases KMT2 (KMT2A-D) activate transcription by opening chromatin through H3K4 methylation. Here we report that KMT2 mutations occur frequently in non-small cell lung cancer (NSCLC) and are associated with high mutation loads and poor survival. KMT2C regulated DNA damage responses (DDR) through direct recruitment to DNA damage sites by Ago2 and small noncoding DNA damage response RNA, where it mediates H3K4 methylation, chromatin relaxation, secondary recruitment of DDR factors, and amplification of DDR signals along chromatin. Furthermore, by disrupting homologous recombination (HR)-mediated DNA repair, KMT2C/D mutations sensitized NSCLC to Poly(ADP-Ribose) Polymerase inhibitors (PARPi), whose efficacy is unclear in NSCLC due to low BRCA1/2 mutation rates. These results demonstrate a novel, transcription-independent role of KMT2C in DDR and identify high-frequency KMT2C/D mutations as much-needed biomarkers for PARPi therapies in NSCLC and other cancers with infrequent BRCA1/2 mutations.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0688
  21. Cancer Lett. 2021 Apr 01. pii: S0304-3835(21)00149-X. [Epub ahead of print]509 1-12
    Proton pump inhibitors suppress DNA damage repair and sensitize treatment resistance in breast cancer by targeting fatty acid synthase.
    Chao J Wang, Deren Li, Jacob A Danielson, Evan H Zhang, Zizheng Dong, Kathy D Miller, Lang Li, Jian-Ting Zhang, Jing-Yuan Liu.
      Human fatty acid synthase (FASN) is the sole cytosolic enzyme responsible for de novo lipid synthesis. FASN is essential for cancer cell survival and contributes to drug and radiation resistance by up-regulating DNA damage repair but not required for most non-lipogenic tissues. Thus, FASN is an attractive target for drug discovery. However, despite decades of effort in targeting FASN, no FASN inhibitors have been approved due to poor pharmacokinetics or toxicities. Here, we show that the FDA-approved proton pump inhibitors (PPIs) effectively inhibit FASN and suppress breast cancer cell survival. PPI inhibition of FASN leads to suppression of non-homologous end joining repair of DNA damages by reducing FASN-mediated PARP1 expression, resulting in apoptosis from oxidative DNA damages and sensitization of cellular resistance to doxorubicin and ionizing radiation. Mining electronic medical records of 6754 breast cancer patients showed that PPI usage significantly increased overall survival and reduced disease recurrence of these patients. Hence, PPIs may be repurposed as anticancer drugs for breast cancer treatments by targeting FASN to overcome drug and radiation resistance.
    Keywords:  DNA damage Repair; Enantiomer; Fatty acid synthase; PARP1; Proton pump inhibitor
    DOI:  https://doi.org/10.1016/j.canlet.2021.03.026
  22. Cell Biosci. 2021 Apr 15. 11(1): 74
    RRM2 silencing suppresses malignant phenotype and enhances radiosensitivity via activating cGAS/STING signaling pathway in lung adenocarcinoma.
    Xueping Jiang, Yangyi Li, Nannan Zhang, Yanping Gao, Linzhi Han, Shuying Li, Jiali Li, Xingyu Liu, Yan Gong, Conghua Xie.
       BACKGROUND: As one of the most common malignancy, lung adenocarcinoma (LUAD) is characterized by low 5-year survival rate. This research aimed to investigate the effects of ribonucleotide reductase regulatory subunit M2 (RRM2) on malignant biological behaviors and activation of cGAS/STING pathway. We also explored the synergistic sensitization mechanisms of RRM2 and radiotherapy.
    METHODS: Bioinformatic tools were used to evaluate the clinical significance of RRM2 in LUAD patients. The roles of RRM2 in malignant phenotype and DNA damage in LUAD cells were investigated with cell proliferation, colony formation, immunofluorescence, modified Boyden chamber and comet assays. The mouse models were used to evaluate the biological significance of RRM2 in vivo. Cytotoxic T cell infiltration was evaluated via flow cytometric analysis and immunohistochemistry staining in C57BL/6 mice. We also explored the synergistic effects of RRM2 silencing and radiation on LUAD cells with apoptosis assay and immunoblotting in vitro.
    RESULTS: Bioinformatic analysis revealed that RRM2 had diagnostic values for LUAD patients. Higher levels of RRM2 predicted worse prognosis. RRM2 silencing inhibited LUAD cell proliferation, invasion and migration. RRM2 knockdown induced S phase arrest and DNA damage. RRM2 silencing induced cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, and the downstream targets were regulated in a STING-dependent manner. Knockdown of RRM2 suppressed tumor growth in the xenograft tumor models. RRM2 deficiency increased CD8 + T cells in the tumor tissues and spleens. Furthermore, RRM2 silencing had synergistic effects with radiation on inhibiting cell proliferation and promoting apoptosis. Meanwhile, this combination promoted the activation of cGAS/STING signaling pathway synergistically, and simultaneously increased expression of IFNβ, CCL5 and CXCL10.
    CONCLUSION: Our results demonstrated that RRM2 silencing had anti-tumor values and activated the cGAS/STING signaling pathway. RRM2 silencing increased CD8 + T cells infiltration. RRM2 silencing cooperated with radiation to inhibit LUAD cell proliferation, promote apoptosis and enhance the activation of cGAS/STING signaling pathway. RRM2 could be a promising target for tumor regression through cancer immunotherapy in LUAD.
    Keywords:  CGAS/STING pathway; Immune responses; Lung adenocarcinoma; RRM2; Radiotherapy
    DOI:  https://doi.org/10.1186/s13578-021-00586-5
  23. Methods Mol Biol. 2021 ;2281 193-207
    Single-Stranded DNA Curtains for Single-Molecule Visualization of Rad51-ssDNA Filament Dynamics.
    Upasana Roy, Eric C Greene.
      Homologous recombination (HR) is a highly conserved DNA repair pathway required for the accurate repair of DNA double-stranded breaks. DNA recombination is catalyzed by the RecA/Rad51 family of proteins, which are conserved from bacteria to humans. The key intermediate catalyzing DNA recombination is the presynaptic complex (PSC), which is a helical filament comprised of Rad51-bound single-stranded DNA (ssDNA). Multiple cellular factors either promote or downregulate PSC activity, and a fine balance between such regulators is required for the proper regulation of HR and maintenance of genomic integrity. However, dissecting the complex mechanisms regulating PSC activity has been a challenge using traditional ensemble methods due to the transient and dynamic nature of recombination intermediates. We have developed a single-molecule assay called ssDNA curtains that allows us to visualize individual DNA intermediates in real-time, using total internal reflection microscopy (TIRFM). This assay has allowed us to study many aspects of HR regulation that involve complex and heterogenous reaction intermediates. Here we describe the procedure for a basic ssDNA curtain assay to study PSC filament dynamics, and explain how to process and analyze the resulting data.
    Keywords:  DNA curtains; Homologous recombination; RPA; Rad51; Single molecule; Single-stranded DNA; Srs2
    DOI:  https://doi.org/10.1007/978-1-0716-1290-3_11
  24. Elife. 2021 Apr 16. pii: e68579. [Epub ahead of print]10
    Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.
    Thomas J Etheridge, Desiree Villahermosa, Eduard Campillo-Funollet, Alex David Herbert, Anja Irmisch, Adam T Watson, Hung Q Dang, Mark A Osborne, Antony W Oliver, Antony M Carr, Johanne M Murray.
      The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.
    Keywords:  S. pombe; chromosomes; gene expression; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.68579
  25. Genetics. 2021 Apr 12. pii: iyab060. [Epub ahead of print]
    Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.
    Elena I Stepchenkova, Anna S Zhuk, Jian Cui, Elena R Tarakhovskaya, Stephanie R Barbari, Polina V Shcherbakova, Dmitrii E Polev, Roman Fedorov, Eugenia Poliakov, Igor B Rogozin, Artem G Lada, Youri I Pavlov.
      Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.
    Keywords:  CDC28; DNA polymerase ε; DNA replication; cyclin-dependent kinase; illegitimate mating; leading strand; mutation rates; yeast
    DOI:  https://doi.org/10.1093/genetics/iyab060
  26. Mol Cell Oncol. 2021 Mar 08. 8(2): 1839294
    Control of S phase duration: a replication capacity model with E2F transcription at its heart.
    Cosetta Bertoli, Robertus A M de Bruin.
      DNA replication capacity, the maximal amount of DNA a cell can synthesize at any given time during S phase, is controlled by E2F-dependent transcription. Controlling replication capacity limits the replication rate and provides a robust mechanism to keep replication fork speed within an optimal range whilst ensuring timely completion of genome duplication.
    Keywords:  DNA replication; DNA replication capacity; E2F-dependent transcription; S phase; genome stability
    DOI:  https://doi.org/10.1080/23723556.2020.1839294
  27. J Cell Biol. 2021 Jun 07. pii: e202011014. [Epub ahead of print]220(6):
    Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe.
    Alexandre T Vessoni, Tianpeng Zhang, Annabel Quinet, Ho-Chang Jeong, Michael Munroe, Matthew Wood, Enzo Tedone, Alessandro Vindigni, Jerry W Shay, Roger A Greenberg, Luis F Z Batista.
      It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.
    DOI:  https://doi.org/10.1083/jcb.202011014
  28. PLoS Genet. 2021 Apr 15. 17(4): e1009329
    Pathways and signatures of mutagenesis at targeted DNA nicks.
    Yinbo Zhang, Luther Davis, Nancy Maizels.
      Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.
    DOI:  https://doi.org/10.1371/journal.pgen.1009329
  29. J Invest Dermatol. 2021 Apr 10. pii: S0022-202X(21)01118-0. [Epub ahead of print]
    ENTPD1(CD39) expression inhibits ultraviolet radiation-induced DNA damage repair via purinergic signaling and is associated with metastasis in human cutaneous squamous cell carcinoma.
    Melodi Javid Whitley, Jutamas Suwanpradid, Chester Lai, Simon Jiang, Jonathan L Cook, Daniel E Zelac, Ross Rudolph, David L Corcoran, Simone Degan, Ivan Spasojevic, Howard Levinson, Detlev Erdmann, Claire Reid, Jennifer Y Zhang, Simon C Robson, Eugene Healy, Wendy L Havran, Amanda S MacLeod.
      Ultraviolet radiation (UVR) and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells (Tregs) promote cSCC carcinogenesis and in other solid tumors, infiltrating Tregs and CD8+ T cells express ENTPD1 (also known as CD39), an ecto-enzyme that catalyzes the rate-limiting step in converting extracellular ATP to extracellular adenosine. We previously demonstrated that extracellular purine nucleotides influence DNA Damage Repair (DDR). Here we investigate whether DDR is modulated via purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared to T cells from blood or non-lesional skin and accordingly, concentrations of derivative extracellular ADP, AMP, and adenosine are increased in tumors compared to normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize compared to those that are non-metastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL27-dependent manner. Finally, increased extracellular adenosine is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DDR secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DDR via purinergic signaling to promote skin carcinogenesis and metastasis.
    DOI:  https://doi.org/10.1016/j.jid.2021.02.753
  30. Cancer Res. 2021 Apr 16. pii: canres.2694.2020. [Epub ahead of print]
    SLFN11 inactivation induces proteotoxic stress and sensitizes cancer cells to ubiquitin activating enzyme inhibitor TAK-243.
    Yasuhisa Murai, Ukhyun Jo, Junko Murai, Lisa Jenkins, Shar-Yin N Huang, Sirisha Chakka, Lu Chen, Ken Cheng, Shinsaku Fukuda, Naoko Takebe, Yves Pommier.
      Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2alpha, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2694
  31. Nat Commun. 2021 04 12. 12(1): 2187
    Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling.
    Guijie Guo, Ming Gao, Xiaochen Gao, Bibo Zhu, Jinzhou Huang, Xinyi Tu, Wootae Kim, Fei Zhao, Qin Zhou, Shouhai Zhu, Zheming Wu, Yuanliang Yan, Yong Zhang, Xiangyu Zeng, Qian Zhu, Ping Yin, Kuntian Luo, Jie Sun, Min Deng, Zhenkun Lou.
      The RNA-sensing pathway contributes to type I interferon (IFN) production induced by DNA damaging agents. However, the potential involvement of RNA sensors in DNA repair is unknown. Here, we found that retinoic acid-inducible gene I (RIG-I), a key cytosolic RNA sensor that recognizes RNA virus and initiates the MAVS-IRF3-type I IFN signaling cascade, is recruited to double-stranded breaks (DSBs) and suppresses non-homologous end joining (NHEJ). Mechanistically, RIG-I interacts with XRCC4, and the RIG-I/XRCC4 interaction impedes the formation of XRCC4/LIG4/XLF complex at DSBs. High expression of RIG-I compromises DNA repair and sensitizes cancer cells to irradiation treatment. In contrast, depletion of RIG-I renders cells resistant to irradiation in vitro and in vivo. In addition, this mechanism suggests a protective role of RIG-I in hindering retrovirus integration into the host genome by suppressing the NHEJ pathway. Reciprocally, XRCC4, while suppressed for its DNA repair function, has a critical role in RIG-I immune signaling through RIG-I interaction. XRCC4 promotes RIG-I signaling by enhancing oligomerization and ubiquitination of RIG-I, thereby suppressing RNA virus replication in host cells. In vivo, silencing XRCC4 in mouse lung promotes influenza virus replication in mice and these mice display faster body weight loss, poorer survival, and a greater degree of lung injury caused by influenza virus infection. This reciprocal regulation of RIG-I and XRCC4 reveals a new function of RIG-I in suppressing DNA repair and virus integration into the host genome, and meanwhile endues XRCC4 with a crucial role in potentiating innate immune response, thereby helping host to prevail in the battle against virus.
    DOI:  https://doi.org/10.1038/s41467-021-22484-7
  32. Nucleic Acids Res. 2021 Apr 13. pii: gkab234. [Epub ahead of print]
    Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.
    Stephen T Hallett, Pascale Schellenberger, Lihong Zhou, Fabienne Beuron, Ed Morris, Johanne M Murray, Antony W Oliver.
      The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.
    DOI:  https://doi.org/10.1093/nar/gkab234
  33. Front Cell Dev Biol. 2021 ;9 617161
    The Enigma of Substrate Recognition and Catalytic Efficiency of APE1-Like Enzymes.
    Anastasiia T Davletgildeeva, Alexander A Ishchenko, Murat Saparbaev, Olga S Fedorova, Nikita A Kuznetsov.
      Despite significant achievements in the elucidation of the nature of protein-DNA contacts that control the specificity of nucleotide incision repair (NIR) by apurinic/apyrimidinic (AP) endonucleases, the question on how a given nucleotide is accommodated by the active site of the enzyme remains unanswered. Therefore, the main purpose of our study was to compare kinetics of conformational changes of three homologous APE1-like endonucleases (insect Drosophila melanogaster Rrp1, amphibian Xenopus laevis xAPE1, and fish Danio rerio zAPE1) during their interaction with various damaged DNA substrates, i.e., DNA containing an F-site (an uncleavable by DNA-glycosylases analog of an AP-site), 1,N 6-ethenoadenosine (εA), 5,6-dihydrouridine (DHU), uridine (U), or the α-anomer of adenosine (αA). Pre-steady-state analysis of fluorescence time courses obtained for the interaction of the APE1-like enzymes with DNA substrates containing various lesions allowed us to outline a model of substrate recognition by this class of enzymes. It was found that the differences in rates of DNA substrates' binding do not lead to significant differences in the cleavage efficiency of DNA containing a damaged base. The results suggest that the formation of enzyme-substrate complexes is not the key factor that limits enzyme turnover; the mechanisms of damage recognition and cleavage efficacy are related to fine conformational tuning inside the active site.
    Keywords:  DNA repair; abasic site; apurinic/apyrimidinic endonuclease; pre-steady state kinetics; target nucleotide recognition
    DOI:  https://doi.org/10.3389/fcell.2021.617161
  34. Genes Cells. 2021 Apr 13.
    UV-induced activation of ATR is mediated by UHRF2.
    Shunsuke Hanaki, Makoto Habara, Midori Shimada.
      UHRF1 (Ubiquitin-like with PHD and ring finger domains 1) regulates DNA methylation and histone modifications, and plays a key role in cell proliferation and the DNA damage response. However, the function of UHRF2, a paralog of UHRF1, in the DNA damage response remains largely unknown. Here, we show that UHRF2 is essential for maintaining cell viability after UV irradiation, as well as for the proliferation of cancer cells. UHRF2 was found to physically interact with ATR in a DNA damage-dependent manner through UHRF2's TTD domain. In addition, phosphorylation of threonine at position 1989, which is required for UV-induced activation of ATR, was impaired in cells depleted of UHRF2, suggesting that UHRF2 is essential in ATR activation. In conclusion, these results suggest a new regulatory mechanism of ATR activation mediated by UHRF2.
    Keywords:  ATR; DNA damage response; UHRF; cancer cell proliferation
    DOI:  https://doi.org/10.1111/gtc.12851
  35. Front Oncol. 2021 ;11 646256
    The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.
    Guochao Liu, David Lim, Zuchao Cai, Wenwen Ding, Zhujun Tian, Chao Dong, Fengmei Zhang, Gongshe Guo, Xiaowei Wang, Pingkun Zhou, Zhihui Feng.
      Ionizing radiation (IR) can induce DNA double-strand breaks (DSBs) in tumor cells during radiotherapy (RT), but the efficiency of RT is limited because of the toxicity to normal cells. Locating an adjuvant treatment to alleviate damage in normal cells while sensitizing tumor cells to IR has attracted much attention. Here, using the 7,12-dimethylbenz[α]anthracene (DMBA)-induced malignant transformed MCF10A cells, we found that valproate (VPA), a histone deacetylase inhibitor (HDACi), radiosensitized transformed cells while alleviated IR-induced damage in normal cells at a safe dose (0.5 mM). We further demonstrated the decrease of homologous recombination (HR)-associated Rad51 in the transformed cells was related to the increase of its ubiquitination regulated by E3 ligase RFWD3 for the radiosensitization, which was opposite to normal cells, indicating that RFWD3-dependent ubiquitination on Rad51 was involved in the VPA-mediated radio-bidirectional effect. Through DMBA-transformed breast cancer rat model, VPA at 200 mg/kg radiosensitized tumor tissue cells by increasing RFWD3 and inhibited Rad51, while radioprotected normal tissue cells by decreasing RFWD3 and enhanced Rad51. In addition, we found high-level Rad51 was associated with tumorigenesis and poor prognosis in breast cancer patients. Our findings uncovered RFWD3-dependent Rad51 ubiquitination was the novel mechanism of VPA-mediated radio-bidirectional effect, VPA is a potential adjuvant treatment for tumor RT.
    Keywords:  RFWD3; Rad51; radioprotection; radiosensitization; ubiquitination; valproate
    DOI:  https://doi.org/10.3389/fonc.2021.646256
  36. ACS Pharmacol Transl Sci. 2021 Apr 09. 4(2): 730-743
    Comparative Activity and Off-Target Effects in Cells of the CHK1 Inhibitors MK-8776, SRA737, and LY2606368.
    Jennifer P Ditano, Alan Eastman.
      DNA damage activates the checkpoint protein CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of CHK1 (CHK1i) enhance damage-induced cell death. Additionally, CHK1i elicits single agent cytotoxicity in some cell lines. We compared three CHK1i that have undergone clinical trials and exhibited different toxicities. Each CHK1i inhibits other targets at higher concentrations, and whether these contribute to the toxicity is unknown. We compared their sensitivity in a panel of cell lines, their efficacy at inhibiting CHK1 and CHK2, and their ability to induce DNA damage and abrogate damage-induced S phase arrest. Published in vitro kinase analyses were a poor predictor of selectivity and potency in cells. LY2606368 was far more potent at inhibiting CHK1 and inducing growth arrest, while all three CHK1i inhibited CHK2 at concentrations 10- (MK-8776 and SRA737) to 100- (LY2606368) fold higher. MK-8776 and SRA737 exhibited similar off-target effects: higher concentrations demonstrated transient protection from growth inhibition, circumvented DNA damage, and prevented checkpoint abrogation, possibly due to inhibition of CDK2. Acquired resistance to LY2606368 resulted in limited cross-resistance to other CHK1i. LY2606368-resistant cells still abrogated DNA damage-induced S phase arrest, which requires low CDK2 activity, whereas inappropriately high CDK2 activity is responsible for sensitivity to CHK1i alone. All three CHK1i inhibited protein synthesis in a sensitive cell line correlating with cell death, whereas resistant cells failed to inhibit protein synthesis and underwent transient cytostasis. LY2606368 appears to be the most selective CHK1i, suggesting that further clinical development of this drug is warranted.
    DOI:  https://doi.org/10.1021/acsptsci.0c00201
  37. Mol Cell Oncol. 2021 ;8(2): 1875804
    Phosphoglycerate mutase 1 (PGAM1) overexpression promotes radio- and chemoresistance in gliomas by activating the DNA damage response.
    Tor-Christian Aase Johannessen, Joydeep Mukherjee.
      The glycolytic enzyme PGAM1 is overexpressed in gliomas where it efficiently facilitates the repair of DNA damage. Mechanistically, PGAM1 prevents inactivation of the ataxia-telangiectasia mutated (ATM) signaling pathway by sequestering the wild-type p53-induced phosphatase 1 (WIP1) in the cytoplasm. Genetic inhibition of PGAM1 expression subsequently sensitizes glioma cells against irradiation and chemotherapy-induced DNA damage.
    Keywords:  DNA damage repair; Glioma; IR; PGAM1; TMZ; WIP1; treatment resistance
    DOI:  https://doi.org/10.1080/23723556.2021.1875804
  38. Nat Commun. 2021 Apr 16. 12(1): 2285
    DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells.
    Lama Tarayrah-Ibraheim, Elital Chass Maurice, Guy Hadary, Sharon Ben-Hur, Alina Kolpakova, Tslil Braun, Yoav Peleg, Keren Yacobi-Sharon, Eli Arama.
      During Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.
    DOI:  https://doi.org/10.1038/s41467-021-22622-1
  39. Methods Mol Biol. 2021 ;2281 265-272
    Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy and Enzymatic Assays.
    Oya Bermek, Grzegorz L Ciesielski.
      The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.
    Keywords:  DNA polymerase γ; Electron microscopy; Mitochondrial DNA replication; Mitochondrial single-stranded DNA-binding protein; Nucleoprotein complexes
    DOI:  https://doi.org/10.1007/978-1-0716-1290-3_16
  40. Nat Rev Immunol. 2021 Apr 15.
    A guide to interrogating immunometabolism.
    Kelsey Voss, Hanna S Hong, Jackie E Bader, Ayaka Sugiura, Costas A Lyssiotis, Jeffrey C Rathmell.
      The metabolic charts memorized in early biochemistry courses, and then later forgotten, have come back to haunt many immunologists with new recognition of the importance of these pathways. Metabolites and the activity of metabolic pathways drive energy production, macromolecule synthesis, intracellular signalling, post-translational modifications and cell survival. Immunologists who identify a metabolic phenotype in their system are often left wondering where to begin and what does it mean? Here, we provide a framework for navigating and selecting the appropriate biochemical techniques to explore immunometabolism. We offer recommendations for initial approaches to develop and test metabolic hypotheses and how to avoid common mistakes. We then discuss how to take things to the next level with metabolomic approaches, such as isotope tracing and genetic approaches. By proposing strategies and evaluating the strengths and weaknesses of different methodologies, we aim to provide insight, note important considerations and discuss ways to avoid common misconceptions. Furthermore, we highlight recent studies demonstrating the power of these metabolic approaches to uncover the role of metabolism in immunology. By following the framework in this Review, neophytes and seasoned investigators alike can venture into the emerging realm of cellular metabolism and immunity with confidence and rigour.
    DOI:  https://doi.org/10.1038/s41577-021-00529-8
  41. Nat Genet. 2021 Apr 12.
    Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.
    Mitchell L Leibowitz, Stamatis Papathanasiou, Phillip A Doerfler, Logan J Blaine, Lili Sun, Yu Yao, Cheng-Zhong Zhang, Mitchell J Weiss, David Pellman.
      Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.
    DOI:  https://doi.org/10.1038/s41588-021-00838-7
  42. Methods Mol Biol. 2021 ;2281 313-322
    Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Binding Protein.
    Ana P C Rodrigues, Marcos T Oliveira.
      Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.
    Keywords:  Mitochondrial DNA replication; P66; Twinkle; dsDNA unwinding assay; mtSSB
    DOI:  https://doi.org/10.1007/978-1-0716-1290-3_20
  43. EBioMedicine. 2021 Apr 13. pii: S2352-3964(21)00125-0. [Epub ahead of print]66 103332
    Metabolomic profiling of pancreatic adenocarcinoma reveals key features driving clinical outcome and drug resistance.
    Abdessamad El Kaoutari, Nicolas A Fraunhoffer, Owen Hoare, Carlos Teyssedou, Philippe Soubeyran, Odile Gayet, Julie Roques, Gwen Lomberk, Raul Urrutia, Nelson Dusetti, Juan Iovanna.
       BACKGROUND: Although significant advances have been made recently to characterize the biology of pancreatic ductal adenocarcinoma (PDAC), more efforts are needed to improve our understanding and to face challenges related to the aggressiveness, high mortality rate and chemoresistance of this disease.
    METHODS: In this study, we perform the metabolomics profiling of 77 PDAC patient-derived tumor xenografts (PDTX) to investigate the relationship of metabolic profiles with overall survival (OS) in PDAC patients, tumor phenotypes and resistance to five anticancer drugs (gemcitabine, oxaliplatin, docetaxel, SN-38 and 5-Fluorouracil).
    FINDINGS: We identified a metabolic signature that was able to predict the clinical outcome of PDAC patients (p < 0.001, HR=2.68 [95% CI: 1.5-4.9]). The correlation analysis showed that this metabolomic signature was significantly correlated with the PDAC molecular gradient (PAMG) (R = 0.44 and p < 0.001) indicating significant association to the transcriptomic phenotypes of tumors. Resistance score established, based on growth rate inhibition metrics using 35 PDTX-derived primary cells, allowed to identify several metabolites related to drug resistance which was globally accompanied by accumulation of several diacy-phospholipids and decrease in lysophospholipids. Interestingly, targeting glycerophospholipid synthesis improved sensitivity to the three tested cytotoxic drugs indicating that interfering with metabolism could be a promising therapeutic strategy to overcome the challenging resistance of PDAC.
    INTERPRETATION: In conclusion, this study shows that the metabolomic profile of pancreatic PDTX models is strongly associated to clinical outcome, transcriptomic phenotypes and drug resistance. We also showed that targeting the lipidomic profile could be used in combinatory therapies against chemoresistance in PDAC.
    Keywords:  Chemosensitivity; FSG67; Metabolic signature; Metabolomics; Pancreatic cancer; Precision medicine; Tumor heterogeneity
    DOI:  https://doi.org/10.1016/j.ebiom.2021.103332
  44. Methods Mol Biol. 2021 ;2281 151-168
    Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.
    Sahiti Kuppa, Nilisha Pokhrel, Elliot Corless, Sofia Origanti, Edwin Antony.
      Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of select DNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-L-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.
    Keywords:  DNA-binding proteins; Fluorescence enhancement through noncanonical amino acids (FEncAA); Protein dynamics; Replication protein A (RPA); Site-specific labeling
    DOI:  https://doi.org/10.1007/978-1-0716-1290-3_9
  45. J Am Chem Soc. 2021 Apr 15.
    Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase.
    Clorice R Reinhardt, Elvira R Sayfutyarova, Jiayun Zhong, Sharon Hammes-Schiffer.
      Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis for all living organisms. It reduces ribonucleotides to the corresponding deoxyribonucleotides by a reversible radical transfer mechanism. The active form of E. coli Ia RNR is composed of two subunits, α and β, which form an active asymmetric α2β2 complex. The radical transfer pathway involves a series of proton-coupled electron transfer (PCET) reactions spanning α and β over ∼32 Å. Herein, quantum mechanical/molecular mechanical free energy simulations of PCET between tyrosine residues Y730 and Y731 are performed on the recently solved cryo-EM structure of the active α2β2 complex, which includes a pre-turnover α/β pair with an ordered PCET pathway and a post-turnover α'/β' pair. The free energy surfaces in both the pre- and post-turnover states are computed. According to the simulations, forward radical transfer from Y731 to Y730 is thermodynamically favored in the pre-turnover state, and backward radical transfer is favored in the post-turnover state, consistent with the reversible mechanism. E623, a glutamate residue that is near these tyrosines only in the pre-turnover state, is discovered to play a key role in facilitating forward radical transfer by thermodynamically stabilizing the radical on Y730 through hydrogen-bonding and electrostatic interactions and lowering the free energy barrier via a proton relay mechanism. Introduction of fluorinated Y731 exhibits expected thermodynamic trends without altering the basic mechanism. These simulations suggest that E623 influences the directionality of PCET between Y731 and Y730 and predict that mutation of E623 will impact catalysis.
    DOI:  https://doi.org/10.1021/jacs.1c02152
  46. Mol Cell Oncol. 2021 ;8(2): 1881395
    It takes three to the DNA damage response tango.
    Sapir Schlam-Babayov, Yael Ziv, Yosef Shiloh.
      The DNA damage response is robustly activated by DNA double-strand breaks and controlled by three apical protein kinases of the PI3-kinase-related protein kinase (PIKK) family: ataxia-telangiectasia, mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). Phosphoproteomic analysis reveals the relative share of these PIKKs in coordinating this network, and compensation by ATR and DNA-PK for ATM absence in the genetic disorder, ataxia-telangiectasia (A-T).
    Keywords:  ATM; Ataxia-telangiectasia; DNA damage response; PIKKs; phosphoproteomic
    DOI:  https://doi.org/10.1080/23723556.2021.1881395
  47. Semin Cell Dev Biol. 2021 Apr 07. pii: S1084-9521(21)00068-9. [Epub ahead of print]
    Genome instability from nuclear catastrophe and DNA damage.
    Anna E Mammel, Emily M Hatch.
      The nuclear envelope compartmentalizes the eukaryotic genome, provides mechanical resistance, and regulates access to the chromatin. However, recent studies have identified several conditions where the nuclear membrane ruptures during interphase, breaking down this compartmentalization leading to DNA damage, chromothripsis, and kataegis. This review discusses three major circumstances that promote nuclear membrane rupture, nuclear deformation, chromatin bridges, and micronucleation, and how each of these nuclear catastrophes results in DNA damage. In addition, we highlight recent studies that demonstrate a single chromosome missegregation can initiate a cascade of events that lead to accumulating damage and even multiple rounds of chromothripsis.
    Keywords:  Chromothripsis; Kataegis; Micronucleus; Nuclear envelope; Nuclear membrane rupture; TREX1
    DOI:  https://doi.org/10.1016/j.semcdb.2021.03.021
  48. J Med Chem. 2021 Apr 12.
    Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.
    Stefano Sainas, Marta Giorgis, Paola Circosta, Valentina Gaidano, Davide Bonanni, Agnese C Pippione, Renzo Bagnati, Alice Passoni, Yaqi Qiu, Carina Florina Cojocaru, Barbara Canepa, Alessandro Bona, Barbara Rolando, Mariia Mishina, Cristina Ramondetti, Barbara Buccinnà, Marco Piccinini, Mohammad Houshmand, Alessandro Cignetti, Enrico Giraudo, Salam Al-Karadaghi, Donatella Boschi, Giuseppe Saglio, Marco L Lolli.
      The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 μM).
    DOI:  https://doi.org/10.1021/acs.jmedchem.0c01549
  49. Biol Chem. 2021 Apr 27. 402(5): 637-644
    Transcription, translation, and DNA repair: new insights from emerging noncanonical substrates of RNA helicases.
    Matthew P Russon, Kirsten M Westerhouse, Elizabeth J Tran.
      RNA helicases are enzymes that exist in all domains of life whose canonical functions include ATP-dependent remodeling of RNA structures and displacement of proteins from ribonucleoprotein complexes (RNPs). These enzymes play roles in virtually all processes of RNA metabolism, including pre-mRNA splicing, rRNA processing, nuclear mRNA export, translation and RNA decay. Here we review emerging noncanonical substrates of RNA helicases including RNA-DNA hybrids (R-loops) and RNA and DNA G-quadruplexes and discuss their biological significance.
    Keywords:  DEAD-box helicase; DEAH-box helicase; G-quadruplexes; R-loops; RNA helicase; RNA-DNA hybrid
    DOI:  https://doi.org/10.1515/hsz-2020-0333
  50. Mol Biol Rep. 2021 Apr 15.
    DNA repair pathways and their roles in drug resistance for lung adenocarcinoma.
    Altan Kara, Aykut Özgür, Sinem Nalbantoğlu, Abdullah Karadağ.
      Lung cancer is the leading cancer type of death rate. The lung adenocarcinoma subtype is responsible for almost half of the total lung cancer deaths. Despite the improvements in cancer treatment in recent years, lung adenocarcinoma patients' overall survival rate remains poor. Immunetherapy and chemotherapy are two of the most widely used options for the treatment of cancer. Although many cancer types initially respond to these treatments, the development of resistance is inevitable. The rapid development of drug resistance mainly characterizes lung adenocarcinoma. Despite being the subject of many studies in recent years, the resistance initiation and progression mechanism is still unclear. In this review, we have examined the role of the primary DNA repair pathways (non-homologous end joining (NHEJ) pathway, homologous-recombinant repair (HR) pathway, base excision repair (BER) pathway, and nucleotide excision repair (NER) pathway and transactivation mechanisms of tumor protein 53 (TP53) in drug resistance development. This review suggests that mentioned pathways have essential roles in developing the resistance against chemotherapy and immunotherapy in lung adenocarcinoma patients.
    Keywords:  Cancer; DNA repair; Drug resistance; Lung; Lung adenocarcinoma; Oncology
    DOI:  https://doi.org/10.1007/s11033-021-06314-z
  51. Mol Cell. 2021 Apr 06. pii: S1097-2765(21)00227-6. [Epub ahead of print]
    Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance.
    Ruben Schep, Eva K Brinkman, Christ Leemans, Xabier Vergara, Robin H van der Weide, Ben Morris, Tom van Schaik, Stefano G Manzo, Daniel Peric-Hupkes, Jeroen van den Berg, Roderick L Beijersbergen, René H Medema, Bas van Steensel.
      DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.
    Keywords:  CRISPR; Chromatin; DNA repair; MMEJ; NHEJ; SSTR; double strand break; heterochromatin; nuclear lamina; reporter assay
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.032
  52. Theranostics. 2021 ;11(11): 5232-5247
    c-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation.
    Ya-Chu Tang, Jenn-Ren Hsiao, Shih-Sheng Jiang, Jang-Yang Chang, Pei-Yi Chu, Ko-Jiunn Liu, Hsun-Lang Fang, Li-Mei Lin, Huang-Hui Chen, Yen-Wen Huang, Yu-Tsen Chen, Fang-Yu Tsai, Su-Fang Lin, Yung-Jen Chuang, Ching-Chuan Kuo.
      Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear. Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2. Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.
    Keywords:  Nuclear factor erythroid 2-related factor 2 (NRF2); c-MYC; glucose-6-phosphate dehydrogenase (G6PD); head and neck squamous cell carcinoma (HNSCC); pentose phosphate pathway (PPP); transketolase (TKT)
    DOI:  https://doi.org/10.7150/thno.53417
  53. Cancer Res. 2021 Apr 16. pii: canres.0340.2021. [Epub ahead of print]
    Targeting adenosine with adenosine deaminase 2 to inhibit growth of solid tumors.
    Lin Wang, Luz M Londono, Jessica Cowell, Ozge Saatci, Mertkaya Aras, Pelin G Ersan, Sara Serra, Hong Pei, Renee Clift, Qiping Zhao, Kim B Phan, Lei Huang, Michael J LaBarre, Xiaoming Li, H Michael Shepard, Silvia Deaglio, Joel Linden, Christopher D Thanos, Ozgur Sahin, Caglar Cekic.
      Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity-dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0340
  54. Mol Cell Oncol. 2021 ;8(2): 1889348
    Structure of Rad5 provides insights into its role in tolerance to replication stress.
    Miaomiao Shen, Nalini Dhingra, Quan Wang, Xiaoxin Gong, Xin Xu, Hengyao Niu, Xiaolan Zhao, Song Xiang.
      The Rad5 family of proteins are critical genome maintenance factors, with helicase-like transcription factor (HLTF) and SNF2 histone linker PHD RING helicase (SHRPH) in humans implicated in several types of cancer. How their multiple activities coordinate has been unclear. Our recent study on Rad5 shed light on this question.
    Keywords:  HIRAN domain; HLTF; Rad5; Replication stress; protein structure
    DOI:  https://doi.org/10.1080/23723556.2021.1889348
  55. Methods Mol Biol. 2021 ;2281 289-301
    Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded DNA-Binding Proteins.
    Fernando Cerrón, Borja Ibarra.
      Optical tweezers can monitor and control the activity of individual DNA polymerase molecules in real time, providing in this way unprecedented insight into the complex dynamics and mechanochemical processes that govern their operation. Here, we describe an optical tweezers-based assay to determine at the single-molecule level the effect of single-stranded DNA-binding proteins (SSB) on the real-time replication kinetics of the human mitochondrial DNA polymerase during the synthesis of the lagging strand.
    Keywords:  DNA replication; Optical tweezers; Protein-protein interactions; Real-time kinetics; Single-molecule manipulation
    DOI:  https://doi.org/10.1007/978-1-0716-1290-3_18
  56. Mol Cancer Res. 2021 Apr 16. pii: molcanres.0791.2020. [Epub ahead of print]
    CHK2 inhibition provides a strategy to suppress hematological toxicity from PARP inhibitors.
    Zhen Xu, Cassandra J Vandenberg, Elizabeth Lieschke, Ladina Di Rago, Clare L Scott, Ian J Majewski.
      Cancer patients treated with poly (ADP-ribose) polymerase inhibitors (PARPi) experience various side effects, with hematological toxicity being most common. Short term treatment of mice with olaparib resulted in depletion of reticulocytes, B cell progenitors and immature thymocytes, whereas longer treatment induced broader myelosuppression. We performed a CRISPR/Cas9 screen that targeted DNA repair genes in Eµ-Myc pre-B lymphoma cell lines as a way to identify strategies to suppress hematological toxicity from PARPi. The screen revealed that sgRNAs targeting the serine/threonine kinase CHK2 were enriched following olaparib treatment. Genetic or pharmacological inhibition of CHK2 blunted PARPi response in lymphoid and myeloid cell lines, and in primary murine pre-B/pro-B cells. Using a Cas9 base editor, we found that blocking CHK2-mediated phosphorylation of p53 also impaired olaparib response. Our results identify the p53 pathway as a major determinant of the acute response to PARPi in normal blood cells and demonstrate that targeting CHK2 can short-circuit this response. Cotreatment with a CHK2 inhibitor did not antagonise olaparib response in ovarian cancer cell lines. Selective inhibition of CHK2 may spare blood cells from the toxic influence of PARPi and broaden the utility of these drugs. Implications: We reveal that genetic or pharmacological inhibition of Checkpoint Kinase 2 (CHK2) may offer a way to alleviate the toxic influence of PARP inhibitors in the hematological system.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0791
  57. ACS Pharmacol Transl Sci. 2021 Apr 09. 4(2): 680-686
    Adenosine Kinase Expression Determines DNA Methylation in Cancer Cell Lines.
    Amir E Wahba, Denise Fedele, Hoda Gebril, Enmar AlHarfoush, Kiran S Toti, Kenneth A Jacobson, Detlev Boison.
      DNA methylation has a major role in cancer, and its inhibitors are used therapeutically. DNA methylation depends on methyl group flux through the transmethylation pathway, which forms adenosine. We hypothesized that an adenosine kinase isoform with nuclear expression (ADK-L) determines global DNA methylation in cancer cells. We quantified ADK-L expression (Western Blot) and global DNA methylation as percent 5-methyldeoxycytidine (5mdC, LC-MS/MS) in three cancer lines (HeLa, HepG2, and U373). ADK-L expression and global DNA methylation correlated positively with the highest levels in HeLa cells compared to U373 and HepG2 cells. To determine whether ADK increases global DNA methylation and to validate its potential therapeutics, we treated HeLa cells with potent ADK inhibitors MRS4203 and MRS4380 (IC50 88 and 140 nM, respectively). Both nucleosides, but not a structurally related poor ADK inhibitor, significantly reduced global DNA methylation in HeLa cells in a concentration-dependent manner. Thus, ADK-L is a potential target for the therapeutic manipulation of DNA methylation levels in cancer.
    DOI:  https://doi.org/10.1021/acsptsci.1c00008
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