bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒04‒04
fifty-five papers selected by
Sean Rudd
Karolinska Institutet
  1. Inhibition of hUNG2 Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-fluorodeoxyuridine (FdU) and Raltitrexed (RTX) but not Fluorouracil (FU).
  2. The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.
  3. Loss of the abasic site sensor HMCES is synthetic lethal with the activity of the APOBEC3A cytosine deaminase in cancer cells.
  4. Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.
  5. SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation.
  6. Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.
  7. RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.
  8. Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.
  9. CST in maintaining genome stability: Beyond telomeres.
  10. Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer's Achilles' Heel.
  11. DNA Damage Response in Xenopus laevis Cell-Free Extracts.
  12. Genomic, Transcriptomic, and Functional Alterations in DNA Damage Response Pathways as Putative Biomarkers of Chemotherapy Response in Ovarian Cancer.
  13. Ubiquitination-mediated degradation of TRDMT1 regulates homologous recombination and therapeutic response.
  14. DNA Damage Response during Replication Correlates with CIN70 Score and Determines Survival in HNSCC Patients.
  15. Molecular Radiobiology and the Origins of the Base Excision Repair Pathway: An Historical Perspective.
  16. Functional Coupling between DNA Replication and Sister Chromatid Cohesion Establishment.
  17. Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein.
  18. Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.
  19. MYBL2 and ATM suppress replication stress in pluripotent stem cells.
  20. Repair of DNA Breaks by Break-Induced Replication.
  21. Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways.
  22. Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay.
  23. Double-strand breaks induce short scale DNA replication and damage amplification in the fully-grown mouse oocytes.
  24. LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair.
  25. Nuclear Translocation of SRPKs Is Associated with 5-FU and Cisplatin Sensitivity in HeLa and T24 Cells.
  26. CRISPR Screens in Synthetic Lethality and Combinatorial Therapies for Cancer.
  27. Breaking the paradigm: early insights from mammalian DNA breakomes.
  28. SLC6A14 and SLC38A5 Drive the Glutaminolysis and Serine-Glycine-One-Carbon Pathways in Cancer.
  29. Loss of Fer Jeopardizes Metabolic Plasticity and Mitochondrial Homeostasis in Lung and Breast Carcinoma Cells.
  30. Gene Co-Expression Analysis of Human RNASEH2A Reveals Functional Networks Associated with DNA Replication, DNA Damage Response, and Cell Cycle Regulation.
  31. Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells.
  32. 20 years of DNA Polymerase μ, the polymerase that still surprises.
  33. Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.
  34. Functions of BLM Helicase in Cells: Is It Acting Like a Double-Edged Sword?
  35. The antiproliferative effects of ataxia-telangiectasia mutated and ATM- and Rad3-related inhibitions and their enhancements with the cytotoxicity of DNA damaging agents in cholangiocarcinoma cells.
  36. New answers to the old RIDDLE: RNF168 and the DNA damage response pathway.
  37. PUMA facilitates EMI1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells.
  38. Regulation of Nuclear Mechanics and the Impact on DNA Damage.
  39. RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.
  40. Human MUS81: A Fence-Sitter in Cancer.
  41. The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin.
  42. Sensitivity of cells to ATR and CHK1 inhibitors requires hyperactivation of CDK2 rather than endogenous replication stress or ATM dysfunction.
  43. Nonhomologous end joining: new accessory factors fine tune the machinery.
  44. The Genome Stability Maintenance DNA Helicase DDX11 and Its Role in Cancer.
  45. Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase.
  46. Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity.
  47. Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.
  48. Inhibition of the DNA damage response to activate immune responses toward tumors.
  49. Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions.
  50. Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair.
  51. Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.
  52. Linking Serine/Glycine Metabolism to Radiotherapy Resistance.
  53. Deaminated purine bypass by DNA polymerase η.
  54. Tailored adjuvant gemcitabine versus 5-fluorouracil/folinic acid based on hENT1 immunohistochemical staining in resected pancreatic ductal adenocarcinoma: A biomarker stratified prospective trial.
  55. SAMHD1 … and Viral Ways around It.
  1. Mol Pharmacol. 2021 Apr 01. pii: MOLPHARM-AR-2020-000191. [Epub ahead of print]
    Inhibition of hUNG2 Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-fluorodeoxyuridine (FdU) and Raltitrexed (RTX) but not Fluorouracil (FU).
    Eric S Christenson, Anthony Gizzi, Junru Cui, Matthew Egleston, Kyle J Seamon, Michael DePasquale, Benjamin Orris, Ben H Park, James T Stivers.
      Previous shRNA knockdown studies have established that depletion of human uracil DNA glycosylase 2 (hUNG2) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here we selectively inhibit the catalytic activity of hUNG2 by lentiviral transduction of UNG inhibitor protein (UGI) into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small molecule targeting of hUNG2. In total, six of eleven colorectal lines showed large increases in FdU potency upon hUNG2 inhibition ("responsive"). This hUNG2 dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates dUTP levels, was only incrementally enhanced upon hUNG2 inhibition, suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than dU. The importance of FdU/G lesions in toxicity is supported by the observation that dT supplementation completely rescued the toxic effects of dU/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms abundant FdU/A and FdU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG2 inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested where the persistence of FU/G base pairs in the absence of hUNG2 activity elicits a unique DNA damage response in a significant fraction of colorectal lines. Significance Statement The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here we show that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme uracil DNA glycosylase (hUNG). The UNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small molecule UNG inhibitor.
    Keywords:  DNA repair; DNA replication; anticancer; nucleosides; uracil
    DOI:  https://doi.org/10.1124/molpharm.120.000191
  2. Nat Commun. 2021 03 29. 12(1): 1940
    The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.
    Man Shang, Huijie Yang, Ran Yang, Tao Chen, Yuan Fu, Yeyi Li, Xianlong Fang, Kangjian Zhang, Jianju Zhang, Hui Li, Xueping Cao, Jinfa Gu, Jianwen Xiao, Qi Zhang, Xinyuan Liu, Qiujing Yu, Ting Wang.
      Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.
    DOI:  https://doi.org/10.1038/s41467-021-22173-5
  3. PLoS Biol. 2021 Mar 31. 19(3): e3001176
    Loss of the abasic site sensor HMCES is synthetic lethal with the activity of the APOBEC3A cytosine deaminase in cancer cells.
    Josep Biayna, Isabel Garcia-Cao, Miguel M Álvarez, Marina Salvadores, Jose Espinosa-Carrasco, Marcel McCullough, Fran Supek, Travis H Stracker.
      Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.
    DOI:  https://doi.org/10.1371/journal.pbio.3001176
  4. Sci Transl Med. 2021 Mar 31. pii: eabg1168. [Epub ahead of print]13(587):
    Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.
    Blandine Roux, Camille Vaganay, Jesse D Vargas, Gabriela Alexe, Chaima Benaksas, Bryann Pardieu, Nina Fenouille, Jana M Ellegast, Edyta Malolepsza, Frank Ling, Gaetano Sodaro, Linda Ross, Yana Pikman, Amy S Conway, Yangzhong Tang, Tony Wu, Daniel J Anderson, Ronan Le Moigne, Han-Jie Zhou, Frédéric Luciano, Christina R Hartigan, Ilene Galinsky, Daniel J DeAngelo, Richard M Stone, Patrick Auberger, Monica Schenone, Steven A Carr, Josée Guirouilh-Barbat, Bernard Lopez, Mehdi Khaled, Kasper Lage, Olivier Hermine, Michael T Hemann, Alexandre Puissant, Kimberly Stegmaier, Lina Benajiba.
      The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
    DOI:  https://doi.org/10.1126/scitranslmed.abg1168
  5. Cell Rep. 2021 Mar 30. pii: S2211-1247(21)00235-7. [Epub ahead of print]34(13): 108921
    SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation.
    Elizabeth V Minten, Priya Kapoor-Vazirani, Chunyang Li, Hui Zhang, Kamakshi Balakrishnan, David S Yu.
      The breast cancer type I susceptibility protein (BRCA1) and BRCA1-associated RING domain protein I (BARD1) heterodimer promote genome integrity through pleiotropic functions, including DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1-BARD1 heterodimerization is required for their mutual stability, HR function, and role in tumor suppression; however, the upstream signaling events governing BRCA1-BARD1 heterodimerization are unclear. Here, we show that SIRT2, a sirtuin deacetylase and breast tumor suppressor, promotes BRCA1-BARD1 heterodimerization through deacetylation. SIRT2 complexes with BRCA1-BARD1 and deacetylates conserved lysines in the BARD1 RING domain, interfacing BRCA1, which promotes BRCA1-BARD1 heterodimerization and consequently BRCA1-BARD1 stability, nuclear retention, and localization to DNA damage sites, thus contributing to efficient HR. Our findings define a mechanism for regulation of BRCA1-BARD1 heterodimerization through SIRT2 deacetylation, elucidating a critical upstream signaling event directing BRCA1-BARD1 heterodimerization, which facilitates HR and tumor suppression, and delineating a role for SIRT2 in directing DSB repair by HR.
    Keywords:  BRCA1; DNA damage response; DNA repair; HR; SIRT2; acetylation; heterodimerization; homologous recombination; sirtuin; stability
    DOI:  https://doi.org/10.1016/j.celrep.2021.108921
  6. Cancers (Basel). 2021 Mar 22. pii: 1442. [Epub ahead of print]13(6):
    Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.
    Jemina Lehto, Anna Huguet Ninou, Dimitrios Chioureas, Jos Jonkers, Nina M S Gustafsson.
      The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.
    Keywords:  CX3CR1; FANCD2; Fanconi anemia pathway; KAND567; replication
    DOI:  https://doi.org/10.3390/cancers13061442
  7. Nucleic Acids Res. 2021 Mar 30. pii: gkab195. [Epub ahead of print]
    RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.
    Bodil Kavli, Tobias S Iveland, Edith Buchinger, Lars Hagen, Nina B Liabakk, Per A Aas, Tobias S Obermann, Finn L Aachmann, Geir Slupphaug.
      Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.
    DOI:  https://doi.org/10.1093/nar/gkab195
  8. Bioorg Med Chem. 2021 Feb 26. pii: S0968-0896(21)00101-2. [Epub ahead of print]37 116093
    Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.
    Adrianne Wallace-Povirk, Nian Tong, Jennifer Wong-Roushar, Carrie O'Connor, Xilin Zhou, Zhanjun Hou, Xun Bao, Gloria E Garcia, Jing Li, Seongho Kim, Charles E Dann, Larry H Matherly, Aleem Gangjee.
      We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRβ, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.
    DOI:  https://doi.org/10.1016/j.bmc.2021.116093
  9. DNA Repair (Amst). 2021 Mar 22. pii: S1568-7864(21)00060-4. [Epub ahead of print]102 103104
    CST in maintaining genome stability: Beyond telomeres.
    Xinxing Lyu, Pau Biak Sang, Weihang Chai.
      The human CST (CTC1-STN1-TEN1) complex is an RPA-like single-stranded DNA binding protein complex. While its telomeric functions have been well investigated, numerous studies have revealed that hCST also plays important roles in maintaining genome stability beyond telomeres. Here, we review and discuss recent discoveries on CST in various global genome maintenance pathways, including findings on the CST supercomplex structure, its functions in unperturbed DNA replication, stalled replication, double-strand break repair, and the ATR-CHK1 activation pathway. By summarizing these recent discoveries, we hope to offer new insights into genome maintenance mechanisms and the pathogenesis of CST mutation-associated diseases.
    Keywords:  CTC1-STN1-TEN1; DNA replication; DSB repair; Fork stalling; Genome stability; Replication stress
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103104
  10. Cancers (Basel). 2021 Mar 19. pii: 1392. [Epub ahead of print]13(6):
    Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer's Achilles' Heel.
    Daniele Caracciolo, Caterina Riillo, Maria Teresa Di Martino, Pierosandro Tagliaferri, Pierfrancesco Tassone.
      Error-prone DNA repair pathways promote genomic instability which leads to the onset of cancer hallmarks by progressive genetic aberrations in tumor cells. The molecular mechanisms which foster this process remain mostly undefined, and breakthrough advancements are eagerly awaited. In this context, the alternative non-homologous end joining (Alt-NHEJ) pathway is considered a leading actor. Indeed, there is experimental evidence that up-regulation of major Alt-NHEJ components, such as LIG3, PolQ, and PARP1, occurs in different tumors, where they are often associated with disease progression and drug resistance. Moreover, the Alt-NHEJ addiction of cancer cells provides a promising target to be exploited by synthetic lethality approaches for the use of DNA damage response (DDR) inhibitors and even as a sensitizer to checkpoint-inhibitors immunotherapy by increasing the mutational load. In this review, we discuss recent findings highlighting the role of Alt-NHEJ as a promoter of genomic instability and, therefore, as new cancer's Achilles' heel to be therapeutically exploited in precision oncology.
    Keywords:  DNA damage; LIG3; PARP1; PolQ; alternative non-homologous end joining pathway (Alt-NHEJ); error-prone DNA repair; genomic instability; synthetic lethality
    DOI:  https://doi.org/10.3390/cancers13061392
  11. Methods Mol Biol. 2021 ;2267 103-144
    DNA Damage Response in Xenopus laevis Cell-Free Extracts.
    Tomas Aparicio Casado, Jean Gautier.
      The DNA damage response (DDR) is a coordinated cellular response to a variety of insults to the genome. DDR initiates the activation of cell cycle checkpoints preventing the propagation of damaged DNA followed by DNA repair, which are both critical in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free extracts derived from Xenopus eggs as a model system to investigate signaling from DNA damage, modulation of DNA replication, checkpoint activation, and ultimately DNA repair. We outline the preparation of cell-free extracts, DNA substrates, and their subsequent use in assays aimed at understanding the cellular response to DNA damage. Cell-free extracts derived from the eggs of Xenopus laevis remain a robust and versatile system to decipher the biochemical steps underlying this essential characteristic of all cells, critical for genome stability.
    Keywords:  Chromatin; DNA damage response; DNA repair; DNA replication; S-phase; Xenopus cell-free extracts
    DOI:  https://doi.org/10.1007/978-1-0716-1217-0_8
  12. Cancers (Basel). 2021 Mar 20. pii: 1420. [Epub ahead of print]13(6):
    Genomic, Transcriptomic, and Functional Alterations in DNA Damage Response Pathways as Putative Biomarkers of Chemotherapy Response in Ovarian Cancer.
    Sweta Sharma Saha, Lucy Gentles, Alice Bradbury, Dominik Brecht, Rebecca Robinson, Rachel O'Donnell, Nicola J Curtin, Yvette Drew.
      Defective DNA damage response (DDR) pathways are enabling characteristics of cancers that not only can be exploited to specifically target cancer cells but also can predict chemotherapy response. Defective Homologous Recombination Repair (HRR) function, e.g., due to BRCA1/2 loss, is a determinant of response to platinum agents and PARP inhibitors in ovarian cancers. Most chemotherapies function by either inducing DNA damage or impacting on its repair but are generally used in the clinic unselectively. The significance of HRR and other DDR pathways in determining response to several other chemotherapy drugs is not well understood. In this study, the genomic, transcriptomic and functional analysis of DDR pathways in a panel of 14 ovarian cancer cell lines identified that defects in DDR pathways could determine response to several chemotherapy drugs. Carboplatin, rucaparib, and topotecan sensitivity were associated with functional loss of HRR (validated in 10 patient-derived primary cultures) and mismatch repair. Two DDR gene expression clusters correlating with treatment response were identified, with PARP10 identified as a novel marker of platinum response, which was confirmed in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Reduced non-homologous end-joining function correlated with increased sensitivity to doxorubicin, while cells with high intrinsic oxidative stress showed sensitivity to gemcitabine. In this era of personalised medicine, molecular/functional characterisation of DDR pathways could guide chemotherapy choices in the clinic allowing specific targeting of ovarian cancers.
    Keywords:  DNA damage repair; biomarker; chemotherapy; ovarian cancer
    DOI:  https://doi.org/10.3390/cancers13061420
  13. NAR Cancer. 2021 Mar;3(1): zcab010
    Ubiquitination-mediated degradation of TRDMT1 regulates homologous recombination and therapeutic response.
    Xiaolan Zhu, Xiangyu Wang, Wei Yan, Haibo Yang, Yufei Xiang, Fengping Lv, Yi Shi, Hong-Yu Li, Li Lan.
      The RNA methyltransferase TRDMT1 has recently emerged as a key regulator of homologous recombination (HR) in the transcribed regions of the genome, but how it is regulated and its relevance in cancer remain unknown. Here, we identified that TRDMT1 is poly-ubiquitinated at K251 by the E3 ligase TRIM28, removing TRDMT1 from DNA damage sites and allowing completion of HR. Interestingly, K251 is adjacent to G155 in the 3D structure, and the G155V mutation leads to hyper ubiquitination of TRDMT1, reduced TRDMT1 levels and impaired HR. Accordingly, a TRDMT1 G155V mutation in an ovarian cancer super responder to platinum treatment. Cells expressing TRDMT1-G155V are sensitive to cisplatin in vitro and in vivo. In contrast, high expression of TRDMT1 in patients with ovarian cancer correlates with platinum resistance. A potent TRDMT1 inhibitor resensitizes TRDMT1-high tumor cells to cisplatin. These results suggest that TRDMT1 is a promising therapeutic target to sensitize ovarian tumors to platinum therapy.
    DOI:  https://doi.org/10.1093/narcan/zcab010
  14. Cancers (Basel). 2021 Mar 10. pii: 1194. [Epub ahead of print]13(6):
    DNA Damage Response during Replication Correlates with CIN70 Score and Determines Survival in HNSCC Patients.
    Ioan T Bold, Ann-Kathrin Specht, Conrad F Droste, Alexandra Zielinski, Felix Meyer, Till S Clauditz, Adrian Münscher, Stefan Werner, Kai Rothkamm, Cordula Petersen, Kerstin Borgmann.
      Aneuploidy is a consequence of chromosomal instability (CIN) that affects prognosis. Gene expression levels associated with aneuploidy provide insight into the molecular mechanisms underlying CIN. Based on the gene signature whose expression was consistent with functional aneuploidy, the CIN70 score was established. We observed an association of CIN70 score and survival in 519 HNSCC patients in the TCGA dataset; the 15% patients with the lowest CIN70 score showed better survival (p = 0.11), but association was statistically non-significant. This correlated with the expression of 39 proteins of the major repair complexes. A positive association with survival was observed for MSH2, XRCC1, MRE11A, BRCA1, BRCA2, LIG1, DNA2, POLD1, MCM2, RAD54B, claspin, a negative for ERCC1, all related with replication. We hypothesized that expression of these factors leads to protection of replication through efficient repair and determines survival and resistance to therapy. Protein expression differences in HNSCC cell lines did not correlate with cellular sensitivity after treatment. Rather, it was observed that the stability of the DNA replication fork determined resistance, which was dependent on the ATR/CHK1-mediated S-phase signaling cascade. This suggests that it is not the expression of individual DNA repair proteins that causes therapy resistance, but rather a balanced expression and coordinated activation of corresponding signaling cascades.
    Keywords:  CIN70 score; Chk1 inhibition; HNSCC; chromosomal instability (CIN); radiosensitization; radiotherapy; replication stress
    DOI:  https://doi.org/10.3390/cancers13061194
  15. Int J Radiat Biol. 2021 Mar 31. 1-40
    Molecular Radiobiology and the Origins of the Base Excision Repair Pathway: An Historical Perspective.
    Susan S Wallace.
      PURPOSE: To demonstrate how the search by the Molecular Radiobiologists for enzymes that could recognize and remove DNA damage produced by ionizing radiation was intertwined with the development of the Base Excision Repair pathway.CONCLUSION: The Base Excision Repair pathway repairs the vast majority of radiation-induced DNA damages including base damages, alkali labile lesions, and single strand breaks. It turns out that Base Excision Repair actually evolved to repair some thirty to forty thousand endogenous lesions formed in each of our cells every day. Thus, this process is extremely efficient and accordingly, at relatively low doses of radiation, the single lesions repaired by base excision repair result in few lethal or mutagenic events. This efficiency is a double-edged sword since ionizing radiation-induced hydroxyl radicals produced along the radiation track form both bistranded and tandem clustered lesions in DNA. These damages are recognized by the efficient Base Excision Repair enzymes, which, during attempted repair, lead to double strand breaks and/or multiple lesions that can collapse replication forks. Double strand breaks and other complex or clustered lesions formed by ionizing radiation present distinct challenges to DNA repair systems compared to the relative ease and efficiency by which isolated lesions are repaired.
    Keywords:  Base damages; Clustered DNA lesions; DNA glycosylases; DNA repair; Radiation damages in DNA; single strand breaks; sites of base loss
    DOI:  https://doi.org/10.1080/09553002.2021.1908639
  16. Int J Mol Sci. 2021 Mar 10. pii: 2810. [Epub ahead of print]22(6):
    Functional Coupling between DNA Replication and Sister Chromatid Cohesion Establishment.
    Ana Boavida, Diana Santos, Mohammad Mahtab, Francesca M Pisani.
      Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.
    Keywords:  DNA replication; cell cycle; cohesin; replication proteins; sister chromatid cohesion
    DOI:  https://doi.org/10.3390/ijms22062810
  17. Structure. 2021 Mar 25. pii: S0969-2126(21)00080-0. [Epub ahead of print]
    Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein.
    Matthew Day, Sarah Parry-Morris, Jack Houghton-Gisby, Antony W Oliver, Laurence H Pearl.
      CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN, a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR.
    Keywords:  DNA damage signaling; checkpoints; protein kinase mechanisms
    DOI:  https://doi.org/10.1016/j.str.2021.03.007
  18. Science. 2021 Apr 02. 372(6537): 91-94
    Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.
    Dylan A Reid, Patrick J Reed, Johannes C M Schlachetzki, Ioana I Nitulescu, Grace Chou, Enoch C Tsui, Jeffrey R Jones, Sahaana Chandran, Ake T Lu, Claire A McClain, Jean H Ooi, Tzu-Wen Wang, Addison J Lana, Sara B Linker, Anthony S Ricciardulli, Shong Lau, Simon T Schafer, Steve Horvath, Jesse R Dixon, Nasun Hah, Christopher K Glass, Fred H Gage.
      Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.
    DOI:  https://doi.org/10.1126/science.abb9032
  19. EMBO Rep. 2021 Mar 28. e51120
    MYBL2 and ATM suppress replication stress in pluripotent stem cells.
    Daniel Blakemore, Nuria Vilaplana-Lopera, Ruba Almaghrabi, Elena Gonzalez, Miriam Moya, Carl Ward, George Murphy, Agnieszka Gambus, Eva Petermann, Grant S Stewart, Paloma García.
      Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand-fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.
    Keywords:  B-MYB; DNA damage; ESCs; iPSC; origin firing
    DOI:  https://doi.org/10.15252/embr.202051120
  20. Annu Rev Biochem. 2021 Apr 01.
    Repair of DNA Breaks by Break-Induced Replication.
    Z W Kockler, B Osia, R Lee, K Musmaker, A Malkova.
      Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-081420-095551
  21. Int J Mol Sci. 2021 Mar 07. pii: 2698. [Epub ahead of print]22(5):
    Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways.
    Vladimir Shafirovich, Nicholas E Geacintov.
      The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifications are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical carcinogens or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER factor XPC-RAD23B to these lesions.
    Keywords:  DNA damage; base excision repair; guanine oxidation; nucleotide excision repair; oxidative stress; reactive oxygen species
    DOI:  https://doi.org/10.3390/ijms22052698
  22. Methods Mol Biol. 2021 ;2267 57-71
    Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay.
    Stephanie Biber, Lisa Wiesmüller.
      DNA replication is a fundamental process of life. Any perturbation of this process by endogenous or exogenous factors impacts on genomic stability and thereby on carcinogenesis. More recently, the replication machinery has been discovered as an interesting target for cancer therapeutic strategies. Given its high biological and clinical relevance, technologies for the analysis of DNA replication have attracted major attention. The so-called DNA fiber spreading technique is a powerful tool to directly monitor various aspects of the replication process by sequential incorporation of halogenated nucleotide analogs which later can be fluorescently stained and analyzed. This chapter outlines the use of the DNA fiber spreading technique for the analysis of replication dynamics and replication structures.
    Keywords:  DNA Fiber Spreading Assay; DNA Replication; DNA damage response; Origin firing; Replication Fork Stalling
    DOI:  https://doi.org/10.1007/978-1-0716-1217-0_4
  23. Genetics. 2021 Apr 01. pii: iyab054. [Epub ahead of print]
    Double-strand breaks induce short scale DNA replication and damage amplification in the fully-grown mouse oocytes.
    Jun-Yu Ma, Xie Feng, Feng-Yun Xie, Sen Li, Lei-Ning Chen, Shi-Ming Luo, Shen Yin, Xiang-Hong Ou.
      Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR are initiated haven't been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2´-deoxyuridine (EdU). These ssBIRs could only be induced in the fully-grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully-grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
    Keywords:  Rad51; break-induced replication; multi-invasion; oocyte
    DOI:  https://doi.org/10.1093/genetics/iyab054
  24. Clin Transl Med. 2021 Mar;11(3): e341
    LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair.
    Lifeng Chen, Jing Hou, Xiangyu Zeng, Qiang Guo, Min Deng, Jake A Kloeber, Xinyi Tu, Fei Zhao, Zheming Wu, Jinzhou Huang, Kuntian Luo, Wootae Kim, Zhenkun Lou.
      PARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR- deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high-selective and effective leucine-rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers.
    Keywords:  HR; LRRK2 inhibitor; PARP inhibitor; Rad51
    DOI:  https://doi.org/10.1002/ctm2.341
  25. Cells. 2021 Mar 30. pii: 759. [Epub ahead of print]10(4):
    Nuclear Translocation of SRPKs Is Associated with 5-FU and Cisplatin Sensitivity in HeLa and T24 Cells.
    Ioanna Sigala, Maria Koutroumani, Anastasia Koukiali, Thomas Giannakouros, Eleni Nikolakaki.
      Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core and anchors the kinases in the cytoplasm is a unique structural feature of SRPKs. Here, we report that exposure of HeLa and T24 cells to DNA damage inducers triggers the nuclear translocation of SRPK1 and SRPK2. Furthermore, we show that nuclear SRPKs did not protect from, but on the contrary, mediated the cytotoxic effects of genotoxic agents, such as 5-fluorouracil (5-FU) and cisplatin. Confirming previous data showing that the kinase activity is essential for the entry of SRPKs into the nucleus, SRPIN340, a selective SRPK1/2 inhibitor, blocked the nuclear accumulation of the kinases, thus diminishing the cytotoxic effects of the drugs. ATR/ATM-dependent phosphorylation of threonine 326 and serine 408 in the spacer domain of SRPK1 was essential for the redistribution of the kinase to the nucleus. Substitution of either of these two residues to alanine or inhibition of ATR/ATM kinase activity abolished nuclear localization of SRPK1 and conferred tolerance to 5-FU treatment. These findings suggest that SRPKs may play an important role in linking cellular signaling to DNA damage in eukaryotic cells.
    Keywords:  5-FU; ATM; ATR; SR protein kinases; SRPK1; SRPK2; cisplatin; drug resistance; fixation for immunofluorescence
    DOI:  https://doi.org/10.3390/cells10040759
  26. Cancers (Basel). 2021 Mar 30. pii: 1591. [Epub ahead of print]13(7):
    CRISPR Screens in Synthetic Lethality and Combinatorial Therapies for Cancer.
    Laia Castells-Roca, Eudald Tejero, Benjamín Rodríguez-Santiago, Jordi Surrallés.
      Cancer is a complex disease resulting from the accumulation of genetic dysfunctions. Tumor heterogeneity causes the molecular variety that divergently controls responses to chemotherapy, leading to the recurrent problem of cancer reappearance. For many decades, efforts have focused on identifying essential tumoral genes and cancer driver mutations. More recently, prompted by the clinical success of the synthetic lethality (SL)-based therapy of the PARP inhibitors in homologous recombinant deficient tumors, scientists have centered their novel research on SL interactions (SLI). The state of the art to find new genetic interactions are currently large-scale forward genetic CRISPR screens. CRISPR technology has rapidly evolved to be a common tool in the vast majority of laboratories, as tools to implement CRISPR screen protocols are available to all researchers. Taking advantage of SLI, combinatorial therapies have become the ultimate model to treat cancer with lower toxicity, and therefore better efficiency. This review explores the CRISPR screen methodology, integrates the up-to-date published findings on CRISPR screens in the cancer field and proposes future directions to uncover cancer regulation and individual responses to chemotherapy.
    Keywords:  CRISPR screen; cancer therapeutic resistance; combinatorial therapy; synthetic lethality
    DOI:  https://doi.org/10.3390/cancers13071591
  27. FEBS J. 2021 Mar 31.
    Breaking the paradigm: early insights from mammalian DNA breakomes.
    Xanita Saayman, Fumiko Esashi.
      DNA double-strand breaks (DSBs) can result from both exogenous and endogenous sources, and are potentially toxic lesions to the human genome. If improperly repaired, DSBs can threaten genome integrity and contribute to premature aging, neurodegenerative disorders and carcinogenesis. Through decades of work on genome stability, it has become evident that certain regions of the genome are inherently more prone to breakage than others, known as genome instability hotspots. Recent advancements in sequencing-based technologies now enable the profiling of genome-wide distributions of DSBs, also known as breakomes, to systematically map these instability hotspots. Here, we review the application of these technologies and their implications for our current understanding of the genomic regions most likely to drive genome instability. These breakomes ultimately highlight both new and established breakage hotspots including actively transcribed regions, loop boundaries and early-replicating regions of the genome. Further, these breakomes challenge the paradigm that DNA breakage primarily occurs in hard-to-replicate regions. With these advancements, we begin to gain insights into the biological mechanisms both invoking and protecting against genome instability.
    Keywords:  DNA breaks; genome instability; next generation sequencing; replication; transcription
    DOI:  https://doi.org/10.1111/febs.15849
  28. Pharmaceuticals (Basel). 2021 Mar 04. pii: 216. [Epub ahead of print]14(3):
    SLC6A14 and SLC38A5 Drive the Glutaminolysis and Serine-Glycine-One-Carbon Pathways in Cancer.
    Tyler Sniegowski, Ksenija Korac, Yangzom D Bhutia, Vadivel Ganapathy.
      The glutaminolysis and serine-glycine-one-carbon pathways represent metabolic reactions that are reprogramed and upregulated in cancer; these pathways are involved in supporting the growth and proliferation of cancer cells. Glutaminolysis participates in the production of lactate, an oncometabolite, and also in anabolic reactions leading to the synthesis of fatty acids and cholesterol. The serine-glycine-one-carbon pathway is involved in the synthesis of purines and pyrimidines and the control of the epigenetic signature (DNA methylation, histone methylation) in cancer cells. Methionine is obligatory for most of the methyl-transfer reactions in the form of S-adenosylmethionine; here, too, the serine-glycine-one-carbon pathway is necessary for the resynthesis of methionine following the methyl-transfer reaction. Glutamine, serine, glycine, and methionine are obligatory to fuel these metabolic pathways. The first three amino acids can be synthesized endogenously to some extent, but the need for these amino acids in cancer cells is so high that they also have to be acquired from extracellular sources. Methionine is an essential amino acid, thus making it necessary for cancer cells to acquire this amino acid solely from the extracellular milieu. Cancer cells upregulate specific amino acid transporters to meet this increased demand for these four amino acids. SLC6A14 and SLC38A5 are the two transporters that are upregulated in a variety of cancers to mediate the influx of glutamine, serine, glycine, and methionine into cancer cells. SLC6A14 is a Na+/Cl- -coupled transporter for multiple amino acids, including these four amino acids. In contrast, SLC38A5 is a Na+-coupled transporter with rather restricted specificity towards glutamine, serine, glycine, and methionine. Both transporters exhibit unique functional features that are ideal for the rapid proliferation of cancer cells. As such, these two amino acid transporters play a critical role in promoting the survival and growth of cancer cells and hence represent novel, hitherto largely unexplored, targets for cancer therapy.
    Keywords:  SLC6A14 and SLC38A5; amino acid transporters; cancer-specific metabolism; glutamine addiction; oncometabolites; one-carbon metabolism
    DOI:  https://doi.org/10.3390/ph14030216
  29. Int J Mol Sci. 2021 Mar 25. pii: 3387. [Epub ahead of print]22(7):
    Loss of Fer Jeopardizes Metabolic Plasticity and Mitochondrial Homeostasis in Lung and Breast Carcinoma Cells.
    Linoy Mehazri, Sally Shpungin, Shai Bel, Uri Nir.
      Metabolic plasticity is a hallmark of the ability of metastatic cancer cells to survive under stressful conditions. The intracellular Fer kinase is a selective constituent of the reprogramed mitochondria and metabolic system of cancer cells. In the current work, we deciphered the modulatory roles of Fer in the reprogrammed metabolic systems of metastatic, lung (H358), non-small cell lung cancer (NSCLC), and breast (MDA-MB-231), triple-negative breast cancer (TNBC), carcinoma cells. We show that H358 cells devoid of Fer (H358ΔFer), strictly depend on glucose for their proliferation and growth, and fail to compensate for glucose withdrawal by oxidizing and metabolizing glutamine. Furthermore, glucose deficiency caused increased reactive oxygen species (ROS) production and induction of a DNA damage response (DDR), accompanied by the onset of apoptosis and attenuated cell-cycle progression. Analysis of mitochondrial function revealed impaired respiratory and electron transport chain (ETC) complex 1 (comp. I) activity in the Fer-deficient H358ΔFer cells. This was manifested by decreased levels of NAD+ and ATP and relatively low abundance of tricarboxylic acid (TCA) cycle metabolites. Impaired electron transport chain comp. I activity and dependence on glucose were also confirmed in Fer-deficient, MDA-MB-231ΔFer cells. Although both H358ΔFer and MDA-MB-231ΔFer cells showed a decreased aspartate level, this seemed to be compensated by the predominance of pyrimidines synthesis over the urea cycle progression. Notably, absence of Fer significantly impeded the growth of H358ΔFer and MDA-MB-231ΔFer xenografts in mice provided with a carb-deficient, ketogenic diet. Thus, Fer plays a key role in the sustention of metabolic plasticity of malignant cells. In compliance with this notion, targeting Fer attenuates the progression of H358 and MDA-MB-231 tumors, an effect that is potentiated by a glucose-restrictive diet.
    Keywords:  Fer; Mitochondrial homeostasis; metabolic plasticity; non-small cell lung cancer; triple-negative breast cancer
    DOI:  https://doi.org/10.3390/ijms22073387
  30. Biology (Basel). 2021 Mar 13. pii: 221. [Epub ahead of print]10(3):
    Gene Co-Expression Analysis of Human RNASEH2A Reveals Functional Networks Associated with DNA Replication, DNA Damage Response, and Cell Cycle Regulation.
    Stefania Marsili, Ailone Tichon, Deepali Kundnani, Francesca Storici.
      Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits, RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings.
    Keywords:  RNASEH; RNASEH2A; cancer; cell cycle; co-expression; genotype-tissue expression
    DOI:  https://doi.org/10.3390/biology10030221
  31. Life Sci Alliance. 2021 Jun;pii: e202101023. [Epub ahead of print]4(6):
    Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells.
    Jone Michelena, Stefania Pellegrino, Vincent Spegg, Matthias Altmeyer.
      DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1's binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.
    DOI:  https://doi.org/10.26508/lsa.202101023
  32. FEBS J. 2021 Mar 31.
    20 years of DNA Polymerase μ, the polymerase that still surprises.
    Dipayan Ghosh, Sathees C Raghavan.
      DNA polymerases are important enzymes involved in DNA replication and repair. Based on sequence homology, DNA polymerases have been grouped into distinct families, which are A, B, X and Y. The Pol X family consists of four members: Pol λ, μ, β and terminal transferase or TdT. Members of the family X are involved in base excision repair, nonhomologous end joining (NHEJ) and V(D)J recombination. One of the most interesting pol X family members is DNA polymerase μ, discovered back in 2000. Subsequent studies established the importance of Pol μ as a repair polymerase in NHEJ and its interactions with the other proteins of the NHEJ machinery. Pol μ has a number of interesting properties which sets it apart from the other known DNA polymerases, including its ability to synthesize DNA from an unpaired primer terminus as well in the complete absence of a template strand (terminal transferase activity). Another standout property of Pol μ is its reduced ability to discriminate between ribonucleotides and deoxyribonucleotides and its ability to utilize both ribonucleotides and deoxyribonucleotides as substrates during the gap filling stage of NHEJ. In this review, we provide a brief overview of Pol μ in double-strand break repair and the current knowledge on its various functional aspects.
    Keywords:  DNA Double-strand break; DSB repair; Error-prone DNA polymerase; Genomic instability; NHEJ; Nonhomologous DNA End joining; Pol Lambda; Pol X family
    DOI:  https://doi.org/10.1111/febs.15852
  33. Int J Mol Sci. 2021 Mar 27. pii: 3481. [Epub ahead of print]22(7):
    Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.
    Afaf Eladl, Yudai Yamaoki, Shoko Hoshina, Haruka Horinouchi, Keiko Kondo, Shou Waga, Takashi Nagata, Masato Katahira.
      Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1413-511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413-511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413-511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.
    Keywords:  DNA replication; G-quadruplex; NMR; origin recognition complex; replication origin; structure
    DOI:  https://doi.org/10.3390/ijms22073481
  34. Front Genet. 2021 ;12 634789
    Functions of BLM Helicase in Cells: Is It Acting Like a Double-Edged Sword?
    Ekjot Kaur, Ritu Agrawal, Sagar Sengupta.
      DNA damage repair response is an important biological process involved in maintaining the fidelity of the genome in eukaryotes and prokaryotes. Several proteins that play a key role in this process have been identified. Alterations in these key proteins have been linked to different diseases including cancer. BLM is a 3'-5' ATP-dependent RecQ DNA helicase that is one of the most essential genome stabilizers involved in the regulation of DNA replication, recombination, and both homologous and non-homologous pathways of double-strand break repair. BLM structure and functions are known to be conserved across many species like yeast, Drosophila, mouse, and human. Genetic mutations in the BLM gene cause a rare, autosomal recessive disorder, Bloom syndrome (BS). BS is a monogenic disease characterized by genomic instability, premature aging, predisposition to cancer, immunodeficiency, and pulmonary diseases. Hence, these characteristics point toward BLM being a tumor suppressor. However, in addition to mutations, BLM gene undergoes various types of alterations including increase in the copy number, transcript, and protein levels in multiple types of cancers. These results, along with the fact that the lack of wild-type BLM in these cancers has been associated with increased sensitivity to chemotherapeutic drugs, indicate that BLM also has a pro-oncogenic function. While a plethora of studies have reported the effect of BLM gene mutations in various model organisms, there is a dearth in the studies undertaken to investigate the effect of its oncogenic alterations. We propose to rationalize and integrate the dual functions of BLM both as a tumor suppressor and maybe as a proto-oncogene, and enlist the plausible mechanisms of its deregulation in cancers.
    Keywords:  BLM helicase; RecQ helicase; neoplastic transformation; oncogene; tumor suppressor
    DOI:  https://doi.org/10.3389/fgene.2021.634789
  35. J Pharm Pharmacol. 2021 Mar 01. 73(1): 40-51
    The antiproliferative effects of ataxia-telangiectasia mutated and ATM- and Rad3-related inhibitions and their enhancements with the cytotoxicity of DNA damaging agents in cholangiocarcinoma cells.
    Benchamart Moolmuang, Mathuros Ruchirawat.
      OBJECTIVE: To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines.METHODS: Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis.
    KEY FINDINGS: The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells.
    CONCLUSIONS: Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.
    Keywords:  ATM inhibition; ATR inhibition; DNA damage response; DNA damaging agents; cholangiocarcinoma
    DOI:  https://doi.org/10.1093/jpp/rgaa050
  36. FEBS J. 2021 Apr 02.
    New answers to the old RIDDLE: RNF168 and the DNA damage response pathway.
    Jessica Kelliher, Gargi Ghosal, Justin Wai Chung Leung.
      The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination plays an integral role in regulating cellular processes including DNA damage signaling and repair. The ubiquitin E3 ligase RNF168 is essential in assembling a cohort of DNA repair proteins at the damaged chromatin via its enzymatic activity. RNF168 ubiquitinates histone H2A(X) at the N-terminus and generates a specific docking scaffold for ubiquitin binding motif-containing proteins. The regulation of RNF168 at damaged chromatin and the mechanistic implication in the recruitment of DNA repair proteins to the damaged sites remains an area of active investigation. Here, we review the function and regulation of RNF168 in the context of ubiquitin-mediated DNA damage signaling and repair. We will also discuss the unanswered questions that require further investigation and how understanding RNF168 targeting specificity could benefit the therapeutic development for cancer treatment.
    Keywords:  DNA double strand break; DNA repair; RIDDLE syndrome; RIDDLIN; ubiquitin
    DOI:  https://doi.org/10.1111/febs.15857
  37. Signal Transduct Target Ther. 2021 Mar 31. 6(1): 129
    PUMA facilitates EMI1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells.
    Jin Wook Kang, Zhiyan Zhan, Guangzhen Ji, Youzhou Sang, Daohong Zhou, Yanxin Li, Haizhong Feng, Tao Cheng.
      Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration, while preventing cell transformation after damage. Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation (IR), while without promoting tumorigenesis in the long-term survivors. This finding suggests that PUMA (p53 upregulated modulator of apoptosis) may have a function other than regulates apoptosis. Here, we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells (PSCs) and immortalized hematopoietic progenitor cells (HPCs) after IR. We found that PUMA-deficient PSCs and HPCs exhibited a significant higher double-strand break (DSB) DNA repair activity via Rad51-mediated homologous recombination (HR). This is because PUMA can be associated with early mitotic inhibitor 1 (EMI1) and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation, thereby inhibiting Rad51 nuclear translocation and HR DNA repair. Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.
    DOI:  https://doi.org/10.1038/s41392-021-00510-w
  38. Int J Mol Sci. 2021 Mar 20. pii: 3178. [Epub ahead of print]22(6):
    Regulation of Nuclear Mechanics and the Impact on DNA Damage.
    Ália Dos Santos, Christopher P Toseland.
      In eukaryotic cells, the nucleus houses the genomic material of the cell. The physical properties of the nucleus and its ability to sense external mechanical cues are tightly linked to the regulation of cellular events, such as gene expression. Nuclear mechanics and morphology are altered in many diseases such as cancer and premature ageing syndromes. Therefore, it is important to understand how different components contribute to nuclear processes, organisation and mechanics, and how they are misregulated in disease. Although, over the years, studies have focused on the nuclear lamina-a mesh of intermediate filament proteins residing between the chromatin and the nuclear membrane-there is growing evidence that chromatin structure and factors that regulate chromatin organisation are essential contributors to the physical properties of the nucleus. Here, we review the main structural components that contribute to the mechanical properties of the nucleus, with particular emphasis on chromatin structure. We also provide an example of how nuclear stiffness can both impact and be affected by cellular processes such as DNA damage and repair.
    Keywords:  DNA; DNA damage; chromatin; cytoskeleton; lamin; mechanics; nucleus
    DOI:  https://doi.org/10.3390/ijms22063178
  39. FASEB J. 2021 May;35(5): e21476
    RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.
    Iryna Kolosenko, Oksana Goroshchuk, Linda Vidarsdottir, Ann-Charlotte Björklund, Steven F Dowdy, Caroline Palm-Apergi.
      Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.
    Keywords:  Polo-like kinase; Prodrugs; RNA interference; cytarabine; pediatric acute myeloid leukemia
    DOI:  https://doi.org/10.1096/fj.202002454RR
  40. Front Cell Dev Biol. 2021 ;9 657305
    Human MUS81: A Fence-Sitter in Cancer.
    Sisi Chen, Xinwei Geng, Madiha Zahra Syeda, Zhengming Huang, Chao Zhang, Songmin Ying.
      MUS81 complex, exhibiting endonuclease activity on specific DNA structures, plays an influential part in DNA repair. Research has proved that MUS81 is dispensable for embryonic development and cell viability in mammals. However, an intricate picture has emerged from studies in which discrepant gene mutations completely alter the role of MUS81 in human cancers. Here, we review the recent understanding of how MUS81 functions in tumors with distinct genetic backgrounds and discuss the potential therapeutic strategies targeting MUS81 in cancer.
    Keywords:  DNA damage response; cancer therapy; chromosomal instability; endonuclease; human MUS81
    DOI:  https://doi.org/10.3389/fcell.2021.657305
  41. Life (Basel). 2021 Mar 24. pii: 267. [Epub ahead of print]11(4):
    The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin.
    Serge Bauwens, Liudmyla Lototska, Stephane Koundrioukoff, Michelle Debatisse, Jing Ye, Eric Gilson, Aaron Mendez-Bermudez.
      Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.
    Keywords:  ORC; TRF2; heterochromatin; pericentromeric DNA; replication; telomeres
    DOI:  https://doi.org/10.3390/life11040267
  42. Sci Rep. 2021 Mar 29. 11(1): 7077
    Sensitivity of cells to ATR and CHK1 inhibitors requires hyperactivation of CDK2 rather than endogenous replication stress or ATM dysfunction.
    Jennifer P Ditano, Katelyn L Donahue, Laura J Tafe, Charlotte F McCleery, Alan Eastman.
      DNA damage activates cell cycle checkpoint proteins ATR and CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of ATR (ATRi) and CHK1 (CHK1i) enhance damage-induced cell death. Intriguingly, both CHK1i and ATRi alone elicit cytotoxicity in some cell lines. Sensitivity has been attributed to endogenous replications stress, but many more cell lines are sensitive to ATRi than CHK1i. Endogenous activation of the DNA damage response also did not correlate with drug sensitivity. Sensitivity correlated with the appearance of γH2AX, a marker of DNA damage, but without phosphorylation of mitotic markers, contradicting suggestions that the damage is due to premature mitosis. Sensitivity to ATRi has been associated with ATM mutations, but dysfunction in ATM signaling did not correlate with sensitivity. CHK1i and ATRi circumvent replication stress by reactivating stalled replicons, a process requiring a low threshold activity of CDK2. In contrast, γH2AX induced by single agent ATRi and CHK1i requires a high threshold activity CDK2. Hence, phosphorylation of different CDK2 substrates is required for cytotoxicity induced by replication stress plus ATRi/CHK1i as compared to their single agent activity. In summary, sensitivity to ATRi and CHK1i as single agents is elicited by premature hyper-activation of CDK2.
    DOI:  https://doi.org/10.1038/s41598-021-86490-x
  43. Trends Genet. 2021 Mar 27. pii: S0168-9525(21)00054-8. [Epub ahead of print]
    Nonhomologous end joining: new accessory factors fine tune the machinery.
    Dipayan Ghosh, Sathees C Raghavan.
      Nonhomologous DNA end joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways in eukaryotes. The well-known critical proteins involved in NHEJ include Ku70/80, DNA-PKcs, Artemis, DNA pol λ/μ, DNA ligase IV-XRCC4, and XLF. Recent studies have added a number of new proteins to the NHEJ repertoire namely paralog of XRCC4 and XLF (PAXX), modulator of retroviral infection (MRI)/ cell cycle regulator of NHEJ (CYREN), transactivation response DNA-binding protein (TARDBP) of 43 kDa (TDP-43), intermediate filament family orphan (IFFO1), ERCC excision repair 6 like 2 (ERCC6L2), and RNase H2. PAXX acts as a stabilizing factor for the main NHEJ components. MRI/CYREN seems to play a dual role stimulating NHEJ in the G1 phase of the cell cycle, while inhibiting the pathway in the S and G2 phases. TDP-43 can recruit the ligase IV-XRCC4 complex to the DSB sites and stimulate ligation in neuronal cells. RNase H2 excises out the ribonucleotides inserted during repair by DNA polymerase μ/TdT. This review provides a brief glimpse into how these new partners were discovered and their contribution to the mechanism and regulation of NHEJ.
    Keywords:  DSB repair; NHEJ machinery; cell cycle; double-strand break; end joining; genomic instability; homologous recombination
    DOI:  https://doi.org/10.1016/j.tig.2021.03.001
  44. Genes (Basel). 2021 Mar 10. pii: 395. [Epub ahead of print]12(3):
    The Genome Stability Maintenance DNA Helicase DDX11 and Its Role in Cancer.
    Mohammad Mahtab, Ana Boavida, Diana Santos, Francesca M Pisani.
      DDX11/ChlR1 is a super-family two iron-sulfur cluster containing DNA helicase with roles in DNA replication and sister chromatid cohesion establishment, and general chromosome architecture. Bi-allelic mutations of the DDX11 gene cause a rare hereditary disease, named Warsaw breakage syndrome, characterized by a complex spectrum of clinical manifestations (pre- and post-natal growth defects, microcephaly, intellectual disability, heart anomalies and sister chromatid cohesion loss at cellular level) in accordance with the multifaceted, not yet fully understood, physiological functions of this DNA helicase. In the last few years, a possible role of DDX11 in the onset and progression of many cancers is emerging. Herein we summarize the results of recent studies, carried out either in tumoral cell lines or in xenograft cancer mouse models, suggesting that DDX11 may have an oncogenic role. The potential of DDX11 DNA helicase as a pharmacological target for novel anti-cancer therapeutic interventions, as inferred from these latest developments, is also discussed.
    Keywords:  DDX11/ChlR1; DNA helicase; cancer; genome stability; human papillomavirus; long noncoding RNAs; oncogene; sister chromatid cohesion; tumor suppressor gene
    DOI:  https://doi.org/10.3390/genes12030395
  45. Cells. 2021 Mar 04. pii: 550. [Epub ahead of print]10(3):
    Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase.
    Indra A Shaltiel, Alba Llopis, Melinda Aprelia, Rob Klompmaker, Apostolos Menegakis, Lenno Krenning, René H Medema.
      Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.
    Keywords:  CDK4; CDK6; CDKs; DNA damage; G1; cell cycle; checkpoint; recovery
    DOI:  https://doi.org/10.3390/cells10030550
  46. Bioorg Chem. 2021 Mar 10. pii: S0045-2068(21)00190-5. [Epub ahead of print]110 104813
    Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity.
    Cheng Peng, Yu-Hsuan Li, Chao-Wu Yu, Ze-Hua Cheng, Jia-Rong Liu, Jui-Ling Hsu, Ling-Wei Hsin, Chen-Tsung Huang, Hsueh-Fen Juan, Ji-Wang Chern, Yi-Sheng Cheng.
      MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.
    Keywords:  8-Oxo-dGTP; Isothermal titration calorimetry; Oxidative DNA damage; Ultra-high-throughput drug screening
    DOI:  https://doi.org/10.1016/j.bioorg.2021.104813
  47. Cell Rep. 2021 Mar 30. pii: S2211-1247(21)00220-5. [Epub ahead of print]34(13): 108906
    Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.
    Antonio Marsella, Elisa Gobbini, Corinne Cassani, Renata Tisi, Elda Cannavo, Giordano Reginato, Petr Cejka, Maria Pia Longhese.
      The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.
    Keywords:  MRX; Mre11-Rad50; Rad53; Rif2; Sae2; Tel1; checkpoint; double-strand breaks
    DOI:  https://doi.org/10.1016/j.celrep.2021.108906
  48. FEBS J. 2021 Apr 02.
    Inhibition of the DNA damage response to activate immune responses toward tumors.
    Sheetal Sharma, Mrinal Srivastava.
      Cancer immunotherapy represents a very encouraging mode of treatment for cancer where one's immune system is utilized to eliminate tumor cells. Wayne et al. explore inhibition of DNA damage response (DDR) pathways with small molecule inhibitors as a means to prime cells with immune response. These findings suggest that a one-size-fits-all approach cannot be used when harnessing immune response via DDR inhibitors and genotoxic agents, which are required ultimately for the success of immunotherapy.
    Keywords:  ATRi; Chk1i; DNA damage response; Wee1i; immunotherapy; innate immune response
    DOI:  https://doi.org/10.1111/febs.15836
  49. Proc Natl Acad Sci U S A. 2021 Apr 06. pii: e2100240118. [Epub ahead of print]118(14):
    Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions.
    Akil Hamza, Leanne Amitzi, Lina Ma, Maureen R M Driessen, Nigel J O'Neil, Philip Hieter.
      Genetic screens can identify synthetic lethal (SL) interactions and uncover potential anticancer therapeutic targets. However, most SL screens have utilized knockout or knockdown approaches that do not accurately mimic chemical inhibition of a target protein. Here, we test whether missense mutations can be utilized as a model for a type of protein inhibition that creates a dominant gain-of-function cytotoxicity. We expressed missense mutations in the FEN1 endonuclease and the replication-associated helicase, CHL1, that inhibited enzymatic activity but retained substrate binding, and found that these mutations elicited a dominant SL phenotype consistent with the generation of cytotoxic protein-DNA or protein-protein intermediates. Genetic screens with nuclease-defective hFEN1 and helicase-deficient yCHL1 captured dominant SL interactions, in which ectopic expression of the mutant form, in the presence of the wild-type form, caused SL in specific mutant backgrounds. Expression of nuclease-defective hFEN1 in yeast elicited DNA binding-dependent dominant SL with homologous recombination mutants. In contrast, dominant SL interactions with helicase-deficient yCHL1 were observed in spindle-associated, Ctf18-alternative replication factor C (Ctf18-RFC) clamp loader complex, and cohesin mutant backgrounds. These results highlight the different mechanisms underlying SL interactions that occur in the presence of an inhibited form of the target protein and point to the utility of modeling trapping mutations in pursuit of more clinically relevant SL interactions.
    Keywords:  Chl1 helicase; DNA trapping; Fen1 endonuclease; dominant missense mutations; dominant synthetic lethality
    DOI:  https://doi.org/10.1073/pnas.2100240118
  50. Genome Res. 2021 Apr 02. pii: gr.271155.120. [Epub ahead of print]
    Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair.
    Vinay Tripuraneni, Gonen Memisoglu, Heather K MacAlpine, Trung Q Tran, Wei Zhu, Alexander J Hartemink, James E Haber, David M MacAlpine.
      We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ~8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.
    DOI:  https://doi.org/10.1101/gr.271155.120
  51. PLoS One. 2021 ;16(3): e0248941
    Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.
    Mona Al-Mugotir, Jeffrey J Lovelace, Joseph George, Mika Bessho, Dhananjaya Pal, Lucas Struble, Carol Kolar, Sandeep Rana, Amarnath Natarajan, Tadayoshi Bessho, Gloria E O Borgstahl.
      Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.
    DOI:  https://doi.org/10.1371/journal.pone.0248941
  52. Cancers (Basel). 2021 Mar 10. pii: 1191. [Epub ahead of print]13(6):
    Linking Serine/Glycine Metabolism to Radiotherapy Resistance.
    Anaís Sánchez-Castillo, Marc Vooijs, Kim R Kampen.
      The activation of de novo serine/glycine biosynthesis in a subset of tumors has been described as a major contributor to tumor pathogenesis, poor outcome, and treatment resistance. Amplifications and mutations of de novo serine/glycine biosynthesis enzymes can trigger pathway activation; however, a large group of cancers displays serine/glycine pathway overexpression induced by oncogenic drivers and unknown regulatory mechanisms. A better understanding of the regulatory network of de novo serine/glycine biosynthesis activation in cancer might be essential to unveil opportunities to target tumor heterogeneity and therapy resistance. In the current review, we describe how the activation of de novo serine/glycine biosynthesis in cancer is linked to treatment resistance and its implications in the clinic. To our knowledge, only a few studies have identified this pathway as metabolic reprogramming of cancer cells in response to radiation therapy. We propose an important contribution of de novo serine/glycine biosynthesis pathway activation to radioresistance by being involved in cancer cell viability and proliferation, maintenance of cancer stem cells (CSCs), and redox homeostasis under hypoxia and nutrient-deprived conditions. Current approaches for inhibition of the de novo serine/glycine biosynthesis pathway provide new opportunities for therapeutic intervention, which in combination with radiotherapy might be a promising strategy for tumor control and ultimately eradication. Further research is needed to gain molecular and mechanistic insight into the activation of this pathway in response to radiation therapy and to design sophisticated stratification methods to select patients that might benefit from serine/glycine metabolism-targeted therapies in combination with radiotherapy.
    Keywords:  DNA repair; PHGDH; PSAT1; PSPH; SHMT; cancer; hypoxia; radiotherapy; redox homeostasis; resistance; serine and glycine metabolism
    DOI:  https://doi.org/10.3390/cancers13061191
  53. Biochem J. 2021 Apr 16. 478(7): 1309-1313
    Deaminated purine bypass by DNA polymerase η.
    Antonina Andreeva.
      A recent work by Jung and colleagues (Biochem J.477, 4797-4810) provides an explanation of how DNA polymerase η replicates through deaminated purine bases such as xanthine and hypoxanthine. This commentary discusses the crystal structures of the polymerase η complexes that implicate the role of tautomerism in the bypass of these DNA lesions.
    DOI:  https://doi.org/10.1042/BCJ20200989
  54. Pancreatology. 2021 Mar 04. pii: S1424-3903(21)00072-7. [Epub ahead of print]
    Tailored adjuvant gemcitabine versus 5-fluorouracil/folinic acid based on hENT1 immunohistochemical staining in resected pancreatic ductal adenocarcinoma: A biomarker stratified prospective trial.
    Dong Woo Shin, Jong-Chan Lee, Jaihwan Kim, Yoo-Seok Yoon, Ho-Seong Han, Haeryoung Kim, Jin-Hyeok Hwang.
      BACKGROUND: The study aimed to evaluate the clinical outcomes of tailored adjuvant chemotherapy according to human equilibrative nucleoside transporter 1 (hENT1) expression in resected pancreatic ductal adenocarcinoma (PDA).METHODS: Patients who underwent pancreatectomy for PDA were enrolled prospectively. According to intra-tumoral hENT1 expression, the high hENT1 (≥50%) group received gemcitabine and the low hENT1 (<50%) group received 5-fluorouracil plus folinic acid (5-FU/FA). The propensity score-matched control consisted of patients who received hENT1-independent adjuvant chemotherapy. The primary outcome was recurrence free survival (RFS) and the secondary outcomes were overall survival (OS) and toxicities.
    RESULTS: Between May 2015 and June 2017, we enrolled 44 patients with resected PDA. During a median follow-up period of 28.5 months, the intention-to-treat population showed much longer median RFS [22.9 (95% CI, 11.3-34.5) vs. 10.9 (95% CI, 6.9-14.9) months, P = 0.043] and median OS [36.2 (95% CI, 26.5-45.9) vs. 22.1 (95% CI, 17.7-26.6) months, P = 0.001] compared to the controls. Among 5 patients in the low hENT1 group who discontinued treatment, 2 patients receiving 5-FU/FA discontinued treatment due to drug toxicities (febrile neutropenia and toxic epidermal necrolysis).
    CONCLUSION: Tailored adjuvant chemotherapy based on hENT1 staining provides excellent clinical outcomes among patients with resected PDA.
    CLINICAL TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT02486497.
    Keywords:  Gemcitabine; Human equilibrative nucleoside transporter 1; Pancreatic cancer
    DOI:  https://doi.org/10.1016/j.pan.2021.02.022
  55. Viruses. 2021 Mar 02. pii: 395. [Epub ahead of print]13(3):
    SAMHD1 … and Viral Ways around It.
    Janina Deutschmann, Thomas Gramberg.
      The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.
    Keywords:  HIV; SAMHD1; Vpx; dNTP hydrolase; herpesviruses; restriction factor; viral antagonism; viral interference; viral kinases
    DOI:  https://doi.org/10.3390/v13030395
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