bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒10‒25
fifty-nine papers selected by
Sean Rudd
Karolinska Institutet

  1. J Clin Invest. 2020 Oct 20. pii: 139929. [Epub ahead of print]
    Huang F, Huffman K, Wang Z, Wang X, Li K, Cai F, Yang C, Cai L, Shih TS, Zacharias LG, Chung AS, Yang Q, Chalishazar MD, Ireland AS, Stewart CA, Cargill KR, Girard L, Liu Y, Ni M, Xu J, Wu X, Zhu H, Drapkin BJ, Byers LA, Oliver TG, Gazdar A, Minna J, DeBerardinis R.
      MYC stimulates both metabolism and protein synthesis, but it is unknown how cells coordinate these complementary programs. Previous work reported that in a subset of small cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here we demonstrated that primary MYCHigh human SCLC tumors also contain abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH links the metabolic and protein synthesis outputs of oncogenic MYC. Co-expression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA Polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functions as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant, MYCHigh SCLC.
    Keywords:  Intermediary metabolism; Lung cancer; Metabolism; Oncogenes; Oncology
  2. Mol Cancer Ther. 2020 Oct 21. pii: molcanther.0259.2020. [Epub ahead of print]
    Shorstova T, Su J, Zhao T, Dahabieh M, Leibovitch M, De Sa Tavares Russo M, Avizonis D, Rajkumar S, Watson IR, Del Rincón SV, Miller WH, Foulkes WD, Witcher M.
      Small Cell Carcinoma of the Ovary, Hypercalcemic Type is a rare but often lethal cancer which is diagnosed at a median age of 24 years. Optimal management of patients is not well defined and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo. Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass Spectrometry, coupled with knockdown of newly-found targets including thymidylate synthetase, revealed that the repression of a panel of proteins involved in nucleotide synthesis, underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.
  3. Proc Natl Acad Sci U S A. 2020 Oct 23. pii: 202009899. [Epub ahead of print]
    Wei X, Yang J, Adair SJ, Ozturk H, Kuscu C, Lee KY, Kane WJ, O'Hara PE, Liu D, Demirlenk YM, Habieb AH, Yilmaz E, Dutta A, Bauer TW, Adli M.
      Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.
    Keywords:  CRISPR screening; cancer genomics and epigenomics; combinatorial drugs targets; pancreatic cancer; synthetic lethality
  4. Nat Commun. 2020 Oct 23. 11(1): 5362
    Gao M, Guo G, Huang J, Kloeber JA, Zhao F, Deng M, Tu X, Kim W, Zhou Q, Zhang C, Yin P, Luo K, Lou Z.
      Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.
  5. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30177-4. [Epub ahead of print]93 102928
    Whitaker AM, Freudenthal BD.
      DNA polymerase β (Pol β) is an essential mammalian enzyme involved in the repair of DNA damage during the base excision repair (BER) pathway. In hopes of faithfully restoring the coding potential to damaged DNA during BER, Pol β first uses a lyase activity to remove the 5'-deoxyribose phosphate moiety from a nicked BER intermediate, followed by a DNA synthesis activity to insert a nucleotide triphosphate into the resultant 1-nucleotide gapped DNA substrate. This DNA synthesis activity of Pol β has served as a model to characterize the molecular steps of the nucleotidyl transferase mechanism used by mammalian DNA polymerases during DNA synthesis. This is in part because Pol β has been extremely amenable to X-ray crystallography, with the first crystal structure of apoenzyme rat Pol β published in 1994 by Dr. Samuel Wilson and colleagues. Since this first structure, the Wilson lab and colleagues have published an astounding 267 structures of Pol β that represent different liganded states, conformations, variants, and reaction intermediates. While many labs have made significant contributions to our understanding of Pol β, the focus of this article is on the long history of the contributions from the Wilson lab. We have chosen to highlight select seminal Pol β structures with emphasis on the overarching contributions each structure has made to the field.
  6. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30163-4. [Epub ahead of print]93 102915
    Shibata A, Jeggo PA.
      In mammalian cells, the mediator protein, 53BP1, exerts distinct impacts on the repair of DNA double strand breaks (DSBs) depending on the setting, for example whether the DSBs arise at telomeres or during replication or class switch recombination. Here, we focus on two roles of 53BP1 in response to ionising radiation (IR)-induced DSBs (IR-DSBs). Canonical DNA non-homologous end-joining (c-NHEJ) is the major DSB repair pathway with homologous recombination (HR) contributing to DSB repair in S/G2 phase. ATM signalling promotes histone modifications and protein assembly in the DSB vicinity, which can be visualised as irradiation induced foci (IRIF). 53BP1 assembles at DSBs in a complex manner involving the formation of nano-domains. In G1 and G2 phase, X- or gamma-ray induced DSBs are repaired with biphasic kinetics. 70-80 % of DSBs are repaired with fast kinetics in both cell cycle phases by c-NHEJ; the remaining DSBs are repaired with slower kinetics in G2 phase via HR and in G1 by a specialised form of c-NHEJ termed Artemis and resection-dependent c-NHEJ, due to a specific requirement for the nuclease, Artemis and resection factors. 53BP1 is essential for the repair of DSBs rejoined with slow kinetics in G1 and G2 phase. This 53BP1 function requires its tandem BRCT domain and interaction with NBS1. As a distinct function, 53BP1 suppresses resection during both HR and Artemis and resection-dependent c-NHEJ. This latter role requires RIF1 and is counteracted by BRCA1. 53BP1 appears to be dispensable for the rejoining of the fast c-NHEJ repair process.
    Keywords:  53BP1; Chromatin; DNA double-strand break repair; Homologous recombination; Ionising radiation; Non-homologous end-joining
  7. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30179-8. [Epub ahead of print]93 102930
    Saville KM, Clark J, Wilk A, Rogers GD, Andrews JF, Koczor CA, Sobol RW.
      The enzymes of the base excision repair (BER) pathway form DNA lesion-dependent, transient complexes that vary in composition based on the type of DNA damage. These protein sub-complexes facilitate substrate/product handoff to ensure reaction completion so as to avoid accumulation of potentially toxic DNA repair intermediates. However, in the mammalian cell, additional signaling molecules are required to fine-tune the activity of the BER pathway enzymes and to facilitate chromatin/histone reorganization for access to the DNA lesion for repair. These signaling enzymes include nicotinamide adenine dinucleotide (NAD+) dependent poly(ADP-ribose) polymerases (PARP1, PARP2) and class III deacetylases (SIRT1, SIRT6) that comprise a key PARP-NAD-SIRT axis to facilitate the regulation and coordination of BER in the mammalian cell. Here, we briefly describe the key nodes in the BER pathway that are regulated by this axis and highlight the cellular and organismal variation in NAD+ bioavailability that can impact BER signaling potential. We discuss how cellular NAD+ is required for BER to maintain genome stability and to mount a robust cellular response to DNA damage. Finally, we consider the dependence of BER on the PARP-NAD-SIRT axis for BER protein complex assembly.
    Keywords:  Base excision repair; Nicotinamide adenine dinucleotide; PARP1; Sirtuin
  8. PLoS One. 2020 ;15(10): e0235103
    Zuilkoski CM, Skibbens RV.
      PCNA sliding clamp binds factors through which histone deposition, chromatin remodeling, and DNA repair are coupled to DNA replication. PCNA also directly binds Eco1/Ctf7 acetyltransferase, which in turn activates cohesins and establishes cohesion between nascent sister chromatids. While increased recruitment thus explains the mechanism through which elevated levels of chromatin-bound PCNA rescue eco1 mutant cell growth, the mechanism through which PCNA instead worsens cohesin mutant cell growth remains unknown. Possibilities include that elevated levels of long-lived chromatin-bound PCNA reduce either cohesin deposition onto DNA or cohesin acetylation. Instead, our results reveal that PCNA increases the levels of both chromatin-bound cohesin and cohesin acetylation. Beyond sister chromatid cohesion, PCNA also plays a critical role in genomic stability such that high levels of chromatin-bound PCNA elevate genotoxic sensitivities and recombination rates. At a relatively modest increase of chromatin-bound PCNA, however, fork stability and progression appear normal in wildtype cells. Our results reveal that even a moderate increase of PCNA indeed sensitizes cohesin mutant cells to DNA damaging agents and in a process that involves the DNA damage response kinase Mec1(ATR), but not Tel1(ATM). These and other findings suggest that PCNA mis-regulation results in genome instabilities that normally are resolved by cohesin. Elevating levels of chromatin-bound PCNA may thus help target cohesinopathic cells linked that are linked to cancer.
  9. Curr Genet. 2020 Oct 20.
    Ononye OE, Sausen CW, Bochman ML, Balakrishnan L.
      PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell.
    Keywords:  DNA repair; DNA replication; G4 resolvase; Lysine acetylation; NuA4 (Esa1); Pif1 helicase; Rpd3
  10. Mol Ther. 2020 Oct 19. pii: S1525-0016(20)30550-5. [Epub ahead of print]
    Saha A, Zhao S, Chen Z, Georgiou G, Stone E, Kidane D, DiGiovanni J.
      Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest forms of cancer with very few available therapeutic options. We previously reported that an engineered human enzyme, Cyst(e)inase, that degrades L-cysteine and cystine, inhibits growth of multiple cancer cells including PDAC both in vitro and in vivo. Here, we show that Cyst(e)inase treatment leads to increased clustered oxidative DNA damage, DNA single strand breaks, apurinic/apyrimidinic sites and DNA double strand breaks (DSBs) in PDAC cells sensitive to intracellular depletion of L-Cys that is associated with reduced survival. BRCA2 deficient PDAC cells exhibited increased DSBs and enhanced sensitivity to Cyst(e)inase. Blocking a second antioxidant pathway (thioredoxin/thioredoxin reductase) using Auranofin or inhibiting DNA repair using the PARP inhibitor, Olaparib, led to significant increases in DSBs following Cyst(e)inase treatment in all PDAC cells examined. Cyst(e)inase plus Olaparib also synergistically inhibited growth of sensitive and resistant PDAC cells in both xenograft and allograft tumor models. Collectively, these results demonstrate an important role for oxidative DNA damage and ultimately DNA DSBs in the anticancer action of Cyst(e)inase. The data further show the potential for combining agents that target alternate antioxidant pathways or by targeting DNA repair pathways or genetic liabilities in DNA repair pathways to enhance the therapeutic action of Cyst(e)inase for PDAC. Cyst(e)inase is an engineered human enzyme shown to inhibit growth of multiple cancer cells. In this study, Saha et al. found that combining agents that target alternate antioxidant pathways or by targeting DNA repair pathways or genetic liabilities in DNA repair pathways improves therapeutic action of Cyst(e)inase in pancreatic cancer.
  11. Cancer Drug Resist. 2020 ;3 572-585
    Miller AL, Garcia PL, Gamblin TL, Vance RB, Yoon KJ.
      Aim: Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces durable remissions of this tumor type. We developed three PDAC patient-derived xenograft (PDX) models with gemcitabine resistance (gemR) acquired in vivo, with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies.Methods: Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly. Tumors initially responded, but regrew on treatment and were designated gemR. We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance [ribonucleotide reductase subunit M1 (RRM1), RRM2, human concentrative nucleoside transporter 1 (hCNT1), human equilibrative nucleoside transporter 1 (hENT1), cytidine deaminase (CDA), and deoxycytidine kinase (dCK)] in gemR and respective gemcitabine-naive parental tumors.
    Results: Parental and gemR tumors did not differ in tumor cell morphology, amount of tumor-associated stroma, or expression of stem cell markers. No consistent pattern of expression of the six gemR marker proteins was observed among the models. Increases in RRM1 and CDA were consistent with in vitro-derived gemR models. However, rather than the expected decreases of hCNT1, hENT1, and dCK, gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.
    Conclusion: These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo. The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo. Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models.
    Keywords:  cytidine deaminase; deoxycytidine kinase; gemcitabine resistance; human concentrative nucleoside transporter 1; human equilibrative nucleoside transporter 1; patient-derived xenograft; ribonucleotide reductase subunit M1; ribonucleotide reductase subunit M2
  12. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30157-9. [Epub ahead of print]93 102909
    Kong M, Beckwitt EC, Van Houten B.
      DNA repair is a highly dynamic process in which the actual damage recognition process occurs through an amazing dance between the DNA duplex containing the lesion and the DNA repair proteins. Single molecule investigations have revealed that DNA repair proteins solve the speed-stability paradox, of rapid search versus stable complex formation, by conformational changes induced in both the damaged DNA and the repair proteins. Using Rad4, XPA, PARP1, APE1, OGG1 and UV-DDB as examples, we have discovered how these repair proteins limit their travel on DNA, once a lesion is encountered through a process of anomalous diffusion. We have also observed how PARP1 and APE1, as well as UV-DDB and OGG1 or APE1, co-localize dynamically at sites near DNA damage. This review highlights how our group has greatly benefited from our productive collaborations with Sam Wilson's research group.
    Keywords:  APE1; Base excision repair; Nucleotide excision repair; OGG1; PARP1; Single molecule; UV-DDB; XPA; XPC
  13. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30156-7. [Epub ahead of print]93 102908
    Sallmyr A, Rashid I, Bhandari SK, Naila T, Tomkinson AE.
      To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair.
    Keywords:  DNA joining; Okazaki fragments; alternative end joining; base excision repair; non-homologous end joining; nucleotide excision repair; single strand break reapir
  14. Nucleic Acids Res. 2020 Oct 17. pii: gkaa897. [Epub ahead of print]
    Eki R, She J, Parlak M, Benamar M, Du KP, Kumar P, Abbas T.
      DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.
  15. J Biol Chem. 2020 Oct 23. pii: jbc.RA120.014179. [Epub ahead of print]
    Tang LY, Thomas A, Zhou M, Zhang YE.
      Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities.  We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with ring finger protein 20 (RNF20) in shaping chromatin landscape and preserving genomic stability.  However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and mass spectrometry analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at S384 by ataxia telangiectasia mutated (ATM) serine/threonine kinase and this phosphorylation is required for its interaction with RNF20.  We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly.  More importantly, mouse embryonic fibroblasts (MEFs) expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared to those expressing wild type SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ATM-induced phosphorylation at S384 constitutes a negative feedback loop that regulates DSB repair.
    Keywords:  ATM; DNA damage response; DNA repair; H2B ubiquitination; RNF20; SMURF2; histone modification; phosphorylation; ubiquitylation (ubiquitination)
  16. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30167-1. [Epub ahead of print]93 102919
    Harrod A, Lane KA, Downs JA.
      Mammalian cells possess multiple closely related SWI/SNF chromatin remodelling complexes. These complexes have been implicated in the cellular response to DNA double strand breaks (DSBs). Evidence suggests that SWI/SNF complexes contribute to successful repair via both the homologous recombination and non-homologous end joining pathways. In addition, repressing transcription near DSBs is dependent on SWI/SNF activity. Understanding these roles is important because SWI/SNF complexes are frequently dysregulated in cancer, and DNA DSB repair defects have the potential to be therapeutically exploited. In this graphical review, we summarise what is known about SWI/SNF contribution to DNA DSB responses in mammalian cells and provide an overview of the SWI/SNF-encoding gene alteration spectrum in human cancers.
    Keywords:  BAF; BRG1; Cancer; Chromatin remodeling; DNA repair; Double strand breaks; SMARCA4; SWI/SNF
  17. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30178-6. [Epub ahead of print]93 102929
    Sertic S, Quadri R, Lazzaro F, Muzi-Falconi M.
      Exonuclease 1 (EXO1) is an evolutionarily well conserved exonuclease. Its ability to resect DNA in the 5'-3' direction has been extensively characterized and shown to be implicated in several genomic DNA metabolic processes such as replication stress response, double strand break repair, mismatch repair, nucleotide excision repair and telomere maintenance. While the processing of DNA is critical for its repair, an excessive nucleolytic activity can lead to secondary lesions, increased genome instability and alterations in cellular functions. It is thus clear that different regulatory layers must be in effect to keep DNA degradation under control. Regulatory events that modulate EXO1 activity have been reported to act at different levels. Here we summarize the different post-translational modifications (PTMs) that affect EXO1 and discuss the implications of PTMs for EXO1 activities and how this regulation may be associated to cancer development.
    Keywords:  Cancer; DNA damage; Exonuclease 1; Post-translational modifications
  18. Cancer Lett. 2020 Oct 16. pii: S0304-3835(20)30501-2. [Epub ahead of print]497 54-65
    Song J, Yu J, Jeong LS, Lee SK.
      Most nucleoside anticancer drugs show a primary resistance to p53-deficient or p53-mutated cancer cells and are limited in the clinic to the treatment of hematological malignancies. However, 2'-fluoro-4'-seleno-ara-C (F-Se-Ara-C), a new generation of cytarabine (Ara-C) analogs, exhibited potent antitumor activity against the p53-deficient prostate cancer cell line PC-3. The distinct activity of F-Se-Ara-C was achieved by targeting the synthetic lethal interaction between p53 and mitogen-activated protein kinase-activated protein kinase-2 (MK2). MK2 is a checkpoint effector for DNA damage responses to drive cell cycle arrest and DNA repair in p53-deficient cancer cells. Therefore, targeting MK2 may be an effective therapeutic strategy that induces apoptosis for cancers deficient in p53. F-Se-Ara-C effectively induced anti-prostate cancer activity in vitro and in vivo by inhibition of MK2 activation in p53-deficient prostate cancer cells. Moreover, combining F-Se-Ara-C with cabozantinib, an anticancer drug currently in clinical use, induced synergistic antitumor activity in p53-deficient prostate cancer cells. Taken together, these data show that F-Se-Ara-C may become great anticancer drug candidate with its unique mechanism of action for overcoming the apoptotic resistance of p53-deficient cells by targeting the synthetic lethal interaction.
    Keywords:  A nucleoside analog F–Se-Ara-C; Mitogen-activated protein kinase-activated protein kinase-2 (MK2) inhibition; Mitotic catastrophe; Synthetic lethality; p53-deficient cancer cells
  19. Nature. 2020 Oct 21.
    Sedlackova H, Rask MB, Gupta R, Choudhary C, Somyajit K, Lukas J.
      Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.
  20. Cells. 2020 Oct 19. pii: E2324. [Epub ahead of print]9(10):
    Chatterjee A, Eshwaran R, Poschet G, Lomada S, Halawa M, Wilhelm K, Schmidt M, Hammes HP, Wieland T, Feng Y.
      Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.
    Keywords:  Ang-2; O-GlcNAcylation; OGA; UDP-GlcNAc; nucleoside diphosphate kinase B
  21. Proc Natl Acad Sci U S A. 2020 Oct 19. pii: 202014176. [Epub ahead of print]
    Layer JV, Debaize L, Van Scoyk A, House NC, Brown AJ, Liu Y, Stevenson KE, Hemann M, Roberts SA, Price BD, Weinstock DM, Day TA.
      Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.
    Keywords:  POLD1; POLD2; nonhomologous end-joining; polymerase δ; translocation
  22. Essays Biochem. 2020 Oct 23. pii: EBC20200012. [Epub ahead of print]
    Newman JA, Gileadi O.
      Helicases are enzymes that use the energy derived from ATP hydrolysis to catalyze the unwinding of DNA or RNA. The RecQ family of helicases is conserved through evolution from prokaryotes to higher eukaryotes and plays important roles in various DNA repair pathways, contributing to the maintenance of genome integrity. Despite their roles as general tumor suppressors, there is now considerable interest in exploiting RecQ helicases as synthetic lethal targets for the development of new cancer therapeutics. In this review, we summarize the latest developments in the structural and mechanistic study of RecQ helicases and discuss their roles in various DNA repair pathways. Finally, we consider the potential to exploit RecQ helicases as therapeutic targets and review the recent progress towards the development of small molecules targeting RecQ helicases as cancer therapeutics.
    Keywords:  DNA damage; DNA synthesis and repair; protein structure; synthetic lethality
  23. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30181-6. [Epub ahead of print]93 102932
    Kaminski AM, Bebenek K, Pedersen LC, Kunkel TA.
      DNA polymerase μ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps arising as intermediates in the process of V(D)J recombination and during processing of accidental double strand breaks. Pol μ is the only known template-dependent polymerase that can repair non-complementary DSBs with unpaired 3´primer termini. Here we review the unique properties of Pol μ that allow it to productively engage such a highly unstable substrate to generate a nick that can be sealed by DNA Ligase IV.
    Keywords:  DNA double strand break repair; DNA polymerase μ; Family X DNA polymerases; Nonhomologous end-joining
  24. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30166-X. [Epub ahead of print]93 102918
    Huang Y, Li GM.
      DNA mismatch repair (MMR) maintains genomic stability primarily by correcting replication errors. Defects in MMR lead to cancers and cause resistance to many chemotherapeutic drugs. Emerging evidence reveals that MMR is coupled with replication and precisely regulated in the context of chromatin; strikingly, tumors defective in MMR are highly responsive to immune checkpoint blockade therapy. As a tribute to Dr. Samuel Wilson for his many scientific contributions to the field of DNA repair and his leadership as Editor-in-Chief of the journal DNA Repair, we summarize recent developments in research on MMR at the chromatin level, its implications for tumorigenesis, and its therapeutic potential.
    Keywords:  Cancer therapy; Chromatin structure; Mimsatch repair; Replication fork
  25. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30160-9. [Epub ahead of print]93 102912
    Lai Y, Beaver JM, Laverde E, Liu Y.
      Trinucleotide repeat (TNR) instability is the cause of over 40 human neurodegenerative diseases and certain types of cancer. TNR instability can result from DNA replication, repair, recombination, and gene transcription. Emerging evidence indicates that DNA base damage and base excision repair (BER) play an active role in regulating somatic TNR instability. These processes may potentially modulate the onset and progression of TNR-related diseases, given that TNRs are hotspots of DNA base damage that are present in mammalian cells with a high frequency. In this review, we discuss the recent advances in our understanding of the molecular mechanisms underlying BER-mediated TNR instability. We initially discuss the roles of the BER pathway and locations of DNA base lesions in TNRs and their interplay with non-B form DNA structures in governing repeat instability. We then discuss how the coordinated activities of BER enzymes can modulate a balance between the removal and addition of TNRs to regulate somatic TNR instability. We further discuss how this balance can be disrupted by the crosstalk between BER and DNA mismatch repair (MMR) machinery resulting in TNR expansion. Finally, we suggest future directions regarding BER-mediated somatic TNR instability and its association with TNR disease prevention and treatment.
    Keywords:  DNA base damage; DNA base excision repair; DNA repair crosstalk; Trinucleotide repeat instability
  26. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30172-5. [Epub ahead of print]93 102923
    Ijsselsteijn R, Jansen JG, de Wind N.
      Canonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are involved in the suppression of mutagenic, and the induction of protective, responses to various types of DNA damage. In this review we describe these non-canonical roles of MMR at different lesion types. Loss of non-canonical MMR gene functions may have important ramifications for the prevention, development and treatment of colorectal cancer associated with inherited MMR gene defects in Lynch syndrome. This graphical review pays tribute to Samuel H. Wilson. Sam not only made seminal contributions to understanding base excision repair, particularly with respect to structure-function relationships in DNA polymerase β but also, as Editor of DNA Repair, has maintained a high standard of the journal.
    Keywords:  Colorectal cancer; DNA damage responses; DNA mismatch repair; Mutagenesis
  27. EMBO J. 2020 Oct 19. e104500
    Glousker G, Briod AS, Quadroni M, Lingner J.
      The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.
    Keywords:  DNA damage response; POT1; R-loops; homologous recombination; telomeric proteome
  28. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30154-3. [Epub ahead of print]93 102906
    Sassa A, Odagiri M.
      8-Oxo-7,8-dihydroguanine (8-oxoG) is the major base damage in the genomic DNA by exposure to reactive oxygen species. Organisms have evolved various DNA repair mechanisms, such as base excision repair (BER) and nucleotide excision repair (NER), to protect the cellular genome from these mutagenic DNA lesions. The efficiency and capacity of BER and NER mechanisms can be modulated by the local sequence and structural contexts in which 8-oxoG is located. This graphical review summarizes the biochemical and structural studies that have provided insights into the impact of the microenvironment around the site of the lesion on oxidative DNA damage repair.
    Keywords:  8-Oxo-7,8-dihydroguanine; Base excision repair; Clustered DNA lesions; DNA glycosylase; Nucleotide excision repair; Ribonucleotide
  29. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30165-8. [Epub ahead of print]93 102917
    London RE.
      X-ray cross complementing protein 1 (XRCC1) is a DNA repair scaffold that supports base excision repair and single strand break repair, and is also a participant in other repair pathways. It also serves as an important co-transporter for several other repair proteins, including aprataxin and PNKP-like factor (APLF), and DNA Ligase 3α (LIG3). By combining highly specialized regions that help to organize specific repair functions with recruitment of additional enzymes whose contribution is dependent on the details of the damaged site, XRCC1 is able to handle an expanded range of problems that may arise as the repair progresses or in connection with other repair pathways with which it interfaces. This review discusses the interplay between these functions and considers some possible interactions that underlie its reported repair activities.
    Keywords:  Base excision repair; DNA repair; Single strand break repair; X-ray cross complementing protein 1; XRCC1
  30. Mol Cancer Ther. 2020 Oct 20. pii: molcanther.0550.2020. [Epub ahead of print]
    McDonald G, Chubukov V, Coco J, Truskowski K, Narayanaswamy R, Choe S, Steadman M, Artin E, Padyana AK, Jin L, Ronseaux S, Locuson C, Fan ZP, Erdmann T, Mann A, Hayes S, Fletcher M, Nellore K, Rao SS, Subramanya H, Kunnam SR, Panigrahi SK, Anthony T, Gopinath S, Sui Z, Nagaraja N, Dang L, Lenz G, Hurov J, Biller SA, Murtie J, Marks KM, Ulanet DB.
      Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA damage response pathways.
  31. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30164-6. [Epub ahead of print]93 102916
    Gustafson MA, Sullivan ED, Copeland WC.
      Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria. The mitochondrial genome is susceptible to damage by both endogenous and exogenous sources. Missense mutations to the nuclear genes encoding the core mtDNA replisome (POLG, POLG2, TWNK, and SSBP1) cause changes to the biochemical functions of their protein products. These protein variants can damage mtDNA and perturb oxidative phosphorylation. Ultimately, these mutations cause a diverse set of diseases that can affect virtually every system in the body. Here, we briefly review the mechanisms of mtDNA damage and the clinical consequences of disease variants of the core mtDNA replisome.
    Keywords:  Mitochondrial DNA; Mitochondrial disease; Mutagenesis; POLG; POLG2; Replication; SSBP1; TWNK
  32. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30161-0. [Epub ahead of print]93 102913
    Sasanuma H, Yamada S, Tsuda M, Takeda S.
      Radiotherapy kills malignant cells by generating double-strand breaks (DSBs). Ionizing- radiation (IR) generates "dirty" DSBs, which associates with blocking chemical adducts at DSB ends. Homologous-directed repair (HDR) efficiently removes IR-induced blocking adducts from both 3' and 5' ends of DSBs. Nonhomologous end-joining (NHEJ) rejoins virtually all DSBs in G1 phase and ∼80 % of DSBs in G2 phase. However, DNA Ligase IV, an essential NHEJ factor, rejoins only "clean" ligatable DSBs carrying 3'-OH and 5'-phosphate DSB ends but not dirty DSBs. Recent studies have identified a number of nucleases, especially the MRE11 nuclease, as key factors performing the removal of blocking chemical adducts to restore clean ligatable DSBs for subsequent NHEJ. This restoration, but not subsequent NHEJ, is the rate-limiting step in the rejoining of IR- induced DSBs. This review describes repair factors that contribute to the restoration of clean DSBs before NHEJ.
    Keywords:  Blocking adducts; DNA double-strand breaks; Etoposide; Ionizing radiation; MRE11; Non-homologous end joining; Radiotherapy
  33. Nucleic Acids Res. 2020 Oct 19. pii: gkaa845. [Epub ahead of print]
    Stewart JA, Schauer G, Bhagwat AS.
      The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.
  34. Nat Commun. 2020 Oct 23. 11(1): 5379
    Dodd T, Botto M, Paul F, Fernandez-Leiro R, Lamers MH, Ivanov I.
      Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.
  35. Biochem Soc Trans. 2020 Oct 21. pii: BST20200713. [Epub ahead of print]
    Sengupta D, Mukhopadhyay A, Sengupta K.
      Lamins are type V intermediate filament proteins which are ubiquitously present in all metazoan cells providing a platform for binding of chromatin and related proteins, thereby serving a wide range of nuclear functions including DNA damage repair. Altered expression of lamins in different subtypes of cancer is evident from researches worldwide. But whether cancer is a consequence of this change or this change is a consequence of cancer is a matter of future investigation. However changes in the expression levels of lamins is reported to have direct or indirect association with cancer progression or have regulatory roles in common neoplastic symptoms like higher nuclear deformability, increased genomic instability and reduced susceptibility to DNA damaging agents. It has already been proved that loss of A type lamin positively regulates cathepsin L, eventually leading to degradation of several DNA damage repair proteins, hence impairing DNA damage repair pathways and increasing genomic instability. It is established in ovarian cancer, that the extent of alteration in nuclear morphology can determine the degree of genetic changes and thus can be utilized to detect low to high form of serous carcinoma. In this review, we have focused on ovarian cancer which is largely caused by genomic alterations in the DNA damage response pathways utilizing proteins like RAD51, BRCA1, 53BP1 which are regulated by lamins. We have elucidated the current understanding of lamin expression in ovarian cancer and its implications in the regulation of DNA damage response pathways that ultimately result in telomere deformation and genomic instability.
    Keywords:  DNA damage response; genome integrity; lamins; ovarian cancer
  36. Trends Mol Med. 2020 Oct 16. pii: S1471-4914(20)30258-6. [Epub ahead of print]
    Burdak-Rothkamm S, Rothkamm K.
    Keywords:  PARP inhibitor; alternative DNA repair pathways; homologous recombination repair
  37. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30159-2. [Epub ahead of print]93 102911
    Lavrik OI.
      Poly(ADP-ribosyl)ation is one of immediate cellular responses to DNA damage and is catalyzed by poly(ADP-ribose) polymerases (PARPs). PARP1 is a well-known regulator of DNA repair. Another member of this family, PARP2, was discovered later. The study of PARP1 and PARP2 functions started a long time ago, and special attention has been given to the role of these enzymes in base excision repair. This review summarizes my lab's data on the functions of PARP1 and PARP2 in base excision repair as well as the results obtained in the course of our collaboration with Dr. Samuel H. Wilson.
    Keywords:  APE1; Base excision repair; PARP1; PARP2; Poly(ADP-ribose); Poly(ADP-ribosyl)ation; Polβ; XRCC1
  38. Cell Rep. 2020 Oct 20. pii: S2211-1247(20)31278-X. [Epub ahead of print]33(3): 108289
    Young SJ, Sebald M, Shah Punatar R, Larin M, Masino L, Rodrigo-Brenni MC, Liang CC, West SC.
      MutSα and MutSβ play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSβ, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSβ binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSβ and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSβ in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease.
    Keywords:  DNA recombination; DNA repair; Holliday junction; MUS81-EME1; SLX1-SLX4; SMX trinuclease; genome stability; resolution
  39. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30162-2. [Epub ahead of print]93 102914
    Vaisman A, Woodgate R.
      With the publication of the first paper describing the biochemical properties of DNA polymerase iota (polɩ), the question immediately arose as to why cells harbor such a low-fidelity enzyme which often violates the Watson-Crick base pairing rules? Yet 20 years after its discovery, the cellular function of polɩ remains unknown. Here, we provide a graphical review of the unique biochemical properties of polɩ and speculate about the cellular pathways in which enigmatic polɩ may participate.
    Keywords:  Mutagenesis; Replicase; Replication fidelity; Translesion DNA synthesis; Y-family DNA polymerase
  40. Essays Biochem. 2020 Oct 23. pii: EBC20200109. [Epub ahead of print]
    Wu Q.
      This collection of reviews aims to summarise our current understanding of a fundamental question: how do we deal with DNA damage? After identifying key players that are important for this process, we are now starting to reveal the dynamic organisation of detecting and repairing DNA damage. Reviews in this issue provide an update on the exciting research progress that is happening now in this field and also initiate discussion about future challenges and directions that we are heading to.
    Keywords:  DNA Repair; DNA damage response; cancer
  41. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30170-1. [Epub ahead of print]93 102921
    Caldecott KW.
      Damage to DNA bases occurs continuously in cells as a result of the intrinsic instability of nucleic acids and because of the presence of intracellular and environmental genotoxins. As a consequence, all living cells possess a highly conserved biochemical pathway by which damaged DNA bases are detected, removed, and replaced with undamaged bases. This pathway is denoted DNA base excision repair (BER) and is critical for genome stability and human health. In this review I summarise the key features of mammalian BER, highlighting both the molecular choreography that coordinates this pathway and its importance for human health.
    Keywords:  Genome stabiity; PARP1; XRCC1
  42. Cancers (Basel). 2020 Oct 15. pii: E2986. [Epub ahead of print]12(10):
    García-González R, Morejón-García P, Campillo-Marcos I, Salzano M, Lazo PA.
      Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM-/- cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).
    Keywords:  DNA-damage response; acetylation; histone H4; nucleosomal histone kinase-1; phosphorylation
  43. Cell Death Dis. 2020 Oct 21. 11(10): 887
    Deville SS, Luft S, Kaufmann M, Cordes N.
      The function of Keap1 (Kelch-like ECH-associated protein 1), a sensor of oxidative and electrophilic stress, in the radiosensitivity of cancer cells remains elusive. Here, we investigated the effects of pharmacological inhibition of Keap1 with ML344 on radiosensitivity, DNA double-strand break (DSB) repair and autophagy in head and neck squamous cell carcinoma (HNSCC) cell lines. Our data demonstrate that Keap1 inhibition enhances HNSCC cell radiosensitivity. Despite elevated, Nrf2-dependent activity of non-homologous end joining (NHEJ)-related DNA repair, Keap1 inhibition seems to impair DSB repair through delayed phosphorylation of DNA-PKcs. Moreover, Keap1 inhibition elicited autophagy and increased p62 levels when combined with X-ray irradiation. Our findings suggest HNSCC cell radiosensitivity, NHEJ-mediated DSB repair, and autophagy to be co-regulated by Keap1.
  44. Cell Rep. 2020 Oct 20. pii: S2211-1247(20)31276-6. [Epub ahead of print]33(3): 108287
    Gobbini E, Casari E, Colombo CV, Bonetti D, Longhese MP.
      Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.
    Keywords:  9-1-1; DNA damage; Dpb11/TopBP1; MRX/MRN; Rad9/53BP1; S. cerevisiae; checkpoint; double-strand breaks; resection
  45. Nat Struct Mol Biol. 2020 Oct 19.
    Chaplin AK, Hardwick SW, Liang S, Kefala Stavridi A, Hnizda A, Cooper LR, De Oliveira TM, Chirgadze DY, Blundell TL.
      DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.
  46. Transl Oncol. 2020 Oct 20. pii: S1936-5233(20)30390-9. [Epub ahead of print]14(1): 100898
    Gilbreath C, Ma S, Yu L, Sonavane R, Roggero CM, Devineni A, Mauck R, Desai NB, Bagrodia A, Kittler R, Raj GV, Yin Y.
      The study of DNA damage repair response (DDR) in prostate cancer is restricted by the limited number of prostate cancer cell lines and lack of surrogates for heterogeneity in clinical samples. Here, we sought to leverage our experience with patient derived explants (PDEs) cultured ex vivo to study dynamics of DDR in primary tumors following application of clinically relevant doses of ionizing radiation (IR) to tumor cells in their native 3-dimensional microenvironment. We compared DDR dynamics between prostate cancer cell lines, PDEs and xenograft derived explants (XDEs) following treatment with IR (2Gy) either alone or in combination with pharmacological modulators of DDR. We have shown that following treatment with 2Gy, DDR can be consistently detected in PDEs from multiple solid tumors, including prostate, kidney, testes, lung and breast, as evidenced by γ-H2AX, 53BP1, phospho-ATM and phospho-DNA-PKcs foci. By examining kinetics of resolution of IR-induced foci, we have shown that DDR in prostate PDEs (complete resolution in 8 h) is much faster than in prostate cancer cell lines (<50% resolution in 8 h). The transcriptional profile of DDR genes following 2Gy IR appears to be distinct between PDEs and cell lines. Pre-treatment with drugs targeting DDR pathways differentially alter the kinetics of DDR in the PDEs and cell lines, as evidenced by altered kinetics of foci resolution. This study highlights the utility of PDEs as a robust model system for short-term evaluation of DDR in primary solid tumors in clinically relevant microenvironment.
    Keywords:  DNA damage response, DNA repair; Ex vivo culture; Tumor microenvironment; patient-derived explant; radiation resistance
  47. ACS Chem Biol. 2020 Oct 22.
    Flamme M, Röthlisberger P, Levi-Acobas F, Chawla M, Oliva R, Cavallo L, Gasser G, Marlière P, Herdewijn P, Hollenstein M.
      The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.
  48. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30158-0. [Epub ahead of print]93 102910
    Beard WA.
      DNA polymerase (dpol) β has served as a model for structural, kinetic, and computational characterization of the DNA synthesis reaction. The laboratory directed by Samuel H. Wilson has utilized a multifunctional approach to analyze the function of this enzyme at the biological, chemical, and molecular levels for nearly 50 years. Over this time, it has become evident that correlating static crystallographic structures of dpol β with solution kinetic measurements is a daunting task. However, aided by computational and spectroscopic approaches, novel and unexpected insights have emerged. While dpols generally insert wrong nucleotides with similar poor efficiencies, their capacity to insert the right nucleotide depends on the identity of the dpol. Accordingly, the ability to choose right from wrong depends on the efficiency of right, rather than wrong, nucleotide insertion. Structures of dpol β in various liganded forms published by the Wilson laboratory, and others, have provided molecular insights into the molecular attributes that hasten correct nucleotide insertion and deter incorrect nucleotide insertion. Computational approaches have bridged the gap between structures of intermediate complexes and provided insights into this basic and essential chemical reaction.
    Keywords:  DNA polymerase β; DNA synthesis; Fidelity; Genome integrity; Structure
  49. Nat Rev Mol Cell Biol. 2020 Oct 19.
    Zhao B, Rothenberg E, Ramsden DA, Lieber MR.
      Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.
  50. J Biol Chem. 2020 Oct 19. pii: jbc.RA120.015390. [Epub ahead of print]
    Sullivan ED, Longley MJ, Copeland WC.
      Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters double stranded (ds) DNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the Pol γ heterotrimer is well adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex forming sequence at the heavy strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV-exposure, and we have found that efficiency of Pol γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
    Keywords:  DNA polymerase; DNA replication; DNA structure; G-quadruplex; Heavy strand promoter; mitochondrial DNA (mtDNA); mitochondrial DNA damage
  51. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30171-3. [Epub ahead of print]93 102922
    Wright G, Gassman NR.
      Base excision repair (BER) addresses the numerous base lesions and strand breaks induced by exogenous and endogenous stressors daily. The complexity and importance of BER requires careful regulation of basal levels of these proteins and inducible responses following DNA damage. Several reports have noted the dysregulation of BER proteins and defects in BER capacity in cancer. Modulated gene and protein expression of several BER proteins, including APE1, PARP1, POL β, and XRCC1, have been observed in breast cancer. Overexpression of these factors has been associated with chemoresistance and cancer aggressiveness, but the regulatory mechanisms that drive overexpression have not been defined. Here, we review the known transcriptional regulators of these key BER proteins and examine potential mechanisms that may drive overexpression in breast cancer.
  52. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30168-3. [Epub ahead of print]93 102920
    Das L, Quintana VG, Sweasy JB.
      Efficient DNA repair is essential to maintain genomic integrity. An average of 30,000 base lesions per cell are removed daily by the DNA glycosylases of the base excision repair machinery. With the advent of whole genome sequencing, many germline mutations in these DNA glycosylases have been identified and associated with various diseases, including cancer. In this graphical review, we discuss the function of the NTHL1 DNA glycosylase and how genomic mutations and altered function of this protein contributes to cancer and aging. We highlight its role in a rare tumor syndrome, NTHL1-associated polyposis (NAP), and summarize various other polymorphisms in NTHL1 that can induce early hallmarks of cancer, including genomic instability and cellular transformation.
    Keywords:  Base excision repair; DNA glycosylase; DNA repair
  53. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30183-X. [Epub ahead of print]93 102934
    Van Houten B.
  54. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30155-5. [Epub ahead of print]93 102907
    Lehmann AR, Fassihi H.
      Xeroderma pigmentosum (XP) is a well-studied disorder of (in most cases) nucleotide excision repair. The establishment in 2010 of a multidisciplinary XP clinic in the UK has enabled us to make a detailed analysis of genotype-phenotype relationships in XP patients and in several instances to make confident prognostic predictions. Splicing mutations in XPA and XPD and a specific amino acid change in XPD are associated with mild phenotypes, and individuals assigned to the XP-F group appear to have reduced pigmentation changes and a lower susceptibility to skin cancer than XPs in other groups. In an XP-C patient with advanced metastatic cancer arising from an angiosarcoma, molecular analysis of the tumour DNA suggested that immunotherapy, not normally recommended for angiosarcomas, might in this case be successful, and indeed the patient showed a dramatic recovery following immunotherapy treatment. These studies show that molecular analyses can improve the management, prognoses and therapy for individuals with XP.
    Keywords:  Immunotherapy; Mutation signature; Neurological abnormalities; Nucleotide excision repair; Skin cancer; Splice mutations; Ultraviolet
  55. Cancer Discov. 2020 Oct 18. pii: CD-20-0282. [Epub ahead of print]
    Binkley MS, Jeon YJ, Nesselbush M, Moding EJ, Nabet BY, Almanza D, Kunder C, Stehr H, Yoo CH, Rhee S, Xiang M, Chabon JJ, Hamilton E, Kurtz DM, Gojenola L, Owen SG, Ko RB, Shin JH, Maxim PG, Lui NS, Backhus LM, Berry MF, Shrager JB, Ramchandran KJ, Padda SK, Das M, Neal JW, Wakelee HA, Alizadeh AA, Loo BW, Diehn M.
      Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that KEAP1 and NFE2L2 mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in KEAP1/NFE2L2 mutation tumors, indicating they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate KEAP1/NFE2L2 mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes KEAP1 mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for KEAP1/NFE2L2 mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations.
  56. DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30180-4. [Epub ahead of print]93 102931
    Bhakat KK, Sengupta S, Mitra S.
      In addition to the key roles of reversible acetylation of histones in chromatin in epigenetic regulation of gene expression, acetylation of nonhistone proteins by histone acetyltransferases (HATs) p300 and CBP is involved in DNA transactions, including repair of base damages and strand breaks. We characterized acetylation of human NEIL1 DNA glycosylase and AP-endonuclease 1 (APE1), which initiate repair of oxidized bases and single-strand breaks (SSBs), respectively. Acetylation induces localized conformation change because of neutralization of the positive charge of specific acetyl-acceptor Lys residues, which are often present in clusters. Acetylation in NEIL1, APE1, and possibly other base excision repair (BER)/SSB repair (SSBR) enzymes by HATs, prebound to chromatin, induces assembly of active repair complexes on the chromatin. In this review, we discuss the roles of acetylation of NEIL1 and APE1 in modulating their activities and complex formation with other proteins for fine-tuning BER in chromatin. Further, the implications of promoter/enhancer-bound acetylated BER protein complexes in the regulation of transcriptional activation, mediated by complex interplay of acetylation and demethylation of histones are discussed.
    Keywords:  APE1; Acetylation; BER complexes; Base excision repair (BER); DNA base damage; Histone acetyltransferases (HATs); NEIL1
  57. NAR Cancer. 2020 Dec;2(4): zcaa029
    Xu YZ, Jenjaroenpun P, Wongsurawat T, Byrum SD, Shponka V, Tannahill D, Chavez EA, Hung SS, Steidl C, Balasubramanian S, Rimsza LM, Kendrick S.
      Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.
  58. Trends Biochem Sci. 2020 Oct 16. pii: S0968-0004(20)30243-7. [Epub ahead of print]
    Zhang X, Blumenthal RM, Cheng X.
      The leading cause of mutation due to oxidative damage is 8-oxo-2'-deoxyguanosine (8-oxoG) mispairing with adenine (Ade), which can occur in two ways. First, guanine of a G:C DNA base pair can be oxidized. If not repaired in time, DNA polymerases can mispair Ade with 8-oxoG in the template. This 8-oxoG:A can be repaired by enzymes that remove Ade opposite to template 8-oxoG, or 8-oxoG opposite to Cyt. Second, free 8-oxo-dGTP can be misincorporated by DNA polymerases into DNA opposite template Ade. However, there is no known repair activity that removes 8-oxoG opposite to template Ade. We suggest that a major role of N6-methyladenine in mammalian DNA is minimizing incorporation of 8-oxoG opposite to Ade by DNA polymerases following adduct formation.
    Keywords:  8-oxoguanine; DNA adenine methylation; MettL3-MettL14; YTHDC1; single-stranded DNA
  59. Nat Commun. 2020 10 20. 11(1): 5288
    Sanford SL, Welfer GA, Freudenthal BD, Opresko PL.
      Telomerase is a specialized reverse transcriptase that adds GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation. We conduct a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the nucleotide reverse transcriptase inhibitors ddITP and AZT-TP to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or therapeutic 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy.