bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒08‒23
thirty-four papers selected by
Sean Rudd
Karolinska Institutet

  1. Sci Rep. 2020 Aug 17. 10(1): 13907
    Silveira SC, Buhagiar-Labarchède G, Onclercq-Delic R, Gemble S, Bou Samra E, Mameri H, Duchambon P, Machon C, Guitton J, Amor-Guéret M.
      Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.
  2. Nat Commun. 2020 Aug 17. 11(1): 4124
    Kong YW, Dreaden EC, Morandell S, Zhou W, Dhara SS, Sriram G, Lam FC, Patterson JC, Quadir M, Dinh A, Shopsowitz KE, Varmeh S, Yilmaz ÖH, Lippard SJ, Reinhardt HC, Hemann MT, Hammond PT, Yaffe MB.
      In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
  3. Nat Struct Mol Biol. 2020 Aug 17.
    Malik R, Kopylov M, Gomez-Llorente Y, Jain R, Johnson RE, Prakash L, Prakash S, Ubarretxena-Belandia I, Aggarwal AK.
      DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.
  4. Mol Cancer Res. 2020 Aug 14. pii: molcanres.0396.2020. [Epub ahead of print]
    Sriramkumar S, Matthews TD, Ghobashi AH, Miller SA, VanderVere-Carozza PS, Pawelczak KS, Nephew KP, Turchi JJ, O'Hagan HM.
      Platinum resistance is a common occurrence in high grade serous ovarian cancer (HGSOC) and a major cause of OC deaths. Platinum agents form DNA crosslinks, which activate nucleotide excision repair (NER), fanconi anemia (FA) and homologous recombination repair (HRR) pathways. Chromatin modifications occur in the vicinity of DNA damage and play an integral role in the DNA damage response (DDR). Chromatin modifiers, including polycomb repressive complex 1 (PRC1) members, and chromatin structure are frequently dysregulated in OC and can potentially contribute to platinum resistance. However, the role of chromatin modifiers in the repair of platinum DNA damage in OC is not well understood. We demonstrate that the PRC1 complex member RING1A mediates monoubiquitination of lysine 119 of phosphorylated H2AX (γH2AXub1) at sites of platinum DNA damage in OC cells. After platinum treatment, our results reveal that NER and HRR both contribute to RING1A localization and γH2AX monoubiquitination. Importantly, replication protein A (RPA), involved in both NER and HRR, mediates RING1A localization to sites of damage. Furthermore, RING1A deficiency impairs the activation of the G2/M DNA damage checkpoint, reduces the ability of OC cells to repair platinum DNA damage and increases sensitivity to platinum. Implications: Elucidating the role of RING1A in the DDR to platinum agents will allow for the identification of therapeutic targets to improve the response of OC to standard chemotherapy regimens.
  5. Biochem Biophys Res Commun. 2020 Aug 15. pii: S0006-291X(20)31519-9. [Epub ahead of print]
    Pernicone N, Grinshpon S, Listovsky T.
      MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a 'closed' active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an 'open' inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus β-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2's C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2's preferential complex formation.
    Keywords:  CDH1(FZR1); MAD2L2; Protein-protein interaction
  6. J Biol Chem. 2020 Aug 14. pii: jbc.RA120.014228. [Epub ahead of print]
    Ha A, Lin Y, Yan S.
      The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand crosslink repair pathways via its DNA glycosylase and/or AP lyase activity, which is considered as canonical roles of NEIL3 in genome integrity. In comparison to other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C-terminus has two highly conserved zinc finger motifs containing GRxF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA) whereas the major AP endonuclease APE1 doesn't. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that NEIL3 Zf-GRF repeat associated with higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein-protein interaction assays show that NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by NEIL3 Zf-GRF repeat while one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a non-canonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.
    Keywords:  APE1; APE2; DNA damage response; DNA endonuclease; DNA enzyme; DNA repair; NEIL3; Zinc finger Zf-GRF motif; genomic instability; ssDNA
  7. Life Sci Alliance. 2020 Oct;pii: e202000668. [Epub ahead of print]3(10):
    Benedict B, van Bueren MA, van Gemert FP, Lieftink C, Guerrero Llobet S, van Vugt MA, Beijersbergen RL, Te Riele H.
      Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.
  8. Sci Signal. 2020 Aug 18. pii: eaba8091. [Epub ahead of print]13(645):
    Kharat SS, Ding X, Swaminathan D, Suresh A, Singh M, Sengodan SK, Burkett S, Marks H, Pamala C, He Y, Fox SD, Buehler EC, Muegge K, Martin SE, Sharan SK.
      Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.
  9. Elife. 2020 Aug 17. pii: e60371. [Epub ahead of print]9
    Deegan TD, Mukherjee PP, Fujisawa R, Polo Rivera C, Labib K.
      The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48 / p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.
    Keywords:  S. cerevisiae; chromosomes; gene expression
  10. Essays Biochem. 2020 Aug 18. pii: EBC20200009. [Epub ahead of print]
    Sriraman A, Debnath TK, Xhemalce B, Miller KM.
      Cells encounter a multitude of external and internal stress-causing agents that can ultimately lead to DNA damage, mutations and disease. A cascade of signaling events counters these challenges to DNA, which is termed as the DNA damage response (DDR). The DDR preserves genome integrity by engaging appropriate repair pathways, while also coordinating cell cycle and/or apoptotic responses. Although many of the protein components in the DDR are identified, how chemical modifications to DNA impact the DDR is poorly understood. This review focuses on our current understanding of DNA methylation in maintaining genome integrity in mammalian cells. DNA methylation is a reversible epigenetic mark, which has been implicated in DNA damage signaling, repair and replication. Sites of DNA methylation can trigger mutations, which are drivers of human diseases including cancer. Indeed, alterations in DNA methylation are associated with increased susceptibility to tumorigenesis but whether this occurs through effects on the DDR, transcriptional responses or both is not entirely clear. Here, we also highlight epigenetic drugs currently in use as therapeutics that target DNA methylation pathways and discuss their effects in the context of the DDR. Finally, we pose unanswered questions regarding the interplay between DNA methylation, transcription and the DDR, positing the potential coordinated efforts of these pathways in genome integrity. While the impact of DNA methylation on gene regulation is widely understood, how this modification contributes to genome instability and mutations, either directly or indirectly, and the potential therapeutic opportunities in targeting DNA methylation pathways in cancer remain active areas of investigation.
    Keywords:  Cancer; DNA damage; DNA methylation; DNA synthesis and repair; epigenetics; genome integrity
  11. Biochim Biophys Acta Gen Subj. 2020 Aug 14. pii: S0304-4165(20)30217-8. [Epub ahead of print] 129705
    Lafont F, Fleury F, Benhelli-Mokrani H.
      BACKGROUND: DNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown.METHODS: Using IP techniques, and HeLa cell line, we evaluated the effect of pharmacological or siOGT mediated O-GlcNAc level modulation on DNA-PKcs O-GlcNAcylation. We used the RPA32 phosphorylation as a DNA-PKcs activity reporter substrate to evaluate the effect of O-GlcNAc modulators.
    RESULTS: We show here that human DNA-PKcs is an O-GlcNAc modified protein and that this new PTM is responsive to the cell O-GlcNAcylation level modulation. Our findings reveal that DNA-PKcs hypo O-GlcNAcylation affects its kinase activity and that the bleomycin-induced Ser2056 phosphorylation, is modulated by DNA-PKcs O-GlcNAcylation.
    CONCLUSIONS: DNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation.
    GENERAL SIGNIFICANCE: Given the essential role of DNA-PKcs Ser2056 phosphorylation in the DDR, this study brings data about the role of cell O-GlcNAc level on genome integrity maintenance.
    Keywords:  DNA repair; DNA-PKcs; HR; NHEJ; O-GlcNAcylation; Phosphorylation
  12. Mol Cell. 2020 Aug 07. pii: S1097-2765(20)30517-7. [Epub ahead of print]
    Reisländer T, Groelly FJ, Tarsounas M.
      Cancer immunotherapies enhance anti-tumor immune responses using checkpoint inhibitors, such as PD-1 or PD-L1 inhibitors. Recent studies, however, have extended the scope of immunotherapeutics by unveiling DNA damage-induced innate immunity as a novel target for cancer treatment. Elucidating the interplay among the DNA damage response (DDR), cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway activation, and anti-tumoral immunity is critical for the development of effective cancer immunotherapies. Here, we discuss the current understanding of the mechanisms by which DNA damage activates immune responses that target and eradicate cancer cells. Yet, understanding how cancer cells can escape this immune surveillance and promote tumor progression represents an outstanding challenge. We highlight the most recent clinical advances, in particular how pharmacological fine-tuning of innate/adaptive immunity and its combination with DDR inhibitors, ionizing radiation (IR), and chemotherapy can be exploited to improve cancer treatment.
  13. Nat Commun. 2020 Aug 21. 11(1): 4196
    Brissett NC, Zabrady K, Płociński P, Bianchi J, Korycka-Machała M, Brzostek A, Dziadek J, Doherty AJ.
      Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.
  14. ChemMedChem. 2020 Aug 19.
    Yusoh NA, Ahmad H, Gill M.
      Platinum drugs are heavily used first-line chemotherapeutic agents for many solid tumours and have stimulated substantial interest in the biological activity of DNA-binding metal complexes. These complexes generate DNA lesions which trigger the activation of DNA damage response (DDR) pathways that are essential to maintain the genomic integrity. Cancer cells exploit this intrinsic DNA repair network to counteract many types of chemotherapies. Therefore, advances in the molecular biology of cancer has paved the way for the development of DDR inhibitors, especially the poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Now, it has emerged that agents which induce high levels of DNA replication stress or single-strand break damage can be combined with PARPi for potent synergy and has potential to improve disease outcomes specifically in advanced cancer patients. In this review, we summarize early-stage, preclinical and clinical findings of work exploring platinum and emerging ruthenium anti-cancer complexes in combination with PARPi.
    Keywords:  Cancer; PARP inhibitors; combination therapy; platinum drugs; ruthenium
  15. Nature. 2020 Aug 19.
    Cannavo E, Sanchez A, Anand R, Ranjha L, Hugener J, Adam C, Acharya A, Weyland N, Aran-Guiu X, Charbonnier JB, Hoffmann ER, Borde V, Matos J, Cejka P.
      During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.
  16. Biochem Pharmacol. 2020 Aug 13. pii: S0006-2952(20)30431-7. [Epub ahead of print] 114195
    Ray U, Raghavan SC.
      Conventional cancer treatment modalities such as radiation and chemotherapy, cause cancer cell death by inducing DNA damage, particularly DNA strand breaks. Over the years, newer avenues have emerged for overcoming radio/chemoresistance by harnessing repair proteins as targets for small molecule inhibitors. Analysis of genome-wide expression data in cancer subtypes and understanding synthetic lethal interactions among repair pathways are important stepping-stones. Several inhibitors targeting DNA strand break repair proteins have yielded good effects in preclinical studies, and have the potential to be developed as therapeutics in cancer as monotherapy or in combination with radiation and chemotherapy. Furthermore, these small molecule inhibitors can aid in precise genome editing (using CRISPR) by harnessing the differential levels of repair inside cells. Shifting the repair balance towards homology-directed repair using inhibitors of NHEJ or stimulators of HR has yielded promising effects alongside CRISPR in cells and several disease models. In short, DNA strand break repair inhibitors are the forerunners in cancer therapy and genome editing, working in concert with the established artillery in the field.
    Keywords:  Cancer therapeutics; Double-strand break; End joining; Gene modification; Genomic instability; NHEJ; Synthetic lethality
  17. Nat Commun. 2020 Aug 19. 11(1): 4154
    Bruhn C, Ajazi A, Ferrari E, Lanz MC, Batrin R, Choudhary R, Walvekar A, Laxman S, Longhese MP, Fabre E, Smolka MB, Foiani M.
      The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.
  18. Chin Clin Oncol. 2020 Aug 19. pii: cco-20-4. [Epub ahead of print]
    Chelariu-Raicu A, Coleman RL.
      The discovery of cancer-causing BRCA1/2 mutations and the emergence of genetic testing have brought precision in patient selection for poly-(ADP)-ribose polymerase inhibitor (PARPi) treatment. Interestingly, patients who are carriers of BRCA1/2 mutations have a higher risk for developing cancer, but respond better to DNA-damaging cytotoxic therapy, such as platinum-based chemotherapy. The distinctive biology of ovarian cancer involves high genomic instability consisting of gene amplification, gene deletion, oncogene hypomethylation, loss of heterozygosity, and tumor suppressor gene promoter hypermethylation in many of the DNA damage response (DDR) genes, including BRCA1/2. Several of these genetic abnormalities can impair high fidelity DNA damage repair increasing the therapeutic audience for PARPi's. This is especially important given the clinical development over the last decade of this group of agents and the dramatic increase in progression free survival among ovarian cancer patients who received PARPi, both in treatment or maintenance setting. In this review, we summarize our current understanding of the role of BRCA1/2 mutations in ovarian cancer and present relevant clinical trials in which BRCA1/2 was investigated as biomarker for therapy. We also outline the role of homologous recombination (HR) deficiency as biomarker by presenting the recent clinical development and recent approvals PARPi for firstline maintenance in ovarian cancer.
    Keywords:  BRCA1/2; biomarker; homologous recombination (HR); ovarian cancer; poly-(ADP)-ribose polymerase inhibitor (PARPi)
  19. J Recept Signal Transduct Res. 2020 Aug 19. 1-8
    Wang Y, Zuo M, Jin H, Lai M, Luo J, Cheng Z.
      BACKGROUND: E74 Like ETS Transcription Factor 3 (ELF3) functions as a transcriptional factor to regulate non-small cell lung cancer (NSCLC) differentiation and progression. Poly(ADP-ribose) polymerase (PARP) inhibitors demonstrate anti-tumor effect in NSCLC. This study aimed to investigate whether ELF3 confers synthetic lethal with PARP inhibitor in NSCLC.MATERIALS AND METHODS: The sensitivity of PARP inhibitor, Olaparib, to different NSCLC cell lines was determined by half maximal inhibitory concentration (IC50). Expression of ELF3 in NSCLC cell lines was evaluated by western blot. The effects of ELF3 on cytotoxicity of Olaparib to NSCLC were investigated by MTT (3-(4,5- di methyl thiazol -2-yl)-2,5-di phenyl tetrazolium bromide) and colony formation assays. The underlying mechanism involved in synthetic lethality with ELF3 and PARP inhibitors in NSCLC were detected by immunofluorescence and Western blot.
    RESULTS: ELF3 was up-regulated in NSCLC cell lines exhibiting resistance to PARP inhibitor, Olaparib. Knock down of ELF3 decreased the sensitivity and enhanced cytotoxicity of Olaparib to NSCLC cells. Moreover, knock down of ELF3 increased S139 phosphorylated histone H2AX (γH2AX), and inhibited homologous recombination activity via down-regulation of DNA repair protein RAD51 homolog 1 (RAD51), thus showing deficiency in DNA damage repair. Over-expression of ELF3 could up-regulate phosphorylation of AKT (Protein kinase B), while knock down of ELF3 regulated homologous recombination-mediated DNA repair via down-regulation of phosphorylation of AKT.
    CONCLUSION: Knock down of ELF3 revealed homologous recombination deficiency via AKT signaling pathway, and synthetic lethality with ELF3 inhibition and PARP inhibitor indicated the clinical significance of PARP inhibitor in ELF3-deficient NSCLC.
    Keywords:  AKT; ELF3; PARP; sensitivity; synthetic lethal
  20. Cell Cycle. 2020 Aug 17. 1-16
    Piekna-Przybylska D, Bambara RA, Maggirwar SB, Dewhurst S.
      Altered telomere maintenance mechanism (TMM) is linked to increased DNA damage at telomeres and telomere uncapping. We previously showed that HIV-1 latent cells have altered TMM and are susceptible to ligands that target G-quadruplexes (G4) at telomeres. Susceptibility of latent cells to telomere targeting could potentially be used to support approaches to eradicate HIV reservoirs. However, G4 ligands also target G-quadruplexes in promoters blocking gene transcription. Since HIV promoter sequence can form G-quadruplexes, we investigated whether G4 ligands interfere with HIV-1 promoter activity and virus reactivation from latency, and whether telomere targeting could be combined with latency reversing agents (LRAs) to promote elimination of HIV reservoirs. Our results indicate that Sp1 binding region in HIV-1 promoter can adopt G4 structures in duplex DNA, and that in vitro binding of Sp1 to G-quadruplex is blocked by G4 ligand, suggesting that agents targeting telomeres interfere with virus reactivation. However, our studies show that G4 agents do not affect HIV-1 promoter activity in cell culture, and do not interfere with latency reversal. Importantly, primary memory CD4 + T cells infected with latent HIV-1 are more susceptible to combined treatment with LRAs and G4 ligands, indicating that drugs targeting TMM may enhance killing of HIV reservoirs. Using a cell-based DNA repair assay, we also found that HIV-1 infected cells have reduced efficiency of DNA mismatch repair (MMR), and base excision repair (BER), suggesting that altered TMM in latently infected cells could be associated with accumulation of DNA damage at telomeres and changes in telomeric caps.
    Keywords:  DNA damage response; DNA repair; HIV-1 latency; TMM; latency reversing agents; telomere maintenance mechanism
  21. Proc Natl Acad Sci U S A. 2020 Aug 19. pii: 202008645. [Epub ahead of print]
    Öz R, Howard SM, Sharma R, Törnkvist H, Ceppi I, Kk S, Kristiansson E, Cejka P, Westerlund F.
      The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.
    Keywords:  CtIP; DNA repair; homologous recombination; nanofluidics; single DNA molecule biophysics
  22. Nucleic Acids Res. 2020 Aug 20. 48(14): 7712-7727
    Tellier M, Zaborowska J, Caizzi L, Mohammad E, Velychko T, Schwalb B, Ferrer-Vicens I, Blears D, Nojima T, Cramer P, Murphy S.
      Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
  23. Int J Mol Sci. 2020 Aug 17. pii: E5896. [Epub ahead of print]21(16):
    Puts G, Jarrett S, Leonard M, Matsangos N, Snyder D, Wang Y, Vincent R, Portney B, Abbotts R, McLaughlin L, Zalzman M, Rassool F, Kaetzel D.
      Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.
    Keywords:  DNA double strand break repair; DNA repair; cancer; homing endonuclease; homologous recombination; melanoma; metastasis; non-homologous end-joining
  24. Genome Biol. 2020 Aug 21. 21(1): 209
    Murat P, Guilbaud G, Sale JE.
      BACKGROUND: Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. However, current studies of DNA synthesis through STRs are restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour and hampering the characterisation of the determinants of their abundance and stability in eukaryotic genomes.RESULTS: We perform a comprehensive analysis of DNA synthesis at all STR permutations and interrogate the impact of STR sequence and secondary structure on their genomic representation and mutability. To do this, we developed a high-throughput primer extension assay that allows monitoring of the kinetics and fidelity of DNA synthesis through 20,000 sequences comprising all STR permutations in different lengths. By combining these measurements with population-scale genomic data, we show that the response of a model replicative DNA polymerase to variously structured DNA is sufficient to predict the complex genomic behaviour of STRs, including abundance and mutational constraints. We demonstrate that DNA polymerase stalling at DNA structures induces error-prone DNA synthesis, which constrains STR expansion.
    CONCLUSIONS: Our data support a model in which STR length in eukaryotic genomes results from a balance between expansion due to polymerase slippage at repeated DNA sequences and point mutations caused by error-prone DNA synthesis at DNA structures.
    Keywords:  DNA secondary structure; Genome evolution; Genome instability; Polymerase stalling; Short tandem repeat
  25. Radiat Oncol. 2020 Aug 14. 15(1): 197
    Xiang K, Jendrossek V, Matschke J.
      Radiotherapy (RT) is applied in 45-60% of all cancer patients either alone or in multimodal therapy concepts comprising surgery, RT and chemotherapy. However, despite technical innovations approximately only 50% are cured, highlight a high medical need for innovation in RT practice. RT is a multidisciplinary treatment involving medicine and physics, but has always been successful in integrating emerging novel concepts from cancer and radiation biology for improving therapy outcome. Currently, substantial improvements are expected from integration of precision medicine approaches into RT concepts.Altered metabolism is an important feature of cancer cells and a driving force for malignant progression. Proper metabolic processes are essential to maintain and drive all energy-demanding cellular processes, e.g. repair of DNA double-strand breaks (DSBs). Consequently, metabolic bottlenecks might allow therapeutic intervention in cancer patients.Increasing evidence now indicates that oncogenic activation of metabolic enzymes, oncogenic activities of mutated metabolic enzymes, or adverse conditions in the tumor microenvironment can result in abnormal production of metabolites promoting cancer progression, e.g. 2-hyroxyglutarate (2-HG), succinate and fumarate, respectively. Interestingly, these so-called "oncometabolites" not only modulate cell signaling but also impact the response of cancer cells to chemotherapy and RT, presumably by epigenetic modulation of DNA repair.Here we aimed to introduce the biological basis of oncometabolite production and of their actions on epigenetic regulation of DNA repair. Furthermore, the review will highlight innovative therapeutic opportunities arising from the interaction of oncometabolites with DNA repair regulation for specifically enhancing the therapeutic effects of genotoxic treatments including RT in cancer patients.
    Keywords:  DNA repair; Epigenetic regulation; Ionizing radiation; Oncometabolites
  26. Cancers (Basel). 2020 Aug 12. pii: E2263. [Epub ahead of print]12(8):
    Brecht K, Schäfer AM, Meyer Zu Schwabedissen HE.
      Solute carrier transporters comprise a large family of uptake transporters involved in the transmembrane transport of a wide array of endogenous substrates such as hormones, nutrients, and metabolites as well as of clinically important drugs. Several cancer therapeutics, ranging from chemotherapeutics such as topoisomerase inhibitors, DNA-intercalating drugs, and microtubule binders to targeted therapeutics such as tyrosine kinase inhibitors are substrates of solute carrier (SLC) transporters. Given that SLC transporters are expressed both in organs pivotal to drug absorption, distribution, metabolism, and elimination and in tumors, these transporters constitute determinants of cellular drug accumulation influencing intracellular drug concentration required for efficacy of the cancer treatment in tumor cells. In this review, we explore the current understanding of members of three SLC families, namely SLC21 (organic anion transporting polypeptides, OATPs), SLC22A (organic cation transporters, OCTs; organic cation/carnitine transporters, OCTNs; and organic anion transporters OATs), and SLC15A (peptide transporters, PEPTs) in the etiology of cancer, in transport of chemotherapeutic drugs, and their influence on efficacy or toxicity of pharmacotherapy. We further explore the idea to exploit the function of SLC transporters to enhance cancer cell accumulation of chemotherapeutics, which would be expected to reduce toxic side effects in healthy tissue and to improve efficacy.
    Keywords:  cancer; chemotherapy; genetic variants; solute carrier transporters; tumor
  27. Molecules. 2020 Aug 13. pii: E3694. [Epub ahead of print]25(16):
    Zakharenko AL, Drenichev MS, Dyrkheeva NS, Ivanov GA, Oslovsky VE, Ilina ES, Chernyshova IA, Lavrik OI, Mikhailov SN.
      Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2',3',5'-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 2',3',5'-Tri-O-benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 μM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50-100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.
    Keywords:  DNA repair; Tdp1 inhibition; nucleosides; topotecan; tyrosyl-DNA phosphodiesterase
  28. Elife. 2020 Aug 18. pii: e58622. [Epub ahead of print]9
    Jaremko MJ, On KF, Thomas DR, Stillman B, Joshua-Tor L.
      Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.
    Keywords:  chromosomes; gene expression; human
  29. DNA Repair (Amst). 2020 Jul 06. pii: S1568-7864(20)30153-1. [Epub ahead of print]94 102905
    Petljak M, Maciejowski J.
      The APOBEC family of cytidine deaminases has been proposed to represent a major enzymatic source of mutations in cancer. Here, we summarize available evidence that links APOBEC deaminases to cancer mutagenesis. We also highlight newly identified human cell models of APOBEC mutagenesis, including cancer cell lines with suspected endogenous APOBEC activity and a cell system of telomere crisis-associated mutations. Finally, we draw on recent data to propose potential causes of APOBEC misregulation in cancer, including the instigating factors, the relevant mutator(s), and the mechanisms underlying generation of the genome-dispersed and clustered APOBEC-induced mutations.
    Keywords:  APOBEC mutations; Cancer cell lines; Cancer mutagenesis; Chromothripsis; Kataegis; Mutational signature
  30. Cancer Lett. 2020 Aug 16. pii: S0304-3835(20)30421-3. [Epub ahead of print]
    Tian D, Tang J, Geng X, Li Q, Wang F, Zhao H, Narla G, Yao X, Zhang Y.
      Kirsten rat sarcoma virus oncogene homolog (KRAS) mutant lung cancer remains a challenge to cure and chemotherapy is the current standard treatment in the clinic. Hence, understanding molecular mechanisms underlying the sensitivity of KRAS mutant lung cancer to chemotherapy could help uncover unique strategies to treat this disease. Here we report a compound library screen and identification of cardiac glycosides as agents that selectively enhance the in vitro and in vivo effects of chemotherapy on KRAS mutant lung cancer. Quantitative mass spectrometry reveals that cardiac glycosides inhibit DNA double strand break (DSB) repair through suppressing the expression of UHRF1, an important DSB repair factor. Inhibition of UHRF1 by cardiac glycosides was mediated by specific suppression of the oncogenic KRAS pathway. Overexpression of UHRF1 rescued DSB repair inhibited by cardiac glycosides and depletion of UHRF1 enhanced glycoside-enhanced chemotherapeutic drug sensitivity in KRAS mutant lung cancer cells. Our study reveals a targetable dependency on UHRF1-stimulated DSB repair in KRAS mutant lung cancer in response to chemotherapy.
    Keywords:  Cardiac glycoside; Chemo sensitizer; Chemotherapy; Compound library screen; DNA damage Response; DNA repair; DSB repair; KRAS mutation; UHRF1
  31. Elife. 2020 Aug 17. pii: e59994. [Epub ahead of print]9
    Lv L, Chen P, Cao L, Li Y, Zeng Z, Cui Y, Wu Q, Li J, Wang JH, Dong MQ, Qi X, Han T.
      Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of a CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that recruits its target protein directly to DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.
    Keywords:  biochemistry; chemical biology; human
  32. Int J Mol Sci. 2020 Aug 14. pii: E5848. [Epub ahead of print]21(16):
    Adam K, Ning J, Reina J, Hunter T.
      The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylation, highlighting the underappreciated protein-histidine kinase activity of NME family members, specifically in human cells. In parallel, information about the structural and functional aspects of the NME family, and the knowns and unknowns of histidine kinase involvement in cell signaling are summarized.
    Keywords:  NME; histidine kinase; phosphorylation
  33. Cells. 2020 Aug 18. pii: E1914. [Epub ahead of print]9(8):
    Alarcón S, Toro MLÁ, Villarreal C, Melo R, Fernández R, Ayuso Sacido A, Uribe D, San Martín R, Quezada C.
      Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5'-nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.
    Keywords:  adenosine; equilibrative nucleoside transporter 1 (ENT1).; glioblastoma; glioblastoma stem-like cells (GSCs)
  34. Cancer Discov. 2020 Aug 14.
      Residual acute myeloid leukemia (AML) cells required bone marrow stromal cell-derived aspartate.