bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒07‒19
38 papers selected by
Sean Rudd
Karolinska Institutet


  1. J Biol Chem. 2020 Jul 16. pii: jbc.RA120.013726. [Epub ahead of print]
    Ito SS, Nakagawa Y, Matsubayashi M, Sakaguchi YM, Kobashigawa S, Matsui TK, Nanaura H, Nakanishi M, Kitayoshi F, Kikuchi S, Kajihara A, Tamaki S, Sugie K, Kashino G, Takahashi A, Hasegawa M, Mori E, Kirita T.
      The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor (ATRi) remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinases inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell cycle checkpoint. Cells lacking the BRCA2 were sensitive to 5-FU in the presence of ATRi. Moreover, the ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the non-homologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.
    Keywords:  DNA damage response; DNA repair; anticancer drug; cell cycle; homologous recombination
    DOI:  https://doi.org/10.1074/jbc.RA120.013726
  2. Nat Commun. 2020 Jul 15. 11(1): 3531
    Berti M, Teloni F, Mijic S, Ursich S, Fuchs J, Palumbieri MD, Krietsch J, Schmid JA, Garcin EB, Gon S, Modesti M, Altmeyer M, Lopes M.
      Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.
    DOI:  https://doi.org/10.1038/s41467-020-17324-z
  3. DNA Repair (Amst). 2020 Jul 08. pii: S1568-7864(20)30174-9. [Epub ahead of print]94 102925
    Neal JA, Dunger K, Geith K, Meek K.
      It has recently been established that the marked sensitivity of ATM deficient cells to topoisomerase poisons like camptothecin (Cpt) results from unrestrained end-joining of DNA ends at collapsed replication forks that is mediated by the non-homologous end joining [NHEJ] pathway and results in the induction of copious numbers of genomic alterations, termed "toxic NHEJ". Ablation of core components of the NHEJ pathway reverses the Cpt sensitivity of ATM deficient cells, but inhibition of DNA-PKcs does not. Here, we show that complete ablation of DNA-PKcs partially reverses the Cpt sensitivity of ATM deficient cells; thus, ATM deficient cells lacking DNA-PKcs are more resistant to Cpt than cells expressing DNA-PKcs. However, the relative sensitivity of DNA-PKcs proficient ATM deficient cells is inversely proportional to DNA-PKcs expression levels. These data suggest that DNA-PK may phosphorylate an ATM target (that contributes to Cpt resistance), explaining partial rescue of Cpt sensitivity in cells expressing high levels of DNA-PKcs. Although crippling NHEJ function by mutagenic blockade of the critical ABCDE autophosphorylation sites in DNA-PKcs also sensitizes cells to Cpt, this sensitization apparently occurs by a distinct mechanism from ATM ablation because blockade of these sites actually rescues ATM deficient cells from toxic NHEJ. These data are consistent with autophosphorylation of the ABCDE sites (and not ATM mediated phosphorylation) in response to Cpt-induced damage. In contrast, blockade of S3205 (an ATM dependent phosphorylation site in DNA-PKcs) that minimally impacts NHEJ, increases Cpt sensitivity. In sum, these data suggest that ATM and DNA-PK cooperate to facilitate Cpt-induced DNA damage, and that ATM phosphorylation of S3205 facilitates appropriate repair at collapsed replication forks.
    Keywords:  DNA dependent protein kinase (DNA-PK); DNA-PK catalytic subunit (DNA-PKcs); Non-homologous end joining (NHEJ); Toxic NHEJ
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102925
  4. Nat Commun. 2020 Jul 14. 11(1): 3503
    Pennycook BR, Vesela E, Peripolli S, Singh T, Barr AR, Bertoli C, de Bruin RAM.
      DNA replication timing is tightly regulated during S-phase. S-phase length is determined by DNA synthesis rate, which depends on the number of active replication forks and their velocity. Here, we show that E2F-dependent transcription, through E2F6, determines the replication capacity of a cell, defined as the maximal amount of DNA a cell can synthesise per unit time during S-phase. Increasing or decreasing E2F-dependent transcription during S-phase increases or decreases replication capacity, and thereby replication rates, thus shortening or lengthening S-phase, respectively. The changes in replication rate occur mainly through changes in fork speed without affecting the number of active forks. An increase in fork speed does not induce replication stress directly, but increases DNA damage over time causing cell cycle arrest. Thus, E2F-dependent transcription determines the DNA replication capacity of a cell, which affects the replication rate, controlling the time it takes to duplicate the genome and complete S-phase.
    DOI:  https://doi.org/10.1038/s41467-020-17146-z
  5. Nucleic Acids Res. 2020 Jul 11. pii: gkaa570. [Epub ahead of print]
    Guarino Almeida E, Renaudin X, Venkitaraman AR.
      The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.
    DOI:  https://doi.org/10.1093/nar/gkaa570
  6. Cell Death Dis. 2020 Jul 16. 11(7): 538
    Choo JAMY, Schlösser D, Manzini V, Magerhans A, Dobbelstein M.
      The integrated stress response (ISR) allows cells to rapidly shutdown most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within 1 h, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or re-activate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops), which interfere with DNA replication. R-loops accumulate upon histone depletion. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses.
    DOI:  https://doi.org/10.1038/s41419-020-2727-2
  7. Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30865-2. [Epub ahead of print]32(2): 107884
    Shan W, Yuan J, Hu Z, Jiang J, Wang Y, Loo N, Fan L, Tang Z, Zhang T, Xu M, Pan Y, Lu J, Long M, Tanyi JL, Montone KT, Fan Y, Hu X, Zhang Y, Zhang L.
      Recurrent copy-number alterations, mutations, and transcript fusions of the genes encoding CDKs/cyclins are characterized in >10,000 tumors. Genomic alterations of CDKs/cyclins are dominantly driven by copy number aberrations. In contrast to cell-cycle-related CDKs/cyclins, which are globally amplified, transcriptional CDKs/cyclins recurrently lose copy numbers across cancers. Although mutations and transcript fusions are relatively rare events, CDK12 exhibits recurrent mutations in multiple cancers. Among the transcriptional CDKs, CDK7 and CDK12 show the most significant copy number loss and mutation, respectively. Their genomic alterations are correlated with increased sensitivities to DNA-damaging drugs. Inhibition of CDK7 preferentially represses the expression of genes in the DNA-damage-repair pathways and impairs the activity of homologous recombination. Low-dose CDK7 inhibitor treatment sensitizes cancer cells to PARP inhibitor-induced DNA damage and cell death. Our analysis provides genomic information for identification and prioritization of drug targets for CDKs and reveals rationales for treatment strategies.
    Keywords:  CDK7 inhibitor; PARP inhibitor; cancer; combination therapy; cyclin-dependent kinase; genomic alteration
    DOI:  https://doi.org/10.1016/j.celrep.2020.107884
  8. DNA Repair (Amst). 2020 Jun 18. pii: S1568-7864(20)30148-8. [Epub ahead of print]94 102900
    Zagnoli-Vieira G, Caldecott KW.
      DNA topoisomerases alleviate the torsional stress that is generated by processes that are central to genome metabolism such as transcription and DNA replication. To do so, these enzymes generate an enzyme intermediate known as the cleavage complex in which the topoisomerase is covalently linked to the termini of a DNA single- or double-strand break. Whilst cleavage complexes are normally transient they can occasionally become abortive, creating protein-linked DNA breaks that threaten genome stability and cell survival; a process promoted and exploited in the cancer clinic by the use of topoisomerase 'poisons'. Here, we review the consequences to genome stability and human health of abortive topoisomerase-induced DNA breakage and the cellular pathways that cells have adopted to mitigate them, with particular focus on an important class of enzymes known as tyrosyl-DNA phosphodiesterases.
    Keywords:  DNA damage; DNA strand break; DSB; PARP; SSB; TDP1; TDP2; TOP1; TOP2; Topoisomerases
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102900
  9. Curr Genet. 2020 Jul 15.
    Lehmann CP, Jiménez-Martín A, Branzei D, Tercero JA.
      Homologous recombination is essential for the maintenance of genome integrity but must be strictly controlled to avoid dangerous outcomes that produce the opposite effect, genomic instability. During unperturbed chromosome replication, recombination is globally inhibited at ongoing DNA replication forks, which helps to prevent deleterious genomic rearrangements. This inhibition is carried out by Srs2, a helicase that binds to SUMOylated PCNA and has an anti-recombinogenic function at replication forks. However, at damaged stalled forks, Srs2 is counteracted and DNA lesion bypass can be achieved by recombination-mediated template switching. In budding yeast, template switching is dependent on Rad5. In the absence of this protein, replication forks stall in the presence of DNA lesions and cells die. Recently, we showed that in cells lacking Rad5 that are exposed to DNA damage or replicative stress, elimination of the conserved Mgs1/WRNIP1 ATPase allows an alternative mode of DNA damage bypass that is driven by recombination and facilitates completion of chromosome replication and cell viability. We have proposed that Mgs1 is important to prevent a potentially harmful salvage pathway of recombination at damaged stalled forks. In this review, we summarize our current understanding of how unwanted recombination is prevented at damaged stalled replication forks.
    Keywords:  DNA damage bypass; DNA recombination; DNA replication forks; Genome stability; Mgs1; Template switching
    DOI:  https://doi.org/10.1007/s00294-020-01095-7
  10. Semin Cell Dev Biol. 2020 Jul 08. pii: S1084-9521(19)30093-X. [Epub ahead of print]
    Tye S, Ronson GE, Morris JR.
      In response to replication hindrances, DNA replication forks frequently stall and are remodelled into a four-way junction. In such a structure the annealed nascent strand is thought to resemble a DNA double-strand break and remodelled forks are vulnerable to nuclease attack by MRE11 and DNA2. Proteins that promote the recruitment, loading and stabilisation of RAD51 onto single-stranded DNA for homology search and strand exchange in homologous recombination (HR) repair and inter-strand cross-link repair also act to set up RAD51-mediated protection of nascent DNA at stalled replication forks. However, despite the similarities of these pathways, several lines of evidence indicate that fork protection is not simply analogous to the RAD51 loading step of HR. Protection of stalled forks not only requires separate functions of a number of recombination proteins, but also utilises nucleases important for the resection steps of HR in alternative ways. Here we discuss how fork protection arises and how its differences with HR give insights into the differing contexts of these two pathways.
    Keywords:  BRCA1; BRCA2; Fork reversal; Homologous recombination; RAD51; Replication fork protection
    DOI:  https://doi.org/10.1016/j.semcdb.2020.07.004
  11. Cells. 2020 Jul 10. pii: E1665. [Epub ahead of print]9(7):
    Carusillo A, Mussolino C.
      DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.
    Keywords:  CRISPR-Cas; DNA damage; DNA damage response (DDR); HDR; NHEJ; cancer; cell-cycle; gene editing; genome integrity
    DOI:  https://doi.org/10.3390/cells9071665
  12. DNA Repair (Amst). 2020 Jun 29. pii: S1568-7864(20)30151-8. [Epub ahead of print]94 102903
    Amidon KM, Eichman BF.
      Abasic (AP) sites are one of the most frequently occurring types of DNA damage. They lead to DNA strand breaks, interstrand DNA crosslinks, and block transcription and replication. Mutagenicity of AP sites arises from translesion synthesis (TLS) by error-prone bypass polymerases. Recently, a new cellular response to AP sites was discovered, in which the protein HMCES (5-hydroxymethlycytosine (5hmC) binding, embryonic stem cell-specific) forms a stable, covalent DNA-protein crosslink (DPC) to AP sites at stalled replication forks. The stability of the HMCES-DPC prevents strand cleavage by endonucleases and mutagenic bypass by TLS polymerases. Crosslinking is carried out by a unique SRAP (SOS Response Associated Peptidase) domain conserved across all domains of life. Here, we review the collection of recently reported SRAP crystal structures from human HMCES and E. coli YedK, which provide a unified basis for SRAP specificity and a putative chemical mechanism of AP site crosslinking. We discuss the structural and chemical basis for the stability of the SRAP DPC and how it differs from covalent protein-DNA intermediates in DNA lyase catalysis of strand scission.
    Keywords:  Abasic site; DNA lyase; DNA-protein crosslink; HMCES; SRAP; Thiazolidine
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102903
  13. Microb Cell. 2020 Apr 24. 7(7): 190-198
    Ortega P, García-Pichardo D, San Martin-Alonso M, Rondón AG, Gómez-González B, Aguilera A.
      The stability and function of eukaryotic genomes is closely linked to histones and to chromatin structure. The state of the chromatin not only affects the probability of DNA to undergo damage but also DNA repair. DNA damage can result in genetic alterations and subsequent development of cancer and other genetic diseases. Here, we identified two mutations in conserved residues of histone H3 and histone H4 (H3E73Q and H4E53A) that increase recombinogenic DNA damage. Our results suggest that the accumulation of DNA damage in these histone mutants is largely independent on transcription and might arise as a consequence of problems occurring during DNA replication. This study uncovers the relevance of H3E73 and H4E53 residues in the protection of genome integrity.
    Keywords:  DNA replication; chromatin; histone mutants; recombination
    DOI:  https://doi.org/10.15698/mic2020.07.723
  14. J Vis Exp. 2020 Jun 26.
    de la Peña Avalos B, Dray E.
      Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance. There are two major ways of visualizing proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or "foci", that can be visualized by indirect immunofluorescence using specific antibodies. Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.
    DOI:  https://doi.org/10.3791/61447
  15. Nat Commun. 2020 Jul 17. 11(1): 3613
    Sarni D, Sasaki T, Irony Tur-Sinai M, Miron K, Rivera-Mulia JC, Magnuson B, Ljungman M, Gilbert DM, Kerem B.
      Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.
    DOI:  https://doi.org/10.1038/s41467-020-17448-2
  16. Int J Mol Sci. 2020 Jul 11. pii: E4910. [Epub ahead of print]21(14):
    Ferri A, Stagni V, Barilà D.
      Glioblastoma multiforme (GBM) is a severe brain tumor whose ability to mutate and adapt to therapies is at the base for the extremely poor survival rate of patients. Despite multiple efforts to develop alternative forms of treatment, advances have been disappointing and GBM remains an arduous tumor to treat. One of the leading causes for its strong resistance is the innate upregulation of DNA repair mechanisms. Since standard therapy consists of a combinatory use of ionizing radiation and alkylating drugs, which both damage DNA, targeting the DNA damage response (DDR) is proving to be a beneficial strategy to sensitize tumor cells to treatment. In this review, we will discuss how recent progress in the availability of the DDR kinase inhibitors will be key for future therapy development. Further, we will examine the principal existing DDR inhibitors, with special focus on those currently in use for GBM clinical trials.
    Keywords:  DDR inhibitors; DNA damage response; glioblastoma
    DOI:  https://doi.org/10.3390/ijms21144910
  17. Cells. 2020 Jul 09. pii: E1657. [Epub ahead of print]9(7):
    Hanscom T, McVey M.
      Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.
    Keywords:  alt-EJ; chromosome rearrangements; microhomology-mediated end joining; polymerase theta; resection
    DOI:  https://doi.org/10.3390/cells9071657
  18. DNA Repair (Amst). 2020 Jul 10. pii: S1568-7864(20)30175-0. [Epub ahead of print]94 102926
    Sun Y, Saha LK, Saha S, Jo U, Pommier Y.
      Topoisomerases play a pivotal role in ensuring DNA metabolisms during replication, transcription and chromosomal segregation. To manage DNA topology, topoisomerases generate break(s) in the DNA backbone by forming transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and topoisomerase catalytic tyrosyl residues. Topoisomerases have been identified as the cellular targets of a variety of anti-cancer drugs (e.g. topotecan, irinotecan, etoposide and doxorubicin, and antibiotics (e.g. ciprofloxacin and levofloxacin). These drugs, as well as other exogenous and endogenous agents, convert the transient TOPcc into persistent TOPcc, which we refer to as topoisomerase DNA-protein crosslinks (TOP-DPC) that challenge genome integrity and lead to cell death if left unrepaired. Proteolysis of the bulky protein component of TOP-DPC (debulking) is a poorly understood repair process employed across eukaryotes. TOP-DPC proteolysis can be achieved either by the ubiquitin-proteasome pathway (UPP) or by non-proteasomal proteases, which are typified by the metalloprotease SPRTN/WSS1. Debulking of TOP-DPC exposes the phosphotyrosyl bonds, hence enables tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) to access and cleave the bonds. In this review, we focus on current knowledge of the protease pathways for debulking TOP-DPC and highlighting recent advances in understanding the mechanisms regulating the proteolytic repair pathways. We also discuss the avenues that are being exploited to target the proteolytic repair pathways for improving the clinical outcome of topoisomerase inhibitors.
    Keywords:  DNA-protein crosslinks; SPRTN; SUMOylation; Topoisomerases; Ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102926
  19. Annu Rev Genet. 2020 Jul 14.
    Bonilla B, Hengel SR, Grundy MK, Bernstein KA.
      Accurate DNA repair and replication are critical for genomic stability and cancer prevention. RAD51 and its gene family are key regulators of DNA fidelity through diverse roles in double-strand break repair, replication stress, and meiosis. RAD51 is an ATPase that forms a nucleoprotein filament on single-stranded DNA. RAD51 has the function of finding and invading homologous DNA sequences to enable accurate and timely DNA repair. Its paralogs, which arose from ancient gene duplications of RAD51, have evolved to regulate and promote RAD51 function. Underscoring its importance, misregulation of RAD51, and its paralogs, is associated with diseases such as cancer and Fanconi anemia. In this review, we focus on mammalian RAD51 structure and function and highlight the use of model systems to enable mechanistic understanding of RAD51 cellular roles. We also discuss how misregulation of the RAD51 gene family members contributes to disease and consider new approaches to pharmacologically inhibit RAD51. Expected final online publication date for the Annual Review of Genetics, Volume 54 is November 23, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-genet-021920-092410
  20. Cancers (Basel). 2020 Jul 14. pii: E1901. [Epub ahead of print]12(7):
    Fernandes SG, Dsouza R, Pandya G, Kirtonia A, Tergaonkar V, Lee SY, Garg M, Khattar E.
      Telomeres are the ends of linear chromosomes comprised of repetitive nucleotide sequences in humans. Telomeres preserve chromosomal stability and genomic integrity. Telomere length shortens with every cell division in somatic cells, eventually resulting in replicative senescence once telomere length becomes critically short. Telomere shortening can be overcome by telomerase enzyme activity that is undetectable in somatic cells, while being active in germline cells, stem cells, and immune cells. Telomeres are bound by a shelterin complex that regulates telomere lengthening as well as protects them from being identified as DNA damage sites. Telomeres are transcribed by RNA polymerase II, and generate a long noncoding RNA called telomeric repeat-containing RNA (TERRA), which plays a key role in regulating subtelomeric gene expression. Replicative immortality and genome instability are hallmarks of cancer and to attain them cancer cells exploit telomere maintenance and telomere protection mechanisms. Thus, understanding the role of telomeres and their associated proteins in cancer initiation, progression and treatment is very important. The present review highlights the critical role of various telomeric components with recently established functions in cancer. Further, current strategies to target various telomeric components including human telomerase reverse transcriptase (hTERT) as a therapeutic approach in human malignancies are discussed.
    Keywords:  cancer; gene expression; genomic stability; telomerase; telomeres; therapeutic strategies
    DOI:  https://doi.org/10.3390/cancers12071901
  21. Nat Commun. 2020 Jul 17. 11(1): 3615
    Kamp JA, van Schendel R, Dilweg IW, Tijsterman M.
      Failure to preserve the integrity of the genome is a hallmark of cancer. Recent studies have revealed that loss of the capacity to repair DNA breaks via homologous recombination (HR) results in a mutational profile termed BRCAness. The enzymatic activity that repairs HR substrates in BRCA-deficient conditions to produce this profile is currently unknown. We here show that the mutational landscape of BRCA1 deficiency in C. elegans closely resembles that of BRCA1-deficient tumours. We identify polymerase theta-mediated end-joining (TMEJ) to be responsible: knocking out polq-1 suppresses the accumulation of deletions and tandem duplications in brc-1 and brd-1 animals. We find no additional back-up repair in HR and TMEJ compromised animals; non-homologous end-joining does not affect BRCAness. The notion that TMEJ acts as an alternative to HR, promoting the genome alteration of HR-deficient cells, supports the idea that polymerase theta is a promising therapeutic target for HR-deficient tumours.
    DOI:  https://doi.org/10.1038/s41467-020-17455-3
  22. Prostate Cancer Prostatic Dis. 2020 Jul 13.
    Ozturk S, Mathur D, Zhou RW, Mulholland D, Parsons R.
      BACKGROUND: The loss of PTEN function presents in up to 50% of late-stage prostate cancers, and is therefore a potential target for therapeutics. PTEN-deficient cells depend on de novo pyrimidine synthesis, a feature that can present a vulnerability.METHODS: We utilized in vitro growth assays and in vivo xenograft models to test the effect of de novo pyrimidine synthesis inhibition on prostate cell lines.
    RESULTS: Here, we demonstrate that PTEN-deficient prostate cancer cell lines are susceptible to inhibition of de novo pyrimidine synthesis by leflunomide. Tumor growth inhibition was observed in vitro and in vivo following leflunomide treatment, and is likely due to an overwhelming accumulation of DNA damage.
    CONCLUSIONS: Our work highlights that synthetic lethality arises upon the combination of PTEN loss and leflunomide treatment in prostate cancer, and may present a therapeutic opportunity for this patient population.
    DOI:  https://doi.org/10.1038/s41391-020-0251-1
  23. J Vis Exp. 2020 Jun 23.
    Meir A, Kong M, Xue C, Greene EC.
      Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.
    DOI:  https://doi.org/10.3791/61320
  24. Methods Mol Biol. 2020 ;2175 79-94
    Mao P, Wyrick JJ.
      Exposure to ultraviolet (UV) radiation is the major risk factor for skin cancers. UV induces helix-distorting DNA damage such as cyclobutane pyrimidine dimers (CPDs). If not repaired, CPDs can strongly block DNA and RNA polymerases and cause mutagenesis or cell death. Nucleotide excision repair (NER) is critical for the removal of UV-induced photolesions including CPDs in the cell. Investigating CPD formation and repair across the genome is important for understanding the mechanisms by which these lesions promote somatic mutations in skin cancers. Here we describe a high-throughput, single nucleotide-resolution damage mapping method named CPD sequencing (CPD-seq) for genome-wide analysis of UV-induced CPDs. Protocols for CPD-seq library preparation in yeast and human cells, as well as bioinformatics identification of the CPD damage site, are detailed below.
    Keywords:  Bioinformatics analysis; DNA damage; DNA repair; Sequencing library; Single base resolution; UV damage sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-0763-3_7
  25. Proc Natl Acad Sci U S A. 2020 Jul 15. pii: 202006231. [Epub ahead of print]
    Yuan Z, Schneider S, Dodd T, Riera A, Bai L, Yan C, Magdalou I, Ivanov I, Stillman B, Li H, Speck C.
      DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.
    Keywords:  ATPase; DNA replication; Mcm2-7 helicase; cryo-EM; origin recognition complex
    DOI:  https://doi.org/10.1073/pnas.2006231117
  26. Cell Death Dis. 2020 Jul 17. 11(7): 543
    Patel H, Sheikh MS, Huang Y.
      Esophageal Cancer-Related Gene 2 (ECRG2) is a recently identified tumor suppressor, its regulation and involvement in DNA damage response are unknown. Here, we show that DNA damage-induced ECRG2 upregulation coincided with p53 activation and occurred in a p53-dependent manner. We identified two p53-binding sites within ECRG2 promoter and found the promoter activity, mRNA, and protein expression to be regulated by p53. We show that DNA damage significantly enhanced p53 binding to ECRG2 promoter at the anticipated p53-binding sites. We identified a novel natural ECRG2 promoter variant harboring a small deletion that exists in the genomes of ~38.5% of world population and showed this variant to be defective in responding to p53 and DNA-damage. ECRG2 overexpression induced cancer cell death; ECRG2 gene disruption enhanced cell survival following anticancer drug treatments even when p53 was induced. We showed that lower expression of ECRG2 in multiple human malignancies correlated with reduced disease-free survival in patients. Collectively, our novel findings indicate that ECRG2 is an important target of p53 during DNA damage-induced response and plays a critical role in influencing cancer cell sensitivity to DNA damage-inducing cancer therapeutics.
    DOI:  https://doi.org/10.1038/s41419-020-2728-1
  27. Metabolites. 2020 Jul 11. pii: E285. [Epub ahead of print]10(7):
    Alfarouk KO, Ahmed SBM, Elliott RL, Benoit A, Alqahtani SS, Ibrahim ME, Bashir AHH, Alhoufie STS, Elhassan GO, Wales CC, Schwartz LH, Ali HS, Ahmed A, Forde PF, Devesa J, Cardone RA, Fais S, Harguindey S, Reshkin SJ.
      The Pentose Phosphate Pathway (PPP) is one of the key metabolic pathways occurring in living cells to produce energy and maintain cellular homeostasis. Cancer cells have higher cytoplasmic utilization of glucose (glycolysis), even in the presence of oxygen; this is known as the "Warburg Effect". However, cytoplasmic glucose utilization can also occur in cancer through the PPP. This pathway contributes to cancer cells by operating in many different ways: (i) as a defense mechanism via the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to prevent apoptosis, (ii) as a provision for the maintenance of energy by intermediate glycolysis, (iii) by increasing genomic material to the cellular pool of nucleic acid bases, (iv) by promoting survival through increasing glycolysis, and so increasing acid production, and (v) by inducing cellular proliferation by the synthesis of nucleic acid, fatty acid, and amino acid. Each step of the PPP can be upregulated in some types of cancer but not in others. An interesting aspect of this metabolic pathway is the shared regulation of the glycolytic and PPP pathways by intracellular pH (pHi). Indeed, as with glycolysis, the optimum activity of the enzymes driving the PPP occurs at an alkaline pHi, which is compatible with the cytoplasmic pH of cancer cells. Here, we outline each step of the PPP and discuss its possible correlation with cancer.
    Keywords:  cancer; enzyme; metabolism; pH; redox
    DOI:  https://doi.org/10.3390/metabo10070285
  28. Cancer Res. 2020 Jul 15. 80(14): 2977-2978
    Cai C.
      Cancer cells with germline deleterious mutations of BRCA1 or BRCA2 are deficient in homologous recombination repair and therefore sensitive to PARP inhibitor treatment. However, wild-type BRCA1/2-expressing cells with defects in other DNA damage repair pathway components may also exhibit "BRCAness," which in combination with PARP inhibition can similarly induce synthetic lethality. In this issue of Cancer Research, Luo and colleagues report a novel mechanism by which BRCA1 protein degradation in response to DNA double-strand breaks is regulated by prolyl isomerase Pin1. Inactivation of Pin1 can establish BRCAness in cancer cells and thus sensitize cells to PARP inhibitor treatment.See related articles by Luo et al., p. 3033.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1451
  29. Methods Mol Biol. 2020 ;2175 95-108
    Poetsch AR.
      DNA is constantly challenged by chemical modification and spontaneous loss of its bases, which results in apurinic sites (AP-sites). In addition to the direct route, modified bases may be converted into AP-sites through enzymatic removal of the base as part of the base excision repair pathway. Here we present the method AP-seq, which allows enriching and sequencing AP-sites genome-wide. Quantification of DNA recovery (AP-quant) allows for relative quantification of global AP-sites, and AP-site pulldown followed by qPCR (AP-qPCR) allows for site-specific damage assessment. Taking advantage of glycosylases that specifically excise modified bases also in vitro, this method allows not only to address the genomic distribution of AP-sites but also to detect base modifications, e.g., 8-oxo-7,8-dihydroguanine (8-oxoG). AP-quant, AP-qPCR, and AP-seq can be applied to investigate quantitatively the relative amount and genome specificity of DNA damage and repair, effects of radiation, as well as multiple other questions around AP-sites and base modifications.
    Keywords:  8-oxoG; AP-sites; Base excision repair; Base modification; DNA damage; DNA repair; Epigenomics; Genomics; Radiation
    DOI:  https://doi.org/10.1007/978-1-0716-0763-3_8
  30. Mol Cell Biol. 2020 Jul 13. pii: MCB.00232-20. [Epub ahead of print]
    Singh M, Bacolla A, Chaudhary S, Hunt CR, Pandita S, Chauhan R, Gupta A, Tainer JA, Pandita TK.
      The DNA and protein complex known as chromatin is subject to post-translational modifications (PTMs), which regulate cellular functions, such that PTM dysregulation can lead to disease including cancer. One critical PTM is acetylation/deacetylation, which is being investigated as a means to develop targeted cancer therapies. The histone acetyl transferase (HAT) family of proteins perform histone acetylation. In humans, MOF (hMOF), a member of the MYST family of HATs, acetylate histone H4 at lysine 16 (H4K16ac). MOF-mediated acetylation plays a critical roles in the DNA damage response (DDR) and embryonic stem cell development. Functionally, MOF is found in two distinct complexes NSL in humans (Non Specific Lethal) and MSL (Male Specific Lethal) in flies. NSL complex is also being able to acetylate additional histone H4 sites. Dysregulation of MOF activity occurs in multiple cancers including ovarian, medulloblastoma, breast, colorectal and lung cancer. Bioinformatics analysis of KAT8, the gene encoding hMOF, indicated it is highly overexpressed in kidney tumors as part of a concerted gene co-expression program that would support high levels of chromosome segregation and cell proliferation. The linkage between MOF and tumor proliferation suggests there are additional functions of MOF that remain to be discovered.
    DOI:  https://doi.org/10.1128/MCB.00232-20
  31. Cells. 2020 Jul 13. pii: E1678. [Epub ahead of print]9(7):
    Rahman S, Canny MD, Buschmann TA, Latham MP.
      The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease. Here, we discuss, in a structural and biochemical context, mutations in each of the three MRN components that have been associated with diseases such as ataxia telangiectasia-like disorder (ATLD), Nijmegen breakage syndrome (NBS), NBS-like disorder (NBSLD) and certain types of cancers. Overall, deepening our understanding of disease-causing mutations of the MRN complex at the structural and biochemical level is foundational to the future aim of treating diseases associated with these aberrations.
    Keywords:  ATLD; DNA double-strand break repair; MRE11-RAD50-NBS1; NBS; cancer mutations
    DOI:  https://doi.org/10.3390/cells9071678
  32. Front Mol Biosci. 2020 ;7 122
    Pećina-Šlaus N, Kafka A, Salamon I, Bukovac A.
      The acquisition of genomic instability is one of the key characteristics of the cancer cell, and microsatellite instability (MSI) is an important segment of this phenomenon. This review aims to describe the mismatch DNA repair (MMR) system whose deficiency is responsible for MSI and discuss the cellular roles of MMR genes. Malfunctioning of the MMR repair pathway increases the mutational burden of specific cancers and is often involved in its etiology, sometimes as an influential bystander and sometimes as the main driving force. Detecting the presence of MSI has for a long time been an important part of clinical diagnostics, but has still not achieved its full potential. The MSI blueprints of specific tumors are useful for precize grading, evaluation of cancer chance and prognosis and to help us understand how and why therapy-resistant cancers arise. Furthermore, evidence indicates that MSI is an important predictive biomarker for the application of immunotherapy.
    Keywords:  MMR; MSI; cancer; genomic instability; microsatellite instability; mismatch repair
    DOI:  https://doi.org/10.3389/fmolb.2020.00122
  33. Int J Mol Sci. 2020 Jul 15. pii: E5004. [Epub ahead of print]21(14):
    Serebrovskaya EO, Podvalnaya NM, Dudenkova VV, Efremova AS, Gurskaya NG, Gorbachev DA, Luzhin AV, Kantidze OL, Zagaynova EV, Shram SI, Lukyanov KA.
      Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.
    Keywords:  DNA damage respose; FRET; PAR; WWE-domai; fluorescent protein; sensor
    DOI:  https://doi.org/10.3390/ijms21145004
  34. Nat Immunol. 2020 Jul 13.
    Vardhana SA, Hwee MA, Berisa M, Wells DK, Yost KE, King B, Smith M, Herrera PS, Chang HY, Satpathy AT, van den Brink MRM, Cross JR, Thompson CB.
      The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.
    DOI:  https://doi.org/10.1038/s41590-020-0725-2
  35. Proc Natl Acad Sci U S A. 2020 Jul 15. pii: 201918517. [Epub ahead of print]
    Hao P, LeBlanc SJ, Case BC, Elston TC, Hingorani MM, Erie DA, Weninger KR.
      DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch.
    Keywords:  MMR; MutS; mismatch repair; smFRET
    DOI:  https://doi.org/10.1073/pnas.1918517117
  36. Cells. 2020 Jul 11. pii: E1671. [Epub ahead of print]9(7):
    Krokidis MG, D'Errico M, Pascucci B, Parlanti E, Masi A, Ferreri C, Chatgilialoglu C.
      Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3-6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.
    Keywords:  CSA; CSB; free radicals; isotope dilution LC-MS/MS; oxidatively generated DNA damage; oxygen concentration
    DOI:  https://doi.org/10.3390/cells9071671
  37. Cell Chem Biol. 2020 Jul 16. pii: S2451-9456(20)30232-4. [Epub ahead of print]27(7): 877-887.e14
    Wigle TJ, Blackwell DJ, Schenkel LB, Ren Y, Church WD, Desai HJ, Swinger KK, Santospago AG, Majer CR, Lu AZ, Niepel M, Perl NR, Vasbinder MM, Keilhack H, Kuntz KW.
      Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
    Keywords:  NanoBRET; PARP7; TIPARP; bioluminescence resonance energy transfer (BRET); cellular biophysics; mono(ADP-ribosylation) (MARylation); nicotinamide adenine dinucleotide (NAD(+)); poly(ADP-ribose) polymerase (PARP); probe displacement; time-resolved fluorescence energy transfer (TR-FRET)
    DOI:  https://doi.org/10.1016/j.chembiol.2020.06.009
  38. J Clin Lab Anal. 2020 Jul 16. e23457
    Attia F, Fathy S, Anani M, Hassan A, Attia F, Ibrahim G, Elazab M.
      BACKGROUND: Several biomarkers of gemcitabine effectiveness have been studied in cancers, but less so in hepatocellular carcinoma (HCC), which is identified as the fifth most common cancer worldwide. Investigation of human equilibrative nucleoside transporter-1 (HENT-1) and deoxycytidine kinase (DCK), genes involved in gemcitabine uptake and metabolism, can be beneficial in the selection of potential cancer patients who could be responding to the treatment.AIM: To study HENT-1 and DCK gene expression in HCC patients with different protocols of treatment.
    METHODS: Using real-time PCR, we analyzed expression levels of HENT-1 and DCK genes from peripheral blood samples of 109 patients (20 controls & 89 HCC patients) between March 2015 and March 2017. All the 89 HCC patients received the antioxidants selenium (Se) and vitamin E (Vit.E) either alone (45 patients) or in combination with gemcitabine (24 patients) or radiofrequency ablation (RFA) (20 patients).
    RESULTS: There was a significant increase in HENT-1 expression levels in HCC patients treated with Se and Vit.E alone as compared to controls (P ˂ .0001), while there was no significant difference between HCC patients treated with gemcitabine or RFA as compared to controls. In contrast, expression of DCK was significantly increased in all groups of HCC patients as compared to controls (P ˂ .0001).
    CONCLUSIONS: HENT-1 and DCK mRNA expressions are important markers of HCC and for GEM effect and GEM sensitivity in patients with HCC. This could be beneficial in the selection of HCC patients sensitive to gemcitabine to avoid subjecting resistant patients to unnecessary chemotherapy.
    Keywords:  deoxycytidine kinase; gemcitabine; hepatocellular carcinoma; human equilibrative nucleoside transporter-1
    DOI:  https://doi.org/10.1002/jcla.23457