bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒06‒06
forty-two papers selected by
Sean Rudd
Karolinska Institutet
  1. ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.
  2. Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing.
  3. SETD2-mediated H3K14 trimethylation promotes ATR activation and stalled replication fork restart in response to DNA replication stress.
  4. Regulation of DNA Replication Licensing and Re-Replication by Cdt1.
  5. Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.
  6. O-GlcNAcylation Affects the Pathway Choice of DNA Double-Strand Break Repair.
  7. RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells.
  8. Chromatin dynamics and DNA replication roadblocks.
  9. Interplay between DNA replication stress, chromatin dynamics and DNA-damage response for the maintenance of genome stability.
  10. X-ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double-strand break repair impacting cell and cancer biology.
  11. The Ubiquitin Proteasome System in Genome Stability and Cancer.
  12. SUMO-Targeted Ubiquitin Ligases and Their Functions in Maintaining Genome Stability.
  13. RAD51AP1 mediates RAD51 activity through nucleosome interaction.
  14. DNA polymerase η is a substrate for calpain: A possible mechanism for pol η retention in UV induced replication foci.
  15. High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures.
  16. Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line.
  17. Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.
  18. A Potential Role for HUWE1 in Modulating Cisplatin Sensitivity.
  19. High Temperature Drives Topoisomerase Mediated Chromosomal Break Repair Pathway Choice.
  20. SIK2 kinase synthetic lethality is driven by spindle assembly defects in FANCA-deficient cells.
  21. Emerging Roles of SKP2 in Cancer Drug Resistance.
  22. A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli.
  23. Acceleration or Brakes: Which Is Rational for Cell Cycle-Targeting Neuroblastoma Therapy?
  24. Inhibition of DNA Repair in Combination with Temozolomide or Dianhydrogalactiol Overcomes Temozolomide-Resistant Glioma Cells.
  25. Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage.
  26. Towards a Structural Mechanism for Sister Chromatid Cohesion Establishment at the Eukaryotic Replication Fork.
  27. LEDGF/p75 Is Required for an Efficient DNA Damage Response.
  28. Metabolic Aspects of Adenosine Functions in the Brain.
  29. Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models.
  30. ATM: Main Features, Signaling Pathways, and Its Diverse Roles in DNA Damage Response, Tumor Suppression, and Cancer Development.
  31. Mitotic syndicates Aurora Kinase B (AURKB) and mitotic arrest deficient 2 like 2 (MAD2L2) in cohorts of DNA damage response (DDR) and tumorigenesis.
  32. Breast Cancer Predisposition Genes and Synthetic Lethality.
  33. Function and molecular mechanisms of APE2 in genome and epigenome integrity.
  34. Recognition and Repair of Oxidatively Generated DNA Lesions in Plasmid DNA by a Facilitated Diffusion Mechanism.
  35. Survival-Critical Genes Associated with Copy Number Alterations in Lung Adenocarcinoma.
  36. Combination of consensus and ensemble docking strategies for the discovery of human dihydroorotate dehydrogenase inhibitors.
  37. Whole-Cell and Mitochondrial dNTP Pool Quantification from Cells and Tissues.
  38. Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma.
  39. Ex vivo analysis of DNA repair targeting in extreme rare cutaneous apocrine sweat gland carcinoma.
  40. Mitochondrial DNA: cellular genotoxic stress sentinel.
  41. Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.
  42. Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment.
  1. Mol Cell. 2021 May 25. pii: S1097-2765(21)00365-8. [Epub ahead of print]
    ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.
    Daniela Di Marcantonio, Esteban Martinez, Joice S Kanefsky, Jacklyn M Huhn, Rashid Gabbasov, Anushk Gupta, John J Krais, Suraj Peri, YinFei Tan, Tomasz Skorski, Adrienne Dorrance, Ramiro Garzon, Aaron R Goldman, Hsin-Yao Tang, Neil Johnson, Stephen M Sykes.
      Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.
    Keywords:  AML; ATF3; ATF4; cell cycle; differentiation; leukemia; metabolism; purines; pyrimidines; serine
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.008
  2. Int J Mol Sci. 2021 May 07. pii: 4959. [Epub ahead of print]22(9):
    Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing.
    Lilas Courtot, Elodie Bournique, Chrystelle Maric, Laure Guitton-Sert, Miguel Madrid-Mencía, Vera Pancaldi, Jean-Charles Cadoret, Jean-Sébastien Hoffmann, Valérie Bergoglio.
      DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.
    Keywords:  DNA damage; DNA replication stress; DNA replication timing; chromatin accessibility
    DOI:  https://doi.org/10.3390/ijms22094959
  3. Proc Natl Acad Sci U S A. 2021 Jun 08. pii: e2011278118. [Epub ahead of print]118(23):
    SETD2-mediated H3K14 trimethylation promotes ATR activation and stalled replication fork restart in response to DNA replication stress.
    Qian Zhu, Qiaoyan Yang, Xiaopeng Lu, Hui Wang, Lili Tong, Zheng Li, Ge Liu, Yantao Bao, Xingzhi Xu, Luo Gu, Jian Yuan, Xiangyu Liu, Wei-Guo Zhu.
      Ataxia telangiectasia and Rad3 related (ATR) activation after replication stress involves a cascade of reactions, including replication protein A (RPA) complex loading onto single-stranded DNA and ATR activator loading onto chromatin. The contribution of histone modifications to ATR activation, however, is unclear. Here, we report that H3K14 trimethylation responds to replication stress by enhancing ATR activation. First, we confirmed that H3K14 monomethylation, dimethylation, and trimethylation all exist in mammalian cells, and that both SUV39H1 and SETD2 methyltransferases can catalyze H3K14 trimethylation in vivo and in vitro. Interestingly, SETD2-mediated H3K14 trimethylation markedly increases in response to replication stress induced with hydroxyurea, a replication stress inducer. Under these conditions, SETD2-mediated H3K14me3 recruited the RPA complex to chromatin via a direct interaction with RPA70. The increase in H3K14me3 levels was abolished, and RPA loading was attenuated when SETD2 was depleted or H3K14 was mutated. Rather, the cells were sensitive to replication stress such that the replication forks failed to restart, and cell-cycle progression was delayed. These findings help us understand how H3K14 trimethylation links replication stress with ATR activation.
    Keywords:  ATR activation; H3K14 trimethylation; RPA; SETD2; replication stress
    DOI:  https://doi.org/10.1073/pnas.2011278118
  4. Int J Mol Sci. 2021 May 14. pii: 5195. [Epub ahead of print]22(10):
    Regulation of DNA Replication Licensing and Re-Replication by Cdt1.
    Hui Zhang.
      In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.
    Keywords:  CRL4Cdt2; Cdt1; Cdt2; DNA re-replication; DNA repair synthesis; DNA replication; PCNA; Replication licensing; genome instability
    DOI:  https://doi.org/10.3390/ijms22105195
  5. Nucleic Acids Res. 2021 Jun 04. pii: gkab460. [Epub ahead of print]
    Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.
    Dan Yu, John R Horton, Jie Yang, Taraneh Hajian, Masoud Vedadi, Cari A Sagum, Mark T Bedford, Robert M Blumenthal, Xing Zhang, Xiaodong Cheng.
      MettL3-MettL14 methyltransferase complex has been studied widely for its role in RNA adenine methylation. This complex is also recruited to UV- and X-ray exposed DNA damaged sites, and its methyltransfer activity is required for subsequent DNA repair, though in theory this could result from RNA methylation of short transcripts made at the site of damage. We report here that MettL3-MettL14 is active in vitro on double-stranded DNA containing a cyclopyrimidine dimer - a major lesion of UV radiation-induced products - or an abasic site or mismatches. Furthermore, N6-methyladenine (N6mA) decreases misincorporation of 8-oxo-guanine (8-oxoG) opposite to N6mA by repair DNA polymerases. When 8-oxoG is nevertheless incorporated opposite N6mA, the methylation inhibits N6mA excision from the template (correct) strand by the adenine DNA glycosylase (MYH), implying that the methylation decreases inappropriate misrepair. Finally, we observed that the N6mA reader domain of YTHDC1, which is also recruited to sites of DNA damage, binds N6mA that is located across from a single-base gap between two canonical DNA helices. This YTHDC1 complex with a gapped duplex is structurally similar to DNA complexes with FEN1 and GEN1 - two members of the nuclease family that act in nucleotide excision repair, mismatch repair and homologous recombination, and which incise distinct non-B DNA structures. Together, the parts of our study provide a plausible mechanism for N6mA writer and reader proteins acting directly on lesion-containing DNA, and suggest in vivo experiments to test the mechanisms involving methylation of adenine.
    DOI:  https://doi.org/10.1093/nar/gkab460
  6. Int J Mol Sci. 2021 May 27. pii: 5715. [Epub ahead of print]22(11):
    O-GlcNAcylation Affects the Pathway Choice of DNA Double-Strand Break Repair.
    Sera Averbek, Burkhard Jakob, Marco Durante, Nicole B Averbeck.
      Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation.
    Keywords:  DNA-DSB repair; O-GlcNAcylation; chromatin remodeling; high LET; ionizing radiation; particle irradiation
    DOI:  https://doi.org/10.3390/ijms22115715
  7. DNA Repair (Amst). 2021 May 18. pii: S1568-7864(21)00093-8. [Epub ahead of print] 103137
    RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells.
    Michael J Cargill, Alicia Morales, Shashidhar Ravishankar, Edus H Warren.
      Recent studies have suggested that human RNA helicase, DDX3X, is important for DNA repair, but little is known about the nuclear activity of this protein. In vitro analysis of nuclear DDX3X interactions and localization with DNA damage pointed to a direct role for DDX3X in the DNA damage response. We aimed to investigate whether DDX3X plays a direct role in the DNA damage response in live cells. In order to track nuclear DDX3X, we generated a nuclear-export deficient DDX3X mutant construct and performed microirradiation in live cells. We found that DDX3X accumulates at sites of microirradiation shortly after DNA damage induction. We further found DDX3X recruitment to be mediated by its intrinsically disordered domains, similar to other RNA binding proteins that are recruited to sites of DNA damage. Inhibition of liquid-liquid phase separation also reduced DDX3X recruitment. CRISPR/Cas9-mediated knockout of PARP1 ablated DDX3X recruitment, which was restored upon transgenic expression of wild-type PARP1 but not catalytically inactive PARP1, suggesting that DDX3X recruitment is PARP1-dependent.
    Keywords:  DDX3X; DNA damage; RNA helicase
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103137
  8. DNA Repair (Amst). 2021 May 23. pii: S1568-7864(21)00096-3. [Epub ahead of print]104 103140
    Chromatin dynamics and DNA replication roadblocks.
    Ian Hammond-Martel, Alain Verreault, Hugo Wurtele.
      A broad spectrum of spontaneous and genotoxin-induced DNA lesions impede replication fork progression. The DNA damage response that acts to promote completion of DNA replication is associated with dynamic changes in chromatin structure that include two distinct processes which operate genome-wide during S-phase. The first, often referred to as histone recycling or parental histone segregation, is characterized by the transfer of parental histones located ahead of replication forks onto nascent DNA. The second, known as de novo chromatin assembly, consists of the deposition of new histone molecules onto nascent DNA. Because these two processes occur at all replication forks, their potential to influence a multitude of DNA repair and DNA damage tolerance mechanisms is considerable. The purpose of this review is to provide a description of parental histone segregation and de novo chromatin assembly, and to illustrate how these processes influence cellular responses to DNA replication roadblocks.
    Keywords:  DNA repair; DNA replication; De novo chromatin assembly; Histone chaperones; Histone modifications
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103140
  9. Mutat Res. 2021 Jan-Jun;787:pii: S1383-5742(20)30066-1. [Epub ahead of print]787 108346
    Interplay between DNA replication stress, chromatin dynamics and DNA-damage response for the maintenance of genome stability.
    Maddalena Mognato, Susanne Burdak-Rothkamm, Kai Rothkamm.
      DNA replication stress is a major source of DNA damage, including double-stranded breaks that promote DNA damage response (DDR) signaling. Inefficient repair of such lesions can affect genome integrity. During DNA replication different factors act on chromatin remodeling in a coordinated way. While recent studies have highlighted individual molecular mechanisms of interaction, less is known about the orchestration of chromatin changes under replication stress. In this review we attempt to explore the complex relationship between DNA replication stress, DDR and genome integrity in mammalian cells, taking into account the role of chromatin disposition as an important modulator of DNA repair. Recent data on chromatin restoration and epigenetic re-establishment after DNA replication stress are reviewed.
    Keywords:  Chromatin remodeling; DNA damage; DNA repair; DNA replication; Genome integrity
    DOI:  https://doi.org/10.1016/j.mrrev.2020.108346
  10. Protein Sci. 2021 May 30.
    X-ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double-strand break repair impacting cell and cancer biology.
    Michal Hammel, John A Tainer.
      Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIVBRCT -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.
    Keywords:  DNA repair; backbone conformation; cancer; dynamic structures; functional dynamics; genome stability; quantitative flexibility; supramolecular structures; unstructured regions
    DOI:  https://doi.org/10.1002/pro.4133
  11. Cancers (Basel). 2021 May 06. pii: 2235. [Epub ahead of print]13(9):
    The Ubiquitin Proteasome System in Genome Stability and Cancer.
    Jonathan J Morgan, Lisa J Crawford.
      Faithful DNA replication during cellular division is essential to maintain genome stability and cells have developed a sophisticated network of regulatory systems to ensure its integrity. Disruption of these control mechanisms can lead to loss of genomic stability, a key hallmark of cancer. Ubiquitination is one of the most abundant regulatory post-translational modifications and plays a pivotal role in controlling replication progression, repair of DNA and genome stability. Dysregulation of the ubiquitin proteasome system (UPS) can contribute to the initiation and progression of neoplastic transformation. In this review we provide an overview of the UPS and summarize its involvement in replication and replicative stress, along with DNA damage repair. Finally, we discuss how the UPS presents as an emerging source for novel therapeutic interventions aimed at targeting genomic instability, which could be utilized in the treatment and management of cancer.
    Keywords:  DNA damage; DNA replication; cancer; genome stability; ubiquitination
    DOI:  https://doi.org/10.3390/cancers13092235
  12. Int J Mol Sci. 2021 May 20. pii: 5391. [Epub ahead of print]22(10):
    SUMO-Targeted Ubiquitin Ligases and Their Functions in Maintaining Genome Stability.
    Ya-Chu Chang, Marissa K Oram, Anja-Katrin Bielinsky.
      Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, mitosis, and transcription. They operate during unperturbed conditions and in response to challenges, such as genotoxic stress. These E3 ubiquitin ligases modify their target substrates by catalyzing ubiquitin chains that form different linkages, resulting in proteolytic or non-proteolytic outcomes. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Furthermore, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. In this review, we focus on the molecular mechanisms by which STUbLs help to maintain genome stability across different species.
    Keywords:  RNF111; RNF4; STUbL; SUMO; Slx5/Slx8; genome stability; ubiquitin
    DOI:  https://doi.org/10.3390/ijms22105391
  13. J Biol Chem. 2021 May 28. pii: S0021-9258(21)00642-6. [Epub ahead of print] 100844
    RAD51AP1 mediates RAD51 activity through nucleosome interaction.
    Elena Pires, Neelam Sharma, Platon Selemenakis, Bo Wu, Yuxin Huang, Dauren S Alimbetov, Weixing Zhao, Claudia Wiese.
      RAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs), the minimum basic unit of chromatin in which ∼2 super-helical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.
    Keywords:  Homologous recombination DNA repair; RAD51; RAD51AP1; chromatin; mono-nucleosomes; synaptic complex assembly
    DOI:  https://doi.org/10.1016/j.jbc.2021.100844
  14. J Cell Sci. 2021 Jun 03. pii: jcs.258637. [Epub ahead of print]
    DNA polymerase η is a substrate for calpain: A possible mechanism for pol η retention in UV induced replication foci.
    Jo-Ann Nettersheim, Régine Janel-Bintz, Lauriane Kuhn, Agnès M Cordonnier.
      DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across ultraviolet radiation-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here we report that pol η interacts with the calpain small subunit-1 (CAPNS1), in a yeast two-hybrid screening. This interaction is functional as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η is found to occur at position 465 leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin results in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.
    Keywords:  DNA damage response; Translesion DNA synthesis; DNA polymerase η; Replication foci; Calpain protease; CAPNS1
    DOI:  https://doi.org/10.1242/jcs.258637
  15. Int J Mol Sci. 2021 May 14. pii: 5201. [Epub ahead of print]22(10):
    High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures.
    Maria Dede, Silvia Napolitano, Anna Melati, Valentina Pirota, Giovanni Maga, Emmanuele Crespan.
      Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.
    Keywords:  RER; RNaseH2; misincorporated ribonucleotides; non-B DNA
    DOI:  https://doi.org/10.3390/ijms22105201
  16. Int J Mol Sci. 2021 May 28. pii: 5814. [Epub ahead of print]22(11):
    Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line.
    Hadar Golan Berman, Pooja Chauhan, Shira Shalev, Hiba Hassanain, Avital Parnas, Sheera Adar.
      Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.
    Keywords:  DNA damage; DNA repair; cancer; chromatin; cisplatin; nucleotide excision repair; resistance; transcription
    DOI:  https://doi.org/10.3390/ijms22115814
  17. Biochemistry. 2021 Jun 03.
    Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.
    Jenna Thomforde, Iwen Fu, Freddys Rodriguez, Suresh S Pujari, Suse Broyde, Natalia Tretyakova.
      DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.
    DOI:  https://doi.org/10.1021/acs.biochem.1c00130
  18. Cells. 2021 May 20. pii: 1262. [Epub ahead of print]10(5):
    A Potential Role for HUWE1 in Modulating Cisplatin Sensitivity.
    Stijn Wenmaekers, Bastiaan J Viergever, Gunjan Kumar, Onno Kranenburg, Peter C Black, Mads Daugaard, Richard P Meijer.
      Cisplatin is a widely used antineoplastic agent, whose efficacy is limited by primary and acquired therapeutic resistance. Recently, a bladder cancer genome-wide CRISPR/Cas9 knock-out screen correlated cisplatin sensitivity to multiple genetic biomarkers. Among the screen's top hits was the HECT domain-containing ubiquitin E3 ligase (HUWE1). In this review, HUWE1 is postulated as a therapeutic response modulator, affecting the collision between platinum-DNA adducts and the replication fork, the primary cytotoxic action of platins. HUWE1 can alter the cytotoxic response to platins by targeting essential components of the DNA damage response including BRCA1, p53, and Mcl-1. Deficiency of HUWE1 could lead to enhanced DNA damage repair and a dysfunctional apoptotic apparatus, thereby inducing resistance to platins. Future research on the relationship between HUWE1 and platins could generate new mechanistic insights into therapy resistance. Ultimately, HUWE1 might serve as a clinical biomarker to tailor cancer treatment strategies, thereby improving cancer care and patient outcomes.
    Keywords:  DNA damage response; apoptosis; biomarkers; chemotherapy; resistance; translational medicine research; ubiquitination
    DOI:  https://doi.org/10.3390/cells10051262
  19. Cancers (Basel). 2021 May 12. pii: 2315. [Epub ahead of print]13(10):
    High Temperature Drives Topoisomerase Mediated Chromosomal Break Repair Pathway Choice.
    Mohamed E Ashour, Walaa Allam, Waheba Elsayed, Reham Atteya, Menattallah Elserafy, Sameh Magdeldin, Mohamed K Hassan, Sherif F El-Khamisy.
      Cancer-causing mutations often arise from inappropriate DNA repair, yet acute exposure to DNA damage is widely used to treat cancer. The challenge remains in how to specifically induce excessive DNA damage in cancer cells while minimizing the undesirable effects of genomic instability in noncancerous cells. One approach is the acute exposure to hyperthermia, which suppresses DNA repair and synergizes with radiotherapy and chemotherapy. An exception, however, is the protective effect of hyperthermia on topoisomerase targeting therapeutics. The molecular explanation for this conundrum remains unclear. Here, we show that hyperthermia suppresses the level of topoisomerase mediated single- and double-strand breaks induced by exposure to topoisomerase poisons. We further uncover that, hyperthermia suppresses hallmarks of genomic instability induced by topoisomerase targeting therapeutics by inhibiting nuclease activities, thereby channeling repair to error-free pathways driven by tyrosyl-DNA phosphodiesterases. These findings provide an explanation for the protective effect of hyperthermia from topoisomerase-induced DNA damage and may help to explain the inverse relationship between cancer incidence and temperature. They also pave the way for the use of controlled heat as a therapeutic adjunct to topoisomerase targeting therapeutics.
    Keywords:  TDP1; TDP2; ageing; cancer; genomic instability; hyperthermia; topoisomerase
    DOI:  https://doi.org/10.3390/cancers13102315
  20. Mol Oncol. 2021 May 31.
    SIK2 kinase synthetic lethality is driven by spindle assembly defects in FANCA-deficient cells.
    Ka-Kui Chan, Zahi Abdul-Sater, Aditya Sheth, Dana K Mitchell, Richa Sharma, Donna M Edwards, Ying He, Grzegorz Nalepa, Steven D Rhodes, D Wade Clapp, Elizabeth A Sierra Potchanant.
      The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here we performed a kinome-wide synthetic lethality screen in FANCA-/- fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA-deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA co-localizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2-M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole-induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA-deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA-disrupted cancers.
    Keywords:  Fanconi anemia pathway; SIK2; cancer; spindle assembly checkpoint; synthetic lethality
    DOI:  https://doi.org/10.1002/1878-0261.13027
  21. Cells. 2021 May 10. pii: 1147. [Epub ahead of print]10(5):
    Emerging Roles of SKP2 in Cancer Drug Resistance.
    Ting Wu, Xinsheng Gu, Hongmei Cui.
      More than half of all cancer patients receive chemotherapy, however, some of them easily acquire drug resistance. Resistance to chemotherapy has become a massive obstacle to achieve high rates of pathological complete response during cancer therapy. S-phase kinase-associated protein 2 (Skp2), as an E3 ligase, was found to be highly correlated with drug resistance and poor prognosis. In this review, we summarize the mechanisms that Skp2 confers to drug resistance, including the Akt-Skp2 feedback loop, Skp2-p27 pathway, cell cycle and mitosis regulation, EMT (epithelial-mesenchymal transition) property, enhanced DNA damage response and repair, etc. We also addressed novel molecules that either inhibit Skp2 expression or target Skp2-centered interactions, which might have vast potential for application in clinics and benefit cancer patients in the future.
    Keywords:  Akt; DNA damage response and repair; EMT; Skp2; cell cycle; drug resistance; inhibitors; mitosis; p27
    DOI:  https://doi.org/10.3390/cells10051147
  22. Proc Natl Acad Sci U S A. 2021 Jun 08. pii: e2026595118. [Epub ahead of print]118(23):
    A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli.
    Yardena Silas, Esti Singer, Koyeli Das, Norbert Lehming, Ophry Pines.
      Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.
    Keywords:  AlkB; DNA damage; fumarase; metabolite signaling; tricarboxylic acid cycle
    DOI:  https://doi.org/10.1073/pnas.2026595118
  23. Biomolecules. 2021 May 18. pii: 750. [Epub ahead of print]11(5):
    Acceleration or Brakes: Which Is Rational for Cell Cycle-Targeting Neuroblastoma Therapy?
    Kiyohiro Ando, Akira Nakagawara.
      Unrestrained proliferation is a common feature of malignant neoplasms. Targeting the cell cycle is a therapeutic strategy to prevent unlimited cell division. Recently developed rationales for these selective inhibitors can be subdivided into two categories with antithetical functionality. One applies a "brake" to the cell cycle to halt cell proliferation, such as with inhibitors of cell cycle kinases. The other "accelerates" the cell cycle to initiate replication/mitotic catastrophe, such as with inhibitors of cell cycle checkpoint kinases. The fate of cell cycle progression or arrest is tightly regulated by the presence of tolerable or excessive DNA damage, respectively. This suggests that there is compatibility between inhibitors of DNA repair kinases, such as PARP inhibitors, and inhibitors of cell cycle checkpoint kinases. In the present review, we explore alterations to the cell cycle that are concomitant with altered DNA damage repair machinery in unfavorable neuroblastomas, with respect to their unique genomic and molecular features. We highlight the vulnerabilities of these alterations that are attributable to the features of each. Based on the assessment, we offer possible therapeutic approaches for personalized medicine, which are seemingly antithetical, but both are promising strategies for targeting the altered cell cycle in unfavorable neuroblastomas.
    Keywords:  11q loss; DNA damage response; MYCN; RAS; cell cycle; checkpoint; inhibitor; neuroblastoma; replication stress; telomere
    DOI:  https://doi.org/10.3390/biom11050750
  24. Cancers (Basel). 2021 May 24. pii: 2570. [Epub ahead of print]13(11):
    Inhibition of DNA Repair in Combination with Temozolomide or Dianhydrogalactiol Overcomes Temozolomide-Resistant Glioma Cells.
    Shigeo Ohba, Kei Yamashiro, Yuichi Hirose.
      Resistance to temozolomide and intratumoral heterogeneity contribute to the poor prognosis of glioma. The mechanisms of temozolomide resistance can vary within a heterogeneous tumor. Temozolomide adds a methyl group to DNA. The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to a futile DNA mismatch repair cycle, formation of double-strand breaks, and eventual cell death when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair (BER). The study aim was to elucidate temozolomide resistance mechanisms and identify methods to overcome temozolomide resistance in glioma. Several temozolomide-resistant clones were analyzed. Increased homologous recombination and mismatch repair system deficiencies contributed to temozolomide resistance. Inhibition of homologous recombination resensitized resistant cells with high homologous recombination efficiency. For the mismatch repair-deficient cells, inhibition of BER by PARP inhibitor potentiated temozolomide-induced cytotoxicity. Dianhydrogalactiol is a bifunctional DNA-targeting agent that forms N7-alkylguanine and inter-strand DNA crosslinks. Dianhydrogalactiol reduced the proliferation of cells independent of MGMT and mismatch repair, inducing DNA double-strand breaks and apoptosis in temozolomide-resistant cells. Further, inhibition of chk1 or homologous recombination enhanced dianhydrogalactiol-induced cytotoxicity in the cells. Selecting treatments most appropriate to the types of resistance mechanisms can potentially improve the prognosis of glioma.
    Keywords:  PAPR inhibitor; dianhydrogalactiol; drug resistance; glioma; temozolomide
    DOI:  https://doi.org/10.3390/cancers13112570
  25. PLoS One. 2021 ;16(6): e0252587
    Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage.
    Yuriko Inomata, Takuya Abe, Masataka Tsuda, Shunichi Takeda, Kouji Hirota.
      Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη-/-, POLι-/-, POLκ-/-, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη-/- cells, but not POLι-/- or POLκ-/- cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη-/- cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.
    DOI:  https://doi.org/10.1371/journal.pone.0252587
  26. Biology (Basel). 2021 May 26. pii: 466. [Epub ahead of print]10(6):
    Towards a Structural Mechanism for Sister Chromatid Cohesion Establishment at the Eukaryotic Replication Fork.
    Sarah S Henrikus, Alessandro Costa.
      Cohesion between replicated chromosomes is essential for chromatin dynamics and equal segregation of duplicated genetic material. In the G1 phase, the ring-shaped cohesin complex is loaded onto duplex DNA, enriching at replication start sites, or "origins". During the same phase of the cell cycle, and also at the origin sites, two MCM helicases are loaded as symmetric double hexamers around duplex DNA. During the S phase, and through the action of replication factors, cohesin switches from encircling one parental duplex DNA to topologically enclosing the two duplicated DNA filaments, which are known as sister chromatids. Despite its vital importance, the structural mechanism leading to sister chromatid cohesion establishment at the replication fork is mostly elusive. Here we review the current understanding of the molecular interactions between the replication machinery and cohesin, which support sister chromatid cohesion establishment and cohesin function. In particular, we discuss how cryo-EM is shedding light on the mechanisms of DNA replication and cohesin loading processes. We further expound how frontier cryo-EM approaches, combined with biochemistry and single-molecule fluorescence assays, can lead to understanding the molecular basis of sister chromatid cohesion establishment at the replication fork.
    Keywords:  DNA replication; cohesin; cryo-EM; microscopy; replisome; sister chromatid cohesion establishment
    DOI:  https://doi.org/10.3390/biology10060466
  27. Int J Mol Sci. 2021 May 30. pii: 5866. [Epub ahead of print]22(11):
    LEDGF/p75 Is Required for an Efficient DNA Damage Response.
    Victoria Liedtke, Christian Schröder, Dirk Roggenbuck, Romano Weiss, Ralf Stohwasser, Peter Schierack, Stefan Rödiger, Lysann Schenk.
      Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.
    Keywords:  CRISPR/Cas9; DNA damage signaling; LEDGF; ubiquitination; γH2AX
    DOI:  https://doi.org/10.3390/ijms22115866
  28. Front Pharmacol. 2021 ;12 672182
    Metabolic Aspects of Adenosine Functions in the Brain.
    Mercedes Garcia-Gil, Marcella Camici, Simone Allegrini, Rossana Pesi, Maria Grazia Tozzi.
      Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5'-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington's disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.
    Keywords:  5′-nucleotidases; S-adenosylhomocysteine hydrolase; adenosine; adenosine deaminase; adenosine kinase; brain; metabolism; nucleoside transporters
    DOI:  https://doi.org/10.3389/fphar.2021.672182
  29. J Clin Invest. 2021 Jun 01. pii: 146256. [Epub ahead of print]131(11):
    Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models.
    Manal Mehibel, Yu Xu, Caiyun G Li, Eui Jung Moon, Kaushik N Thakkar, Anh N Diep, Ryan K Kim, Joshua D Bloomstein, Yiren Xiao, Julien Bacal, Joshua C Saldivar, Quynh-Thu Le, Karlene A Cimprich, Erinn B Rankin, Amato J Giaccia.
      Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.
    Keywords:  Cancer; Cell Biology; DNA repair; Hypoxia; Oncology
    DOI:  https://doi.org/10.1172/JCI146256
  30. Genes (Basel). 2021 May 30. pii: 845. [Epub ahead of print]12(6):
    ATM: Main Features, Signaling Pathways, and Its Diverse Roles in DNA Damage Response, Tumor Suppression, and Cancer Development.
    Liem Minh Phan, Abdol-Hossein Rezaeian.
      ATM is among of the most critical initiators and coordinators of the DNA-damage response. ATM canonical and non-canonical signaling pathways involve hundreds of downstream targets that control many important cellular processes such as DNA damage repair, apoptosis, cell cycle arrest, metabolism, proliferation, oxidative sensing, among others. Of note, ATM is often considered a major tumor suppressor because of its ability to induce apoptosis and cell cycle arrest. However, in some advanced stage tumor cells, ATM signaling is increased and confers remarkable advantages for cancer cell survival, resistance to radiation and chemotherapy, biosynthesis, proliferation, and metastasis. This review focuses on addressing major characteristics, signaling pathways and especially the diverse roles of ATM in cellular homeostasis and cancer development.
    Keywords:  ATM; DNA damage repair; DNA damage response; autophagy; cancer; cellular homeostasis; hypoxia; mitophagy; oxidative sensing; pexophagy
    DOI:  https://doi.org/10.3390/genes12060845
  31. Mutat Res. 2021 Jan-Jun;787:pii: S1383-5742(21)00013-2. [Epub ahead of print]787 108376
    Mitotic syndicates Aurora Kinase B (AURKB) and mitotic arrest deficient 2 like 2 (MAD2L2) in cohorts of DNA damage response (DDR) and tumorigenesis.
    Rahaba Marima, Rodney Hull, Clement Penny, Zodwa Dlamini.
      Aurora Kinase B (AURKB) and Mitotic Arrest Deficient 2 Like 2 (MAD2L2) are emerging anticancer therapeutic targets. AURKB and MAD2L2 are the least well studied members of their protein families, compared to AURKA and MAD2L1. Both AURKB and MAD2L2 play a critical role in mitosis, cell cycle checkpoint, DNA damage response (DDR) and normal physiological processes. However, the oncogenic roles of AURKB and MAD2L2 in tumorigenesis and genomic instability have also been reported. DDR acts as an arbitrator for cell fate by either repairing the damage or directing the cell to self-destruction. While there is strong evidence of interphase DDR, evidence of mitotic DDR is just emerging and remains largely unelucidated. To date, inhibitors of the DDR components show effective anti-cancer roles. Contrarily, long-term resistance towards drugs that target only one DDR target is becoming a challenge. Targeting interactions between protein-protein or protein-DNA holds prominent therapeutic potential. Both AURKB and MAD2L2 play critical roles in the success of mitosis and their emerging roles in mitotic DDR cannot be ignored. Small molecule inhibitors for AURKB are in clinical trials. A few lead compounds towards MAD2L2 inhibition have been discovered. Targeting mitotic DDR components and their interaction is emerging as a potent next generation anti-cancer therapeutic target. This can be done by developing small molecule inhibitors for AURKB and MAD2L2, thereby targeting DDR components as anti-cancer therapeutic targets and/or targeting mitotic DDR. This review focuses on AURKB and MAD2L2 prospective synergy to deregulate the p53 DDR pathway and promote favourable conditions for uncontrolled cell proliferation.
    Keywords:  Aurora Kinase B (AURKB); DNA damage response (DDR); Mitotic arrest deficient 2 like 2 (MAD2L2); Small molecule inhibitor; Spindle assembly checkpoint (SAC); p53
    DOI:  https://doi.org/10.1016/j.mrrev.2021.108376
  32. Int J Mol Sci. 2021 May 25. pii: 5614. [Epub ahead of print]22(11):
    Breast Cancer Predisposition Genes and Synthetic Lethality.
    Hannah E Neiger, Emily L Siegler, Yihui Shi.
      BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.
    Keywords:  BRCA1/BRCA2; CPGs; DNA repair; PARPi; hereditary breast cancer; synthetic lethality
    DOI:  https://doi.org/10.3390/ijms22115614
  33. Mutat Res. 2021 Jan-Jun;787:pii: S1383-5742(20)30067-3. [Epub ahead of print]787 108347
    Function and molecular mechanisms of APE2 in genome and epigenome integrity.
    Yunfeng Lin, Anne McMahon, Garrett Driscoll, Sharon Bullock, Jianjun Zhao, Shan Yan.
      APE2 is a rising vital player in the maintenance of genome and epigenome integrity. In the past several years, a series of studies have shown the critical roles and functions of APE2. We seek to provide the first comprehensive review on several aspects of APE2 in genome and epigenome integrity. We first summarize the distinct functional domains or motifs within APE2 including EEP (endonuclease/exonuclease/phosphatase) domain, PIP box and Zf-GRF motifs from eight species (i.e., Homo sapiens, Mus musculus, Xenopus laevis, Ciona intestinalis, Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Trypanosoma cruzi). Then we analyze various APE2 nuclease activities and associated DNA substrates, including AP endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-5' exonuclease activities. We also examine several APE2 interaction proteins, including PCNA, Chk1, APE1, Myh1, and homologous recombination (HR) factors such as Rad51, Rad52, BRCA1, BRCA2, and BARD1. Furthermore, we provide insights into the roles of APE2 in various DNA repair pathways (base excision repair, single-strand break repair, and double-strand break repair), DNA damage response (DDR) pathways (ATR-Chk1 and p53-dependent), immunoglobulin class switch recombination and somatic hypermutation, as well as active DNA demethylation. Lastly, we summarize critical functions of APE2 in growth, development, and diseases. In this review, we provide the first comprehensive perspective which dissects all aspects of the multiple-function protein APE2 in genome and epigenome integrity.
    Keywords:  APE2; ATR-Chk1 pathway; DNA demethylation; DNA repair; Genome and epigenome integrity; Immune response
    DOI:  https://doi.org/10.1016/j.mrrev.2020.108347
  34. Biochem J. 2021 Jun 01. pii: BCJ20210095. [Epub ahead of print]
    Recognition and Repair of Oxidatively Generated DNA Lesions in Plasmid DNA by a Facilitated Diffusion Mechanism.
    Marina Kolbanovskiy, Abraham Aharonoff, Ana Helena Sales, Nicholas E Geacintov, Vladimir Shafirovich.
      The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1]>>[SpDNApl]) in contrast to monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with  NEIL1-DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1-DNA complexes.  The amplitude of the burst phase is limited in amplitude because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ » 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1-DNA complexes (τ-ns » 8 s). The estimated average DNA translocation distance of NEIL1 is ~80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of ~ 10.
    Keywords:  Base excision repair; DNA damage; DNA glycosylase; oxidative stress
    DOI:  https://doi.org/10.1042/BCJ20210095
  35. Cancers (Basel). 2021 May 25. pii: 2586. [Epub ahead of print]13(11):
    Survival-Critical Genes Associated with Copy Number Alterations in Lung Adenocarcinoma.
    Chinthalapally V Rao, Chao Xu, Mudassir Farooqui, Yuting Zhang, Adam S Asch, Hiroshi Y Yamada.
      Chromosome Instability (CIN) in tumors affects carcinogenesis, drug resistance, and recurrence/prognosis. Thus, it has a high impact on outcomes in clinic. However, how CIN occurs in human tumors remains elusive. Although cells with CIN (i.e., pre/early cancer cells) are proposed to be removed by apoptosis and/or a surveillance mechanism, this surveillance mechanism is poorly understood. Here we employed a novel data-mining strategy (Gene Expression to Copy Number Alterations [CNA]; "GE-CNA") to comprehensively identify 1578 genes that associate with CIN, indicated by genomic CNA as its surrogate marker, in human lung adenocarcinoma. We found that (a) amplification/insertion CNA is facilitated by over-expressions of DNA replication stressor and suppressed by a broad range of immune cells (T-, B-, NK-cells, leukocytes), and (b) deletion CNA is facilitated by over-expressions of mitotic regulator genes and suppressed predominantly by leukocytes guided by leukocyte extravasation signaling. Among the 39 CNA- and survival-associated genes, the purine metabolism (PPAT, PAICS), immune-regulating CD4-LCK-MEC2C and CCL14-CCR1 axes, and ALOX5 emerged as survival-critical pathways. These findings revealed a broad role of the immune system in suppressing CIN/CNA and cancer development in lung, and identified components representing potential targets for future chemotherapy, chemoprevention, and immunomodulation approaches for lung adenocarcinoma.
    Keywords:  genomic instability; lung tumor; tumor genomics; tumor heterogeneity
    DOI:  https://doi.org/10.3390/cancers13112586
  36. Sci Rep. 2021 Jun 01. 11(1): 11417
    Combination of consensus and ensemble docking strategies for the discovery of human dihydroorotate dehydrogenase inhibitors.
    Garri Chilingaryan, Narek Abelyan, Arsen Sargsyan, Karen Nazaryan, Andre Serobian, Hovakim Zakaryan.
      The inconsistencies in the performance of the virtual screening (VS) process, depending on the used software and structural conformation of the protein, is a challenging issue in the drug design and discovery field. Varying performance, especially in terms of early recognition of the potential hit compounds, negatively affects the whole process and leads to unnecessary waste of the time and resources. Appropriate application of the ensemble docking and consensus-scoring approaches can significantly increase reliability of the VS results. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway. It is considered as a valuable therapeutic target in cancer, autoimmune and viral diseases. Based on the conducted benchmark study and analysis of the effect of different combinations of the applied methods and approaches, here we suggested a structure-based virtual screening (SBVS) workflow that can be used to increase the reliability of VS.
    DOI:  https://doi.org/10.1038/s41598-021-91069-7
  37. Methods Mol Biol. 2021 ;2276 143-151
    Whole-Cell and Mitochondrial dNTP Pool Quantification from Cells and Tissues.
    Juan C Landoni, Liya Wang, Anu Suomalainen.
      Deoxynucleoside 5'-triphosphates (dNTPs) are the molecular building blocks for DNA synthesis, and their balanced concentration in the cell is fundamental for health. dNTP imbalance can lead to genomic instability and other metabolic disturbances, resulting in devastating mitochondrial diseases.The accurate and efficient measurement of dNTPs from different biological samples and cellular compartments is vital to understand the mechanisms behind these diseases and develop and scrutinize their possible treatments. This chapter describes an update on the most recent development of the traditional radiolabeled polymerase extension method and its adaptation for the measurement of whole-cell and mitochondrial dNTP pools from cultured cells and tissue samples. The solid-phase reaction setting enables an increase in efficiency, accuracy, and measurement scale.
    Keywords:  Mitochondrial DNA depletion syndrome; Nucleotide pools; Solid-phase detection; dNTP; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_10
  38. Nat Cancer. 2021 Feb;2(2): 189-200
    Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma.
    Shuai Wu, Takeshi Fukumoto, Jianhuang Lin, Timothy Nacarelli, Yemin Wang, Dionzie Ong, Heng Liu, Nail Fatkhutdinov, Joseph A Zundell, Sergey Karakashev, Wei Zhou, Lauren E Schwartz, Hsin-Yao Tang, Ronny Drapkin, Qin Liu, David G Huntsman, Andrew V Kossenkov, David W Speicher, Zachary T Schug, Chi Van Dang, Rugang Zhang.
      Alterations in components of the SWI/SNF chromatin-remodeling complex occur in ~20% of all human cancers. For example, ARID1A is mutated in up to 62% of clear cell ovarian carcinoma (OCCC), a disease currently lacking effective therapies. Here we show that ARID1A mutation creates a dependence on glutamine metabolism. SWI/SNF represses glutaminase (GLS1) and ARID1A inactivation upregulates GLS1. ARID1A inactivation increases glutamine utilization and metabolism through the tricarboxylic acid cycle to support aspartate synthesis. Indeed, glutaminase inhibitor CB-839 suppresses the growth of ARID1A mutant, but not wildtype, OCCCs in both orthotopic and patient-derived xenografts. In addition, glutaminase inhibitor CB-839 synergizes with immune checkpoint blockade anti-PDL1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation. Our data indicate that pharmacological inhibition of glutaminase alone or in combination with immune checkpoint blockade represents an effective therapeutic strategy for cancers involving alterations in the SWI/SNF complex such as ARID1A mutations.
    DOI:  https://doi.org/10.1038/s43018-020-00160-x
  39. Oncotarget. 2021 May 25. 12(11): 1100-1109
    Ex vivo analysis of DNA repair targeting in extreme rare cutaneous apocrine sweat gland carcinoma.
    Rami Mäkelä, Ville Härmä, Nibal Badra Fajardo, Greg Wells, Zoi Lygerou, Olle Sangfelt, Juha Kononen, Juha K Rantala.
      Cutaneous apocrine carcinoma is an extreme rare malignancy derived from a sweat gland. Histologically sweat gland cancers resemble metastatic mammary apocrine carcinomas, but the genetic landscape remains poorly understood. Here, we report a rare metastatic case with a PALB2 aberration identified previously as a familial susceptibility gene for breast cancer in the Finnish population. As PALB2 exhibits functions in the BRCA1/2-RAD51-dependent homologous DNA recombination repair pathway, we sought to use ex vivo functional screening to explore sensitivity of the tumor cells to therapeutic targeting of DNA repair. Drug screening suggested sensitivity of the PALB2 deficient cells to BET-bromodomain inhibition, and modest sensitivity to DNA-PKi, ATRi, WEE1i and PARPi. A phenotypic RNAi screen of 300 DNA repair genes was undertaken to assess DNA repair targeting in more detail. Core members of the HR and MMEJ pathways were identified to be essential for viability of the cells. RNAi inhibition of RAD52-dependent HR on the other hand potentiated the efficacy of a novel BETi ODM-207. Together these results describe the first ever CAC case with a BRCAness genetic background, evaluate combinatorial DNA repair targeting, and provide a data resource for further analyses of DNA repair targeting in PALB2 deficient cancers.
    Keywords:  DNA repair; PALB2; cutaneous apocrine sweat gland carcinoma; ex vivo drug screening ; rare cancer
    DOI:  https://doi.org/10.18632/oncotarget.27961
  40. Trends Biochem Sci. 2021 Jun 01. pii: S0968-0004(21)00106-7. [Epub ahead of print]
    Mitochondrial DNA: cellular genotoxic stress sentinel.
    Zheng Wu, Alva G Sainz, Gerald S Shadel.
      High copy number, damage prone, and lean on repair mechanisms are unique features of mitochondrial DNA (mtDNA) that are hard to reconcile with its essentiality for oxidative phosphorylation, the primary function ascribed to this maternally inherited component of our genome. We propose that mtDNA is also a genotoxic stress sentinel, as well as a direct second messenger of this type of cellular stress. Here, we discuss existing evidence for this sentinel/effector role through the ability of mtDNA to escape the confines of the mitochondrial matrix and activate nuclear DNA damage/repair responses via interferon-stimulated gene products and other downstream effectors. However, this arrangement may come at a cost, leading to cancer chemoresistance and contributing to inflammation, disease pathology, and aging.
    Keywords:  DNA repair; cGAS-STING; chemoresistance; interferon-stimulated gene (ISG); mtDNA; retrograde signaling
    DOI:  https://doi.org/10.1016/j.tibs.2021.05.004
  41. J Pharm Biomed Anal. 2021 May 19. pii: S0731-7085(21)00263-6. [Epub ahead of print]202 114152
    Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.
    Xiaomin Wang, Wouter H J Vaes, Esther van Duijn, Irene Nooijen, Zeen Tong, Daniel Lopes de Menezes, Stephen E Maxwell.
      We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (∼ 3.7 × 105) and BM (∼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/μg) DNA and 0.22 pgEq/μg RNA, and for BM analysis were 1.7 pgEq/μg DNA and 0.22 pgEq/μg RNA. A linear relationship (i.e., ∼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10-11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as ∼5 mL whole blood and ∼3 mL of BM from patients.
    Keywords:  Accelerator mass spectrometry; Azacitidine; DNA; Quantification; RNA
    DOI:  https://doi.org/10.1016/j.jpba.2021.114152
  42. Trends Cancer. 2021 May 29. pii: S2405-8033(21)00100-X. [Epub ahead of print]
    Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment.
    Anna M Chiarella, Yun K Ryu, Gulam A Manji, Anil K Rustgi.
      ATP hydrolysis and downstream signaling pathways in the extracellular space have a major impact upon tumor progression and metastasis. The complexity and interdependence of various cell types in the extracellular space have been increasingly appreciated in recent years. With increased awareness of the importance of this signaling pathway in the pathogenic development and progression of malignancies, there has been attention to therapeutic strategies targeting extracellular adenosine metabolism and signaling. This review summarizes the molecular and physiologic roles of extracellular ATP and adenosine in normal and disease states, and potential therapeutic applications.
    Keywords:  CD39; CD73; cancer; extracellular ATP; extracellular adenosine; purinergic signaling
    DOI:  https://doi.org/10.1016/j.trecan.2021.04.008
This page is being maintained by Thomas Krichel. It was last updated on 2021‒07‒23 at 10:23:05.