bims-numges 2021-01-31 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–01–31
28 papers selected by
Sean Rudd, Karolinska Institutet

Targeting IGF perturbs global replication through ribonucleotide reductase dysfunction.
Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair.
Single-strand DNA breaks cause replisome disassembly.
Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.
DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.
Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β.
The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.
SOX2-dependent expression of dihydroorotate dehydrogenase regulates oral squamous cell carcinoma cell proliferation.
Targeting Non-Oncogene Addiction for Cancer Therapy.
A novel role of DNA polymerase λ in translesion synthesis in conjunction with DNA polymerase ζ.
Histone Ubiquitination: An Integrative Signaling Platform in Genome Stability.
Apurinic/Apyrimidinic Endonuclease 1 and Tyrosyl-DNA Phosphodiesterase 1 Prevent Suicidal Covalent DNA-Protein Crosslink at Apurinic/Apyrimidinic Site.
A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors.
DNA damage-how and why we age?
On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability.
A comparative analysis of the mutagenicity of platinum-containing chemotherapeutic agents reveals direct and indirect mutagenic mechanisms.
Prospect of reprogramming replication licensing for cancer drug development.
PRPS1-mediated purine biosynthesis is critical for pluripotent stem cell survival and stemness.
A pilot clinical trial of oral tetrahydrouridine/decitabine for noncytotoxic epigenetic therapy of chemoresistant lymphoid malignancies.
Bloom syndrome DNA helicase deficiency is associated with oxidative stress and mitochondrial network changes.
DNA damage response and breast cancer development: Possible therapeutic applications of ATR, ATM, PARP, BRCA1 inhibition.
On the relevance of hydroxyl radical to purine DNA damage.
Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.
Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication.
A multi-omics approach to Epstein-Barr virus immortalization of B-cells reveals EBNA1 chromatin pioneering activities targeting nucleotide metabolism.
The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.
The ins and outs of serine and glycine metabolism in cancer.
PHGDH as a mechanism for resistance in metabolically-driven cancers.

Cancer Res. 2021 Jan 28. pii: canres.2860.2020. [Epub ahead of print]

Targeting IGF perturbs global replication through ribonucleotide reductase dysfunction.

Guillaume Rieunier, Xiaoning Wu, Letitia E Harris, Jack V Mills, Ashwin Nandakumar, Laura Colling, Elena Seraia, Stephanie B Hatch, Daniel V Ebner, Lisa K Folkes, Ulrike Weyer-Czernilofsky, Thomas Bogenrieder, Anderson J Ryan, Valentine M Macaulay.

Inhibition of IGF receptor (IGF-1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF-1R influences endogenous DNA damage. Here we demonstrate that IGF-1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF-1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF-1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF-1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF-1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF-1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM null cells and spheroids and caused regression of ATM null xenografts. This synthetic lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF-1R in alleviating replication stress, and the reciprocal IGF:ATM co-dependence we identify provides an approach to exploit this effect in ATM-deficient cancers.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-2860
PLoS Genet. 2021 Jan 25. 17(1): e1009322

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair.

Natasha C Koussa, Duncan J Smith.

DNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.

DOI: https://doi.org/10.1371/journal.pgen.1009322
Mol Cell. 2021 Jan 15. pii: S1097-2765(20)30960-6. [Epub ahead of print]

Single-strand DNA breaks cause replisome disassembly.

Kyle B Vrtis, James M Dewar, Gheorghe Chistol, R Alex Wu, Thomas G W Graham, Johannes C Walter.

  DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.

Keywords:  CMG; DNA repair; DNA replication; double-strand break; fork collapse; homologous recombination; single molecule; single-strand break

DOI:  https://doi.org/10.1016/j.molcel.2020.12.039
Blood. 2021 Jan 28. 137(4): 513-523

Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.

Jithma P Abeykoon, Xiaosheng Wu, Kevin E Nowakowski, Surendra Dasari, Jonas Paludo, S John Weroha, Chunling Hu, Xiaonan Hou, Jann N Sarkaria, Ann C Mladek, Jessica L Phillips, Andrew L Feldman, Aishwarya Ravindran, Rebecca L King, Justin Boysen, Mary J Stenson, Ryan M Carr, Michelle K Manske, Julian R Molina, Prashant Kapoor, Sameer A Parikh, Shaji Kumar, Steven I Robinson, Jia Yu, Judy C Boughey, Liewei Wang, Matthew P Goetz, Fergus J Couch, Mrinal M Patnaik, Thomas E Witzig.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

DOI: https://doi.org/10.1182/blood.2020009013
Blood Adv. 2021 Jan 26. 5(2): 438-450

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Kensuke Kayamori, Yurie Nagai, Cheng Zhong, Satoshi Kaito, Daisuke Shinoda, Shuhei Koide, Wakako Kuribayashi, Motohiko Oshima, Yaeko Nakajima-Takagi, Masayuki Yamashita, Naoya Mimura, Hans Jiro Becker, Kiyoko Izawa, Satoshi Yamazaki, Satoshi Iwano, Atsushi Miyawaki, Ryoji Ito, Kaoru Tohyama, William Lennox, Josephine Sheedy, Marla Weetall, Emiko Sakaida, Koutaro Yokote, Atsushi Iwama.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

DOI: https://doi.org/10.1182/bloodadvances.2020001461
J Biol Chem. 2020 Feb 07. pii: S0021-9258(17)49860-7. [Epub ahead of print]295(6): 1613-1622

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β.

Mallory R Smith, Khadijeh S Alnajjar, Nicole M Hoitsma, Joann B Sweasy, Bret D Freudenthal.

  During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Keywords:  8-oxoguanine (8-oxo-G); DNA damage; DNA polymerase; DNA repair; DNA replication; mutagenic nucleobase; nucleotidyl transferase reaction; oxidized ribonucleotide; r8-oxo-G lesion; structural biology

DOI:  https://doi.org/10.1074/jbc.RA119.011569
Nat Commun. 2021 01 26. 12(1): 585

The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.

Ann-Marie K Shorrocks, Samuel E Jones, Kaima Tsukada, Carl A Morrow, Zoulikha Belblidia, Johanna Shen, Iolanda Vendrell, Roman Fischer, Benedikt M Kessler, Andrew N Blackford.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

DOI: https://doi.org/10.1038/s41467-020-20818-5
Int J Oral Sci. 2021 Jan 29. 13(1): 3

SOX2-dependent expression of dihydroorotate dehydrogenase regulates oral squamous cell carcinoma cell proliferation.

Xuemei Qiu, Sheng Jiang, Yanxuan Xiao, Yumin He, Tao Ren, Lu Jiang, Rui Liu, Qianming Chen.

Oral squamous cell carcinoma (OSCC) become a heavy burden of public health, with approximately 300 000 newly diagnosed cases and 145 000 deaths worldwide per year. Nucleotide metabolism fuel DNA replication and RNA synthesis, which is indispensable for cell proliferation. But how tumor cells orchestrate nucleotide metabolic enzymes to support their rapid growth is largely unknown. Here we show that expression of pyrimidine metabolic enzyme dihydroorotate dehydrogenase (DHODH) is upregulated in OSCC tissues, compared to non-cancerous adjacent tissues. Enhanced expression of DHODH is correlated with a shortened patient survival time. Inhibition of DHODH by either shRNA or selective inhibitors impairs proliferation of OSCC cells and growth of tumor xenograft. Further, loss of functional DHODH imped de novo pyrimidine synthesis, and disrupt mitochondrial respiration probably through destabilizing the MICOS complex. Mechanistic study shows that transcriptional factor SOX2 plays an important role in the upregulation of DHODH in OSCC. Our findings add to the knowledge of how cancer cells co-opt nucleotide metabolism to support their rapid growth, and thereby highlight DHODH as a potential prognostic and therapeutic target for OSCC treatment.

DOI: https://doi.org/10.1038/s41368-020-00109-x
Biomolecules. 2021 Jan 20. pii: E129. [Epub ahead of print]11(2):

Targeting Non-Oncogene Addiction for Cancer Therapy.

Hae Ryung Chang, Eunyoung Jung, Soobin Cho, Young-Jun Jeon, Yonghwan Kim.

  While Next-Generation Sequencing (NGS) and technological advances have been useful in identifying genetic profiles of tumorigenesis, novel target proteins and various clinical biomarkers, cancer continues to be a major global health threat. DNA replication, DNA damage response (DDR) and repair, and cell cycle regulation continue to be essential systems in targeted cancer therapies. Although many genes involved in DDR are known to be tumor suppressor genes, cancer cells are often dependent and addicted to these genes, making them excellent therapeutic targets. In this review, genes implicated in DNA replication, DDR, DNA repair, cell cycle regulation are discussed with reference to peptide or small molecule inhibitors which may prove therapeutic in cancer patients. Additionally, the potential of utilizing novel synthetic lethal genes in these pathways is examined, providing possible new targets for future therapeutics. Specifically, we evaluate the potential of TONSL as a novel gene for targeted therapy. Although it is a scaffold protein with no known enzymatic activity, the strategy used for developing PCNA inhibitors can also be utilized to target TONSL. This review summarizes current knowledge on non-oncogene addiction, and the utilization of synthetic lethality for developing novel inhibitors targeting non-oncogenic addiction for cancer therapy.

Keywords:  DNA damage response; DNA repair; cancer therapy; non-oncogene addiction

DOI:  https://doi.org/10.3390/biom11020129
Life Sci Alliance. 2021 Apr;pii: e202000900. [Epub ahead of print]4(4):

A novel role of DNA polymerase λ in translesion synthesis in conjunction with DNA polymerase ζ.

Jung-Hoon Yoon, Debashree Basu, Karthi Sellamuthu, Robert E Johnson, Satya Prakash, Louise Prakash.

By extending synthesis opposite from a diverse array of DNA lesions, DNA polymerase (Pol) ζ performs a crucial role in translesion synthesis (TLS). In yeast and cancer cells, Rev1 functions as an indispensable scaffolding component of Polζ and it imposes highly error-prone TLS upon Polζ. However, for TLS that occurs during replication in normal human cells, Rev1 functions instead as a scaffolding component of Pols η, ι, and κ and Rev1-dependent TLS by these Pols operates in a predominantly error-free manner. The lack of Rev1 requirement for Polζ function in TLS in normal cells suggested that some other protein substitutes for this Rev1 role. Here, we identify a novel role of Polλ as an indispensable scaffolding component of Polζ. TLS studies opposite a number of DNA lesions support the conclusion that as an integral component, Polλ adapts Polζ-dependent TLS to operate in a predominantly error-free manner in human cells, essential for genome integrity and cellular homeostasis.

DOI: https://doi.org/10.26508/lsa.202000900
Trends Genet. 2021 Jan 20. pii: S0168-9525(20)30335-8. [Epub ahead of print]

Histone Ubiquitination: An Integrative Signaling Platform in Genome Stability.

Francesca Mattiroli, Lorenza Penengo.

  Complex mechanisms are in place to maintain genome stability. Ubiquitination of chromatin plays a central role in these mechanisms. The ever-growing complexity of the ubiquitin (Ub) code and of chromatin modifications and dynamics challenges our ability to fully understand how histone ubiquitination regulates genome stability. Here we review the current knowledge on specific, low-abundant histone ubiquitination events that are highly regulated within the cellular DNA damage response (DDR), with particular emphasis on the latest discovery of Ub phosphorylation as a novel regulator of the DDR signaling pathway. We discuss players involved and potential implications of histone (phospho)ubiquitination on chromatin structure, and we highlight exciting open questions for future research.

Keywords:  DDR; DNA damage response; H2AK15ub; RNF8/RNF168/USP51; chromatin modifications; pUbT12; ubiquitin phosphorylation

DOI:  https://doi.org/10.1016/j.tig.2020.12.005
Front Cell Dev Biol. 2020 ;8 617301

Apurinic/Apyrimidinic Endonuclease 1 and Tyrosyl-DNA Phosphodiesterase 1 Prevent Suicidal Covalent DNA-Protein Crosslink at Apurinic/Apyrimidinic Site.

Natalia A Lebedeva, Nadejda I Rechkunova, Anton V Endutkin, Olga I Lavrik.

  Bifunctional 8-oxoguanine-DNA glycosylase (OGG1), a crucial DNA-repair enzyme, removes from DNA 8-oxo-7,8-dihydroguanine (8-oxoG) with following cleavage of the arising apurinic/apyrimidinic (AP) site. The major enzyme in eukaryotic cells that catalyzes the cleavage of AP sites is AP endonuclease 1 (APE1). Alternatively, AP sites can be cleaved by tyrosyl-DNA phosphodiesterase 1 (TDP1) to initiate APE1-independent repair, thus expanding the ability of the base excision repair (BER) process. Poly(ADP-ribose) polymerase 1 (PARP1) is a regulatory protein of DNA repair. PARP2 is also activated in response to DNA damage and can be regarded as the BER participant. Here we analyze PARP1 and PARP2 interactions with DNA intermediates of the initial stages of the BER process (8-oxoG and AP-site containing DNA) and their interplay with the proteins recognizing and processing these DNA structures focusing on OGG1. OGG1 as well as PARP1 and PARP2 form covalent complex with AP site-containing DNA without borohydride reduction. AP site incision by APE1 or TDP1 removal of protein adducts but not proteins' PARylation prevent DNA-protein crosslinks.

Keywords:  8-oxoguanine-DNA glycosylase; AP endonuclease 1; DNA-protein crosslinks; apurinic/apyrimidinic site; poly(ADP-ribose) polymerases; tyrosyl-DNA phosphodiesterase 1

DOI:  https://doi.org/10.3389/fcell.2020.617301
Elife. 2021 Jan 25. pii: e64131. [Epub ahead of print]10

A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors.

Negar Afshar, Bilge Argunhan, Maierdan Palihati, Goki Taniguchi, Hideo Tsubouchi, Hiroshi Iwasaki.

  Homologous recombination (HR) is essential for maintaining genome stability. Although Rad51 is the key protein that drives HR, multiple auxiliary factors interact with Rad51 to potentiate its activity. Here, we present an interdisciplinary characterization of the interactions between Rad51 and these factors. Through structural analysis, we identified an evolutionarily conserved acidic patch of Rad51. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52. Furthermore, biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54, indicating that a single motif is important for the interaction with multiple auxiliary factors. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair.

Keywords:  DNA repair; Rad51; RecA; S. pombe; biochemistry; chemical biology; chromosomes; gene expression; genome stability; recombination; yeast

DOI:  https://doi.org/10.7554/eLife.64131
Elife. 2021 Jan 29. pii: e62852. [Epub ahead of print]10

DNA damage-how and why we age?

Matt Yousefzadeh, Chathurika Henpita, Rajesh Vyas, Carolina Soto-Palma, Paul Robbins, Laura Niedernhofer.

  Aging is a complex process that results in loss of the ability to reattain homeostasis following stress, leading, thereby, to increased risk of morbidity and mortality. Many factors contribute to aging, such as the time-dependent accumulation of macromolecular damage, including DNA damage. The integrity of the nuclear genome is essential for cellular, tissue, and organismal health. DNA damage is a constant threat because nucleic acids are chemically unstable under physiological conditions and vulnerable to attack by endogenous and environmental factors. To combat this, all organisms possess highly conserved mechanisms to detect and repair DNA damage. Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. The drawback is that these cancer avoidance mechanisms promote aging. Here, we review evidence that DNA damage plays a causal role in aging. We also provide evidence that genotoxic stress is linked to other cellular processes implicated as drivers of aging, including mitochondrial and metabolic dysfunction, altered proteostasis and inflammation. These links between damage to the genetic code and other pillars of aging support the notion that DNA damage could be the root of aging.

Keywords:  Aging; DNA damage; DNA repair; genetics; genome instability; genomics; progeria

DOI:  https://doi.org/10.7554/eLife.62852
J Biol Chem. 2020 Mar 27. pii: S0021-9258(17)48744-8. [Epub ahead of print]295(13): 4134-4170

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability.

Alexandra N Khristich, Sergei M Mirkin.

  Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?

Keywords:  DNA recombination; DNA repair; DNA replication; DNA structure; G-quadruplex; Huntington disease; R-loop; S-DNA; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); gene expression; genomic instability; hairpin; trinucleotide repeat disease; triplex H-DNA

DOI:  https://doi.org/10.1074/jbc.REV119.007678
Mutagenesis. 2021 Jan 27. pii: geab005. [Epub ahead of print]

A comparative analysis of the mutagenicity of platinum-containing chemotherapeutic agents reveals direct and indirect mutagenic mechanisms.

Bernadett Szikriszt, Ádám Póti, Eszter Németh, Nnennaya Kanu, Charles Swanton, Dávid Szüts.

  Platinum-based drugs are a mainstay of cancer chemotherapy. However, their mutagenic effect can increase tumour heterogeneity, contribute to the evolution of treatment resistance, and also induce secondary malignancies. We coupled whole genome sequencing with phenotypic investigations on two cell line models to compare the magnitude and examine the mechanism of mutagenicity of cisplatin, carboplatin and oxaliplatin. Cisplatin induced significantly more base substitution mutations than carboplatin or oxaliplatin when used at equitoxic concentrations on human TK6 or chicken DT40 cells, and also induced the highest number of short insertions and deletions. The analysis of base substitution spectra revealed that all three tested platinum drugs elicit both a direct mutagenic effect at purine dinucleotides, and an indirect effect of accelerating endogenous mutagenic processes. Whereas the direct mutagenic effect appeared to correlate with the level of DNA damage caused as assessed through histone H2AX phosphorylation and single cell agarose gel electrophoresis, the indirect mutagenic effects were equal. The different mutagenicity and DNA damaging effect of equitoxic platinum drug treatments suggests that DNA damage independent mechanisms significantly contribute to their cytotoxicity. Thus, the comparatively high mutagenicity of cisplatin should be taken into account in the design of chemotherapeutic regimens.

Keywords:  DNA adducts; DNA damage; carboplatin; cisplatin; mutation spectra; oxaliplatin

DOI:  https://doi.org/10.1093/mutage/geab005
Biomed Pharmacother. 2021 Jan 23. pii: S0753-3322(20)31383-4. [Epub ahead of print]136 111190

Prospect of reprogramming replication licensing for cancer drug development.

Isaac Kyei Barffour, Desmond Omane Acheampong.

  Eukaryotic chromosomal DNA replication is preceded by replication licensing which involves the identification of the origin of replication by origin recognition complex (ORC). The ORC loads pre-replication complexes (pre-RCs) through a series of tightly regulated mechanisms where the ORC interacts with Cdc6 to recruit cdt1-MCM2-7 complexes to the origin of replication. In eukaryotes, adherence to regulatory mechanisms of the replication program is required to ensure that all daughter cells carry the exact copy of genetic material as the parent cell. Failure of which leads to the development of genome instability syndromes like cancer, diabetes, etc. In an event of such occurrence, preventing cells from carrying the defaulted genetic material and passing it to other cells hinges on the regulation of chromosomal DNA replication. Thus, understanding the mechanisms underpinning chromosomal DNA replication and particularly replication licensing can expose druggable enzymes, effector molecules, and secondary messengers that can be targeted for diagnosis and therapeutic purposes. Effectively drugging these molecular markers to reprogram pre-replication events can be used to control the fate of chromosomal DNA replication for the treatment of genome instability disorders and in this case, cancer. This review discusses available knowledge of replication licensing in the contest of molecular drug discovery for the treatment of cancer.

Keywords:  Chromosomal DNA; Eukaryotic; Origin recognition complexes; Pre-replication complexes; Replication licensing

DOI:  https://doi.org/10.1016/j.biopha.2020.111190
Aging (Albany NY). 2021 Jan 20. 12

PRPS1-mediated purine biosynthesis is critical for pluripotent stem cell survival and stemness.

Yi Yang, Lili Song, Xia Huang, Yanan Feng, Yingwen Zhang, Yanfeng Liu, Shanshan Li, Zhiyan Zhan, Liang Zheng, Haizhong Feng, Yanxin Li.

  Pluripotent stem cells (PSCs) have a unique energetic and biosynthetic metabolism compared with typically differentiated cells. However, the metabolism profiling of PSCs and its underlying mechanism are still unclear. Here, we report PSCs metabolism profiling and identify the purine synthesis enzymes, phosphoribosyl pyrophosphate synthetase 1/2 (PRPS1/2), are critical for PSCs stemness and survival. Ultra-high performance liquid chromatography/mass spectroscopy (UHPLC-MS) analysis revealed that purine synthesis intermediate metabolite levels in PSCs are higher than that in somatic cells. Ectopic expression of PRPS1/2 did not improve purine biosynthesis, drug resistance, or stemness in PSCs. However, knockout of PRPS1 caused PSCs DNA damage and apoptosis. Depletion of PRPS2 attenuated PSCs stemness and assisted PSCs differentiation. Our finding demonstrates that PRPS1/2-mediated purine biosynthesis is critical for pluripotent stem cell stemness and survival.

Keywords:  PRPS1/2; apoptosis; pluripotent stem cells; purine biosynthesis; stemness

DOI:  https://doi.org/10.18632/aging.202372
Semin Hematol. 2021 Jan;pii: S0037-1963(20)30072-X. [Epub ahead of print]58(1): 35-44

A pilot clinical trial of oral tetrahydrouridine/decitabine for noncytotoxic epigenetic therapy of chemoresistant lymphoid malignancies.

Brian Hill, Deepa Jagadeesh, Brad Pohlman, Robert Dean, Neetha Parameswaran, Joel Chen, Tomas Radivoyevitch, Ashley Morrison, Sherry Fada, Meredith Dever, Shelley Robinson, Daniel Lindner, Mitchell Smith, Yogen Saunthararajah.

One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

DOI: https://doi.org/10.1053/j.seminhematol.2020.11.008
Sci Rep. 2021 Jan 25. 11(1): 2157

Bloom syndrome DNA helicase deficiency is associated with oxidative stress and mitochondrial network changes.

Veena Subramanian, Brian Rodemoyer, Vivek Shastri, Lene J Rasmussen, Claus Desler, Kristina H Schmidt.

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.

DOI: https://doi.org/10.1038/s41598-021-81075-0
DNA Repair (Amst). 2020 Dec 17. pii: S1568-7864(20)30292-5. [Epub ahead of print]98 103032

DNA damage response and breast cancer development: Possible therapeutic applications of ATR, ATM, PARP, BRCA1 inhibition.

Mohammad Mirza-Aghazadeh-Attari, Maria José Recio, Saber Ghazizadeh Darband, Mojtaba Kaviani, Amin Safa, Ainaz Mihanfar, Shirin Sadighparvar, Ansar Karimian, Forough Alemi, Maryam Majidinia, Bahman Yousefi.

  Breast cancer is the most common and significant cancers in females regarding the loss of life quality. Similar to other cancers, one of the etiologic factors in breast cancer is DNA damage. A plethora of molecules are responsible for sensing DNA damage and mediating actions which lead to DNA repair, senescence, cell cycle arrest and if damage is unbearable to apoptosis. In each of these, aberrations leading to unrepaired damage was resulted in uncontrolled proliferation and cancer. Another cellular function is autophagy defined as a process eliminating of unnecessary proteins in stress cases involved in pathogenesis of cancer. Knowing their role in cancer, scholars have tried to develop strategies in order to target DDR and autophagy. Further, the interactions of DDR and autophagy plus their regulatory role on each other have been focused simultaneously. The present review study has aimed to illustrate the importance of DDR and autophagy in breast cancer according to the related studies and uncover the relation between DDR and autophagy and its significance in breast cancer therapy.

Keywords:  ATG; Autophagy; Beclin-1; Breast cancer; DNA repair

DOI:  https://doi.org/10.1016/j.dnarep.2020.103032
Free Radic Res. 2021 Jan 26. 1-21

On the relevance of hydroxyl radical to purine DNA damage.

Chryssostomos Chatgilialoglu, Carla Ferreri, Marios G Krokidis, Annalisa Masi, Michael A Terzidis.

  Hydroxyl radical (HO•) is the most reactive toward DNA among the reactive oxygen species (ROS) generated in aerobic organisms by cellular metabolisms. HO• is generated also by exogenous sources such as ionizing radiations. In this review we focus on the purine DNA damage by HO• radicals. In particular, emphasis is given on mechanistic aspects for the various lesion formation and their interconnections. Although the majority of the purine DNA lesions like 8-oxo-purine (8-oxo-Pu) are generated by various ROS (including HO•), the formation of 5',8-cyclopurine (cPu) lesions in vitro and in vivo relies exclusively on the HO• attack. Methodologies generally utilized for the purine lesions quantification in biological samples are reported and critically discussed. Recent results on cPu and 8-oxo-Pu lesions quantification in various types of biological specimens associated with the cellular repair efficiency as well as with distinct pathologies are presented, providing some insights on their biological significance.

Keywords:  DNA oxidation; Hydroxyl radical; cyclopurines; oxidative damage; reactive oxygen species (ROS)

DOI:  https://doi.org/10.1080/10715762.2021.1876855
J Biol Chem. 2020 Feb 07. pii: S0021-9258(17)49866-8. [Epub ahead of print]295(6): 1685-1693

Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

Adam Z Thelen, Patrick J O'Brien.

  The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine (ϵA) and 1,N2-ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.

Keywords:  DNA alkylation; DNA damage; DNA repair; alkyladenine DNA glycosylase; base excision repair (BER); enzyme kinetics; ethenoguanine; substrate specificity

DOI:  https://doi.org/10.1074/jbc.RA119.011459
Int J Mol Sci. 2021 Jan 23. pii: 1113. [Epub ahead of print]22(3):

Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication.

Céline Duc, Christophe Thiriet.

  The doubling of genomic DNA during the S-phase of the cell cycle involves the global remodeling of chromatin at replication forks. The present review focuses on the eviction of nucleosomes in front of the replication forks to facilitate the passage of replication machinery and the mechanism of replication-coupled chromatin assembly behind the replication forks. The recycling of parental histones as well as the nuclear import and the assembly of newly synthesized histones are also discussed with regard to the epigenetic inheritance.

Keywords:  chromatin; histones; replication

DOI:  https://doi.org/10.3390/ijms22031113
PLoS Pathog. 2021 Jan 26. 17(1): e1009208

A multi-omics approach to Epstein-Barr virus immortalization of B-cells reveals EBNA1 chromatin pioneering activities targeting nucleotide metabolism.

R Jason Lamontagne, Samantha S Soldan, Chenhe Su, Andreas Wiedmer, Kyoung Jae Won, Fang Lu, Aaron R Goldman, Jayamanna Wickramasinghe, Hsin-Yao Tang, David W Speicher, Louise Showe, Andrew V Kossenkov, Paul M Lieberman.

Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers.

DOI: https://doi.org/10.1371/journal.ppat.1009208
J Biol Chem. 2020 Feb 07. pii: S0021-9258(17)49857-7. [Epub ahead of print]295(6): 1575-1586

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Zhihua Qin, Serena Bonifati, Corine St Gelais, Tai-Wei Li, Sun-Hee Kim, Jenna M Antonucci, Bijan Mahboubi, Jacob S Yount, Yong Xiong, Baek Kim, Li Wu.

  Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-β, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-β mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Keywords:  NF-kB transcription factor; NF-kappa B (NF-KB); NF-κB activation; SAM domain and HD domain-containing protein 1 (SAMHD1); SAMHD1; cell differentiation; dNTPase activity; inflammation; innate immune responses; innate immunity; interferon; monocyte; monocytic cells; nuclear localization; type I interferon (IFN-I); viral restriction factor

DOI:  https://doi.org/10.1074/jbc.RA119.010360
Nat Metab. 2021 Jan 28.

The ins and outs of serine and glycine metabolism in cancer.

Shauni L Geeraerts, Elien Heylen, Kim De Keersmaecker, Kim R Kampen.

Cancer cells reprogramme their metabolism to support unrestrained proliferation and survival in nutrient-poor conditions. Whereas non-transformed cells often have lower demands for serine and glycine, several cancer subtypes hyperactivate intracellular serine and glycine synthesis and become addicted to de novo production. Copy-number amplifications of serine- and glycine-synthesis genes and genetic alterations in common oncogenes and tumour-suppressor genes enhance serine and glycine synthesis, resulting in high production and secretion of these oncogenesis-supportive metabolites. In this Review, we discuss the contribution of serine and glycine synthesis to cancer progression. By relying on de novo synthesis pathways, cancer cells are able to enhance macromolecule synthesis, neutralize high levels of oxidative stress and regulate methylation and tRNA formylation. Furthermore, we discuss the immunosuppressive potential of serine and glycine, and the essentiality of both amino acids to promoting survival of non-transformed neighbouring cells. Finally, we point to the emerging data proposing moonlighting functions of serine- and glycine-synthesis enzymes and examine promising small molecules targeting serine and glycine synthesis.

DOI: https://doi.org/10.1038/s42255-020-00329-9
Cancer Drug Resist. 2020 ;3 762-774

PHGDH as a mechanism for resistance in metabolically-driven cancers.

Richa Rathore, Charles R Schutt, Brian A Van Tine.

  At the forefront of cancer research is the rapidly evolving understanding of metabolic reprogramming within cancer cells. The expeditious adaptation to metabolic inhibition allows cells to evolve and acquire resistance to targeted treatments, which makes therapeutic exploitation complex but achievable. 3-phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme of de novo serine biosynthesis and is highly expressed in a variety of cancers, including breast cancer, melanoma, and Ewing's sarcoma. This review will investigate the role of PHGDH in normal biological processes, leading to the role of PHGDH in the progression of cancer. With an understanding of the molecular mechanisms by which PHGDH expression advances cancer growth, we will highlight the known mechanisms of resistance to cancer therapeutics facilitated by PHGDH biology and identify avenues for combatting PHGDH-driven resistance with inhibitors of PHGDH to allow for the development of effective metabolic therapies.

Keywords:  PHGDH; cancer; drug resistance; folate cycle; metabolism; one-carbon metabolism; serine

DOI:  https://doi.org/10.20517/cdr.2020.46