bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒02‒28
twenty-nine papers selected by
Sean Rudd
Karolinska Institutet

  1. Dev Cell. 2021 Feb 22. pii: S1534-5807(21)00069-1. [Epub ahead of print]56(4): 461-477.e7
      Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.
    Keywords:  BRCA1/2; cancer evolution; genome instability; homology-directed repair; hypoxia; nascent DNA degradation; reactive oxygen species; replication stress; ribonucleotide reductase; translesion DNA synthesis
  2. Cell Rep. 2021 Feb 23. pii: S2211-1247(21)00088-7. [Epub ahead of print]34(8): 108775
      In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.
    Keywords:  DNA repair; cGAS; genomic instability; transcription
  3. J Biol Chem. 2021 Feb 23. pii: S0021-9258(21)00228-3. [Epub ahead of print] 100455
      The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 and/or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a "mini-ETAA1" protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuculei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization are important for optimal ATR signaling and genome stability.
    Keywords:  ATR; ATR activation domain; DNA damage response; ETAA1; RPA; TOPBP1; cell cycle checkpoint; phosphatidylinositol 3-kinase related protein kinase; replication stress response
  4. Trends Cell Biol. 2021 Feb 18. pii: S0962-8924(21)00023-4. [Epub ahead of print]
      Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome.
    Keywords:  DNA damage; DNA repair; DNA replication; genome stability; replication stress; ubiquitin
  5. J Biol Chem. 2021 Feb 19. pii: S0021-9258(21)00217-9. [Epub ahead of print] 100444
      Unrepaired DNA-protein crosslinks, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein crosslinks can be cleaved into DNA-peptide crosslinks, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can crosslink the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, crosslinked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA crosslink was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, 10-fold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the crosslinked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.
    Keywords:  DNA alkylation; DNA crosslink repair; DNA cross‐ink; DNA damage; DNA polymerase; DNA replication; DNA-protein interaction
  6. EMBO J. 2021 Feb 26. e106018
      The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.
    Keywords:  BRCA2; DNA double-strand breaks; DNA-RNA hybrids; R-loops; homologous recombination
  7. Elife. 2021 Feb 22. pii: e65471. [Epub ahead of print]10
      The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2‑7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.
    Keywords:  S. cerevisiae; cell biology; chromosomes; gene expression
  8. Nat Commun. 2021 Feb 26. 12(1): 1342
      Bulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. We find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.
  9. Mol Cell Biol. 2021 Feb 22. pii: MCB.00324-20. [Epub ahead of print]
      Regulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast ribosomal RNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the amount of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB, but led to homologous recombination-dependent rDNA instability. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.
  10. Front Pharmacol. 2020 ;11 619458
      Ectonucleotidases are extracellular enzymes with a pivotal role in inflammation that hydrolyse extracellular purine and pyrimidine nucleotides, e.g., ATP, UTP, ADP, UDP, AMP and NAD+. Ectonucleotidases, expressed by virtually all cell types, immune cells included, either as plasma membrane-associated or secreted enzymes, are classified into four main families: 1) nucleoside triphosphate diphosphohydrolases (NTPDases), 2) nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1), 3) ecto-5'-nucleotidase (NT5E), and 4) ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs). Concentration of ATP, UTP and NAD+ can be increased in the extracellular space thanks to un-regulated, e.g., cell damage or cell death, or regulated processes. Regulated processes include secretory exocytosis, connexin or pannexin hemichannels, ATP binding cassette (ABC) transporters, calcium homeostasis modulator (CALMH) channels, the ATP-gated P2X7 receptor, maxi-anion channels (MACs) and volume regulated ion channels (VRACs). Hydrolysis of extracellular purine nucleotides generates adenosine, an important immunosuppressant. Extracellular nucleotides and nucleosides initiate or dampen inflammation via P2 and P1 receptors, respectively. All these agents, depending on their level of expression or activation and on the agonist concentration, are potent modulators of inflammation and key promoters of host defences, immune cells activation, pathogen clearance, tissue repair and regeneration. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases. A selection of these pathologies will be briefly discussed here.
    Keywords:  ATP; acute inflammation; chronic inflammatory diseases; ecto-nucleotidases; immune cells; purinergic receptors; tumors
  11. Nucleic Acids Res. 2021 Feb 22. pii: gkab082. [Epub ahead of print]
      In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.
  12. Bioessays. 2021 Feb 23. e2100011
      We highlight a recent study exploring the hand-off of UV damage to several key nucleotide excision repair (NER) proteins in the cascade: UV-DDB, XPC and TFIIH. The delicate dance of DNA repair proteins is choreographed by the dynamic hand-off of DNA damage from one recognition complex to another damage verification protein or set of proteins. These DNA transactions on chromatin are strictly chaperoned by post-translational modifications (PTM). This new study examines the role that ubiquitylation and subsequent DDB2 degradation has during this process. In total, this study suggests an intricate cellular timer mechanism that under normal conditions DDB2 helps recruit and ubiquitylate XPC, stabilizing XPC at damaged sites. If DDB2 persists at damaged sites too long, it is turned over by auto-ubiquitylation and removed from DNA by the action of VCP/p97 for degradation in the 26S proteosome.
    Keywords:  DNA damage; global genome repair; nucleotide excision repair; post-translational modifications; ubiquitylation
  13. Front Pharmacol. 2020 ;11 629266
      DNA repair pathways are triggered to maintain genetic stability and integrity when mammalian cells are exposed to endogenous or exogenous DNA-damaging agents. The deregulation of DNA repair pathways is associated with the initiation and progression of cancer. As the primary anti-cancer therapies, ionizing radiation and chemotherapeutic agents induce cell death by directly or indirectly causing DNA damage, dysregulation of the DNA damage response may contribute to hypersensitivity or resistance of cancer cells to genotoxic agents and targeting DNA repair pathway can increase the tumor sensitivity to cancer therapies. Therefore, targeting DNA repair pathways may be a potential therapeutic approach for cancer treatment. A better understanding of the biology and the regulatory mechanisms of DNA repair pathways has the potential to facilitate the development of inhibitors of nuclear and mitochondria DNA repair pathways for enhancing anticancer effect of DNA damage-based therapy.
    Keywords:  DNA damage; DNA repair pathways; cancer therapy; drug resistance; mitochondrial DNA
  14. Curr Opin Cell Biol. 2021 Feb 18. pii: S0955-0674(21)00004-1. [Epub ahead of print]70 91-99
      Micronuclei are small membrane-bounded compartments with a DNA content encapsulated by a nuclear envelope and spatially separated from the primary nucleus. Micronuclei have long been linked to chromosome instability, genome rearrangements, and mutagenesis. They are frequently found in cancers, during senescence, and after genotoxic stress. Compromised integrity of the micronuclear envelope delays or disrupts DNA replication, inhibits DNA repair, and exposes micronuclear DNA directly to cytoplasm. Micronuclei play a central role in tumorigenesis, with micronuclear DNA being a source of complex genome rearrangements (including chromothripsis) and promoting a cyclic GMP-AMP synthase (cGAS)-mediated cellular immune response that may contribute to cancer metastasis. Here, we discuss recent findings on how micronuclei are generated, what the consequences are, and what cellular mechanisms can be applied to protect against micronucleation.
  15. Sci Rep. 2021 Feb 26. 11(1): 4774
      Poly (ADP-ribose) polymerase (PARP) inhibitors offer a significant clinical benefit for triple-negative breast cancers (TNBCs) with BRCA1/2 mutation. However, the narrow clinical indication limits the development of PARP inhibitors. Phosphoinositide 3-kinase (PI3K) inhibition sensitizes BRCA-proficient TNBC to PARP inhibition, which broadens the indication of PARP inhibitors. Previously researches have reported that PI3K inhibition induced the defect of homologous recombination (HR) mediated repair by downregulating the expression of BRCA1/2 and Rad51. However, the mechanism for their synergistic effects in the treatment of TNBC is still unclear. Herein, we focused on DNA damage, DNA single-strand breaks (SSBs) repair and DNA double-strand breaks (DSBs) repair three aspects to investigate the mechanism of dual PI3K and PARP inhibition in DNA damage response. We found that dual PI3K and PARP inhibition with BKM120 and olaparib significantly reduced the proliferation of BRCA-proficient TNBC cell lines MDA-MB-231 and MDA231-LM2. BKM120 increased cellular ROS to cause DNA oxidative damage. Olaparib resulted in concomitant gain of PARP1, forkhead box M1 (FOXM1) and Exonuclease 1 (Exo1) while inhibited the activity of PARP. BKM120 downregulated the expression of PARP1 and PARP2 to assist olaparib in blocking PARP mediated repair of DNA SSBs. Meanwhile, BKM120 inhibited the expression of BRAC1/2 and Rad51/52 to block HR mediated repair through the PI3K/Akt/NFκB/c-Myc signaling pathway and PI3K/Akt/ FOXM1/Exo1 signaling pathway. BKM120 induced HR deficiency expanded the application of olaparib to HR proficient TNBCs. Our findings proved that PI3K inhibition impaired the repair of both DNA SSBs and DNA DSBs. FOXM1 and Exo1 are novel therapeutic targets that serves important roles in DNA damage response.
  16. EMBO Rep. 2021 Feb 23. e51749
      During DNA replication, the deubiquitinating enzyme USP1 limits the recruitment of translesion polymerases by removing ubiquitin marks from PCNA to allow specific regulation of the translesion synthesis (TLS) pathway. USP1 activity depends on an allosteric activator, UAF1, and this is tightly controlled. In comparison to paralogs USP12 and USP46, USP1 contains three defined inserts and lacks the second WDR20-mediated activation step. Here we show how inserts L1 and L3 together limit intrinsic USP1 activity and how this is relieved by UAF1. Intriguingly, insert L1 also conveys substrate-dependent increase in USP1 activity through DNA and PCNA interactions, in a process that is independent of UAF1-mediated activation. This study establishes insert L1 as an important regulatory hub within USP1 necessary for both substrate-mediated activity enhancement and allosteric activation upon UAF1 binding.
    Keywords:  allostery; deubiquitinating enzyme; enzyme activity; quantitative kinetic modelling; translesion synthesis
  17. Sci Rep. 2021 Feb 24. 11(1): 4420
      The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.
  18. Biochem Biophys Res Commun. 2021 Feb 18. pii: S0006-291X(21)00210-2. [Epub ahead of print]547 162-168
      Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.
    Keywords:  Acute myeloid leukemia; Differentiation therapy; Mitochondrial ETC complex III; Pyrimidine biosynthesis
  19. DNA Repair (Amst). 2021 Feb 13. pii: S1568-7864(21)00026-4. [Epub ahead of print]100 103070
      The classical small Rho GTPase (Rho, Rac, and Cdc42) protein family is mainly responsible for regulating cell motility and polarity, membrane trafficking, cell cycle control, and gene transcription. Cumulative recent evidence supports important roles for these proteins in the maintenance of genomic stability. Indeed, DNA damage response (DDR) and repair mechanisms are some of the prime biological processes that underlie several disease phenotypes, including genetic disorders, cancer, senescence, and premature aging. Many reports guided by different experimental approaches and molecular hypotheses have demonstrated that, to some extent, direct modulation of Rho GTPase activity, their downstream effectors, or actin cytoskeleton regulation contribute to these cellular events. Although much attention has been paid to this family in the context of canonical actin cytoskeleton remodeling, here we provide a contextualized review of the interplay between Rho GTPase signaling pathways and the DDR and DNA repair signaling components. Interesting questions yet to be addressed relate to the spatiotemporal dynamics of this collective response and whether it correlates with different subcellular pools of Rho GTPases. We highlight the direct and indirect targets, some of which still lack experimental validation data, likely associated with Rho GTPase activation that provides compelling evidence for further investigation in DNA damage-associated events and with potential therapeutic applications in translational medicine.
    Keywords:  Actin cytoskeleton; Cdc42; Genome stability; Rac; Rho; Rho GTPases
  20. Cancer Biol Med. 2021 Feb 15. 18(1): 120-138
      Objective: RECQL4 (a member of the RECQ helicase family) upregulation has been reported to be associated with tumor progression in several malignancies. However, whether RECQL4 sustains esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we determined the functional role for RECQL4 in ESCC progression.Methods: RECQL4 expression in clinical samples of ESCC was examined by immunohistochemistry. Cell proliferation, cellular senescence, the epithelial-mesenchymal transition (EMT), DNA damage, and reactive oxygen species in ESCC cell lines with RECQL4 depletion or overexpression were analyzed. The levels of proteins involved in the DNA damage response (DDR), cell cycle progression, survival, and the EMT were determined by Western blot analyses.
    Results: RECQL4 was highly expressed in tumor tissues when compared to adjacent non-tumor tissues in ESCC (P < 0.001) and positively correlated with poor differentiation (P = 0.011), enhanced invasion (P = 0.033), and metastasis (P = 0.048). RECQL4 was positively associated with proliferation and migration in ESCC cells. Depletion of RECQL4 also inhibited growth of tumor xenografts in vivo. RECQL4 depletion induced G0/G1 phase arrest and cellular senescence. Importantly, the levels of DNA damage and reactive oxygen species were increased when RECQL4 was depleted. DDR, as measured by the activation of ATM, ATR, CHK1, and CHK2, was impaired. RECQL4 was also shown to promote the activation of AKT, ERK, and NF-kB in ESCC cells.
    Conclusions: The results indicated that RECQL4 was highly expressed in ESCC and played critical roles in the regulation of DDR, redox homeostasis, and cell survival.
    Keywords:  DNA damage response; ESCC; RECQL4; redox; senescence
  21. Cell. 2021 Feb 17. pii: S0092-8674(21)00091-X. [Epub ahead of print]
      End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.
    Keywords:  DNA homologous recombination; RNA polymerase III; RNA-DNA hyrbid; dsDNA break repair; end resection; genomic instability
  22. Cell Metab. 2021 Feb 17. pii: S1550-4131(21)00057-7. [Epub ahead of print]
      Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.
    Keywords:  asparaginase; asparagine; cancer metabolism; cancer treatment; dietary restriction; metformin; respiration
  23. Biochem Soc Trans. 2021 Feb 22. pii: BST20190008. [Epub ahead of print]
      One-carbon metabolism (1C-metabolism), also called folate metabolism because the carbon group is attached to folate-derived tetrahydrofolate, is crucial in metabolism. It is at the heart of several essential syntheses, particularly those of purine and thymidylate. After a short reminder of the organization of 1C-metabolism, I list its salient features as reported in the literature. Then, using flux balance analysis, a core model of central metabolism and the flux constraints for an 'average cancer cell metabolism', I explore the fundamentals underlying 1C-metabolism and its relationships with the rest of metabolism. Some unreported properties of 1C-metabolism emerge, such as its potential roles in mitochondrial NADH exchange with cytosolic NADPH, participation in NADH recycling, and optimization of cell proliferation.
    Keywords:  average cancer cell; cancer cells’ metabolism; flux balance analysis; metabolic model; one-carbon metabolism
  24. Nature. 2021 Feb 24.
      Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.
  25. Genes Cells. 2021 Feb 23.
      SIRT2 and SIRT3 protein deacetylases maintain genome integrity and stability. However, their mechanisms for maintaining the genome remain unclear. To examine the roles of SIRT2 and SIRT3 in DSB repair, I-SceI-based GFP reporter assays for HR, single-strand annealing (SSA), and non-homologous end joining (NHEJ) repair were performed under SIRT2- or SIRT3-depleted conditions. SIRT2 or SIRT3 depletion inhibited HR repair equally to RAD52 depletion, but did not affect SSA and NHEJ repairs. SIRT2 or SIRT3 depletion disturbed the recruitment of RAD51 to DSB sites, an essential step for RAD51-dependent HR repair, but not directly through RAD52 deacetylation. SIRT2 or SIRT3 depletion decreased the colocalization of γH2AX foci with RPA1, and thus they might be involved in initiating DSB end resection for the recruitment of RAD51 to DSB sites at an early step in HR repair. These results reveal the novel underlying mechanism of the SIRT2 and SIRT3 functions in HR for genome stability.
  26. Mol Cancer. 2021 Feb 25. 20(1): 40
      DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.
    Keywords:  G-quadruplex; acute myeloid leukemia; cancer progression; cancer therapy; genome instability; malignant melanoma; pancreatic cancer
  27. Nat Commun. 2021 Feb 26. 12(1): 1296
      Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.
  28. Bioessays. 2021 Feb 25. e2000309
      The topology of DNA duplexes changes during replication and also after deproteinization in vitro. Here we describe these changes and then discuss for the first time how the distribution of superhelical stress affects the DNA topology of replication intermediates, taking into account the progression of replication forks. The high processivity of Topo IV to relax the left-handed (+) supercoiling that transiently accumulates ahead of the forks is not essential, since DNA gyrase and swiveling of the forks cooperate with Topo IV to accomplish this task in vivo. We conclude that despite Topo IV has a lower processivity to unlink the right-handed (+) crossings of pre-catenanes and fully replicated catenanes, this is indeed its main role in vivo. This would explain why in the absence of Topo IV replication goes-on, but fully replicated sister duplexes remain heavily catenated.
    Keywords:  DNA chirality; DNA topology; catenation; pre-catenation; replication; supercoiling
  29. Front Pharmacol. 2021 ;12 632622
    Keywords:  adenine-based compounds; glioblastoma multiforme (GBM); purine metabolizing enzymes; purinergic receptors; purinome