bims-numges 2021-02-14 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–02–14
thirty-six papers selected by
Sean Rudd, Karolinska Institutet

NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia.
Adenylate kinase 2 expression and addiction in T-ALL.
The eukaryotic replisome tolerates leading-strand base damage by replicase switching.
Small molecule inhibitor of OGG1 blocks oxidative DNA damage repair at telomeres and potentiates methotrexate anticancer effects.
Replication dynamics of recombination-dependent replication forks.
Targeting the DNA replication stress phenotype of KRAS mutant cancer cells.
Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair.
Role of Base Excision Repair Pathway in the Processing of Complex DNA Damage Generated by Oxidative Stress and Anticancer Drugs.
Loss of TGFβ signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types.
Roles of SDE2 and TIMELESS at active and stalled DNA replication forks.
PARP Inhibitors in Small-Cell Lung Cancer: Rational Combinations to Improve Responses.
DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining.
Overcoming PARPi resistance: Preclinical and clinical evidence in ovarian cancer.
The DNA ligands Arg47 and Arg76 are crucial for catalysis by human PrimPol.
Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells.
Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.
SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.
Tyrosine kinase inhibitors protect the salivary gland from radiation damage by increasing DNA double strand break repair.
USP11 mediates repair of DNA-protein crosslinks by deubiquitinating SPRTN metalloprotease.
DNA damage response inhibitors: An avenue for TNBC treatment.
Rtt109 slows replication speed by histone N-terminus acetylation.
Inducing DNA damage through R-loops to kill cancer cells.
Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway.
Solid tumours hijack the histone variant network.
Pharmacological targeting of differential DNA repair, radio-sensitizes WRN-deficient cancer cells in vitro and in vivo.
DDB2 regulates DNA replication through PCNA-independent degradation of CDT2.
Targeting drug transporters to prevent chemotherapy-induced peripheral neuropathy.
Topoisomerase I prevents transcription-replication conflicts at transcription termination sites.
Regulation of ATR-dependent DNA damage response by nitric oxide.
The uracil-DNA glycosylase UNG protects the fitness of normal and cancer B cells expressing AID.
Glutamine Synthetase as a Therapeutic Target for Cancer Treatment.
Biomarkers of nucleic acid oxidation - A summary state-of-the-art.
SAMHD1 can suppress lung adenocarcinoma progression through the negative regulation of STING.
Deubiquitinase USP47-stabilized splicing factor IK regulates the splicing of ATM pre-mRNA.
Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.
Targeting the purinergic pathway in breast cancer and its therapeutic applications.

Sci Adv. 2021 Jan;pii: eabc9781. [Epub ahead of print]7(5):

NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia.

Shashank Srivastava, Umakant Sahu, Yalu Zhou, Ann K Hogan, Kizhakke Mattada Sathyan, Justin Bodner, Jiehuan Huang, Kelvin A Wong, Natalia Khalatyan, Jeffrey N Savas, Panagiotis Ntziachristos, Issam Ben-Sahra, Daniel R Foltz.

Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) is the most divergent member of UBR box-containing E3 ubiquitin ligases/recognins that mediate the proteasomal degradation of its substrates through the N-end rule. Here, we used a proteomic approach and found phosphoribosyl pyrophosphate synthetases (PRPSs), the essential enzymes for nucleotide biosynthesis, as strong interacting partners of UBR7. UBR7 stabilizes PRPS catalytic subunits by mediating the polyubiquitination-directed degradation of PRPS-associated protein (PRPSAP), the negative regulator of PRPS. Loss of UBR7 leads to nucleotide biosynthesis defects. We define UBR7 as a transcriptional target of NOTCH1 and show that UBR7 is overexpressed in NOTCH1-driven T cell acute lymphoblastic leukemia (T-ALL). Impaired nucleotide biosynthesis caused by UBR7 depletion was concomitant with the attenuated cell proliferation and oncogenic potential of T-ALL. Collectively, these results establish UBR7 as a critical regulator of nucleotide metabolism through the regulation of the PRPS enzyme complex and uncover a metabolic vulnerability in NOTCH1-driven T-ALL.

DOI: https://doi.org/10.1126/sciadv.abc9781
Blood Adv. 2021 Feb 09. 5(3): 700-710

Adenylate kinase 2 expression and addiction in T-ALL.

Nabih Maslah, Mehdi Latiri, Vahid Asnafi, Mélanie Féroul, Nawel Bedjaoui, Thomas Steimlé, Emmanuelle Six, Els Verhoyen, Elizabeth Macintyre, Chantal Lagresle-Peyrou, Ludovic Lhermitte, Guillaume P Andrieu.

T-cell acute lymphoblastic leukemia (T-ALL) represents the malignant expansion of immature T cells blocked in their differentiation. T-ALL is still associated with a poor prognosis, mainly related to occurrence of relapse or refractory disease. A critical medical need therefore exists for new therapies to improve the disease prognosis. Adenylate kinase 2 (AK2) is a mitochondrial kinase involved in adenine nucleotide homeostasis recently reported as essential in normal T-cell development, as defective AK2 signaling pathway results in a severe combined immunodeficiency with a complete absence of T-cell differentiation. In this study, we show that AK2 is constitutively expressed in T-ALL to varying levels, irrespective of the stage of maturation arrest or the underlying oncogenetic features. T-ALL cell lines and patient T-ALL-derived xenografts present addiction to AK2, whereas B-cell precursor ALL cells do not. Indeed, AK2 knockdown leads to early and massive apoptosis of T-ALL cells that could not be rescued by the cytosolic isoform AK1. Mechanistically, AK2 depletion results in mitochondrial dysfunction marked by early mitochondrial depolarization and reactive oxygen species production, together with the depletion of antiapoptotic molecules (BCL-2 and BCL-XL). Finally, T-ALL exposure to a BCL-2 inhibitor (ABT-199 [venetoclax]) significantly enhances the cytotoxic effects of AK2 depletion. We also show that AK2 depletion disrupts the oxidative phosphorylation pathway. Combined with pharmaceutical inhibition of glycolysis, AK2 silencing prevents T-ALL metabolic adaptation, resulting in dramatic apoptosis. Altogether, we pinpoint AK2 as a genuine and promising therapeutic target in T-ALL.

DOI: https://doi.org/10.1182/bloodadvances.2020002700
EMBO J. 2021 Feb 08. e107037

The eukaryotic replisome tolerates leading-strand base damage by replicase switching.

Thomas A Guilliam, Joseph Tp Yeeles.

  The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low-fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading-strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase-polymerase uncoupling a switch from Pol ε, the canonical leading-strand replicase, to the lagging-strand replicase Pol δ, facilitates rapid, efficient and error-free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8-oxoguanine. We propose that replicase switching may promote continued leading-strand synthesis whenever the replisome encounters leading-strand damage that is bypassed more efficiently by Pol δ than by Pol ε.

Keywords:  DNA damage tolerance; DNA polymerase; DNA replication; replisome; translesion synthesis

DOI:  https://doi.org/10.15252/embj.2020107037
Sci Rep. 2021 Feb 10. 11(1): 3490

Small molecule inhibitor of OGG1 blocks oxidative DNA damage repair at telomeres and potentiates methotrexate anticancer effects.

Juan Miguel Baquero, Carlos Benítez-Buelga, Varshni Rajagopal, Zhao Zhenjun, Raúl Torres-Ruiz, Sarah Müller, Bishoy M F Hanna, Olga Loseva, Olov Wallner, Maurice Michel, Sandra Rodríguez-Perales, Helge Gad, Torkild Visnes, Thomas Helleday, Javier Benítez, Ana Osorio.

The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.

DOI: https://doi.org/10.1038/s41598-021-82917-7
Nat Commun. 2021 02 10. 12(1): 923

Replication dynamics of recombination-dependent replication forks.

Karel Naiman, Eduard Campillo-Funollet, Adam T Watson, Alice Budden, Izumi Miyabe, Antony M Carr.

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3' single strand in the context of increased resection.

DOI: https://doi.org/10.1038/s41467-021-21198-0
Sci Rep. 2021 Feb 11. 11(1): 3656

Targeting the DNA replication stress phenotype of KRAS mutant cancer cells.

Tara Al Zubaidi, O H Fiete Gehrisch, Marie-Michelle Genois, Qi Liu, Shan Lu, Jong Kung, Yunhe Xie, Jan Schuemann, Hsiao-Ming Lu, Aaron N Hata, Lee Zou, Kerstin Borgmann, Henning Willers.

Mutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

DOI: https://doi.org/10.1038/s41598-021-83142-y
Mol Cell. 2021 Feb 04. pii: S1097-2765(21)00041-1. [Epub ahead of print]

Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair.

Karl E Zahn, Ryan B Jensen, Richard D Wood, Sylvie Doublié.

  Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.

Keywords:  DNA polymerase; Double-strand break repair; dideoxynucleotides; endonuclease; theta-mediated end joining (TMEJ)

DOI:  https://doi.org/10.1016/j.molcel.2021.01.021
Front Cell Dev Biol. 2020 ;8 617884

Role of Base Excision Repair Pathway in the Processing of Complex DNA Damage Generated by Oxidative Stress and Anticancer Drugs.

Yeldar Baiken, Damira Kanayeva, Sabira Taipakova, Regina Groisman, Alexander A Ishchenko, Dinara Begimbetova, Bakhyt Matkarimov, Murat Saparbaev.

  Chemical alterations in DNA induced by genotoxic factors can have a complex nature such as bulky DNA adducts, interstrand DNA cross-links (ICLs), and clustered DNA lesions (including double-strand breaks, DSB). Complex DNA damage (CDD) has a complex character/structure as compared to singular lesions like randomly distributed abasic sites, deaminated, alkylated, and oxidized DNA bases. CDD is thought to be critical since they are more challenging to repair than singular lesions. Although CDD naturally constitutes a relatively minor fraction of the overall DNA damage induced by free radicals, DNA cross-linking agents, and ionizing radiation, if left unrepaired, these lesions cause a number of serious consequences, such as gross chromosomal rearrangements and genome instability. If not tightly controlled, the repair of ICLs and clustered bi-stranded oxidized bases via DNA excision repair will either inhibit initial steps of repair or produce persistent chromosomal breaks and consequently be lethal for the cells. Biochemical and genetic evidences indicate that the removal of CDD requires concurrent involvement of a number of distinct DNA repair pathways including poly(ADP-ribose) polymerase (PARP)-mediated DNA strand break repair, base excision repair (BER), nucleotide incision repair (NIR), global genome and transcription coupled nucleotide excision repair (GG-NER and TC-NER, respectively), mismatch repair (MMR), homologous recombination (HR), non-homologous end joining (NHEJ), and translesion DNA synthesis (TLS) pathways. In this review, we describe the role of DNA glycosylase-mediated BER pathway in the removal of complex DNA lesions.

Keywords:  DNA glycosylase; Fanconi anemia; base excision repair; bulky DNA adduct; inter-strand DNA crosslink; nucleotide excision repair

DOI:  https://doi.org/10.3389/fcell.2020.617884
Sci Transl Med. 2021 Feb 10. pii: eabc4465. [Epub ahead of print]13(580):

Loss of TGFβ signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types.

Qi Liu, Luis Palomero, Jade Moore, Ines Guix, Roderic Espín, Alvaro Aytés, Jian-Hua Mao, Amanda G Paulovich, Jeffrey R Whiteaker, Richard G Ivey, George Iliakis, Daxian Luo, Anthony J Chalmers, John Murnane, Miquel Angel Pujana, Mary Helen Barcellos-Hoff.

Among the pleotropic roles of transforming growth factor-β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFβ and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology.

DOI: https://doi.org/10.1126/scitranslmed.abc4465
Mol Cell Oncol. 2021 ;8(1): 1855053

Roles of SDE2 and TIMELESS at active and stalled DNA replication forks.

Natalie Lo, Julie Rageul, Hyungjin Kim.

  The fork protection complex (FPC), comprising the TIMELESS (TIM)-TIPIN heterodimer, acts as a scaffold of the replisome to support seamless DNA replication. We recently showed that SDE2, a PCNA-associated DNA replication stress regulator, maintains the integrity of the FPC, and together with TIM, protects stalled replication forks from nucleolytic degradation.

Keywords:  BRCA1/BRCA2; DNA replication stress; Genome instability; SDE2; TIMELESS; fork protection complex

DOI:  https://doi.org/10.1080/23723556.2020.1855053
Cancers (Basel). 2021 Feb 10. pii: 727. [Epub ahead of print]13(4):

PARP Inhibitors in Small-Cell Lung Cancer: Rational Combinations to Improve Responses.

Erik H Knelson, Shetal A Patel, Jacob M Sands.

  Despite recent advances in first-line treatment for small-cell lung cancer (SCLC), durable responses remain rare. The DNA repair enzyme poly-(ADP)-ribose polymerase (PARP) was identified as a therapeutic target in SCLC using unbiased preclinical screens and confirmed in human and mouse models. Early trials of PARP inhibitors, either alone or in combination with chemotherapy, showed promising but limited responses, suggesting that selecting patient subsets and treatment combinations will prove critical to further clinical development. Expression of SLFN11 and other components of the DNA damage response (DDR) pathway appears to select for improved responses. Combining PARP inhibitors with agents that damage DNA and inhibit DDR appears particularly effective in preclinical and early trial data, as well as strategies that enhance antitumor immunity downstream of DNA damage. A robust understanding of the mechanisms of DDR in SCLC, which exhibits intrinsic replication stress, will improve selection of agents and predictive biomarkers. The most effective combinations will target multiple nodes in the DNA damage/DDR/immune activation cascade to minimize toxicity from synthetic lethality.

Keywords:  DDR; ICB; PARP; SCLC; SLFN11; STING; synthetic lethality

DOI:  https://doi.org/10.3390/cancers13040727
J Cell Sci. 2021 Feb 08. pii: jcs.249706. [Epub ahead of print]

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining.

Matteo Cabrini, Marco Roncador, Alessandro Galbiati, Lina Cipolla, Antonio Maffia, Fabio Iannelli, Simone Sabbioneda, Fabrizio d'Adda di Fagagna, Sofia Francia.

  The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.

Keywords:  DNA DAMAGE; DROSHA; RNA

DOI:  https://doi.org/10.1242/jcs.249706
Drug Resist Updat. 2021 Jan 16. pii: S1368-7646(21)00002-9. [Epub ahead of print] 100744

Overcoming PARPi resistance: Preclinical and clinical evidence in ovarian cancer.

M Chiappa, F Guffanti, F Bertoni, I Colombo, G Damia.

  Ovarian cancer is the fifth cause of cancer-related deaths in women with high grade serous carcinoma (HGSOC) representing the most common histological subtype. Approximately 50 % of HGSOC are characterized by deficiency in homologous recombination (HR), one of the main cellular pathways to repair DNA double strand breaks and one of the well-described mechanisms is the loss of function of the BRCA1 or BRCA2 genes. Inhibition of the poly-ADP-ribose polymerase (PARP) is synthetic lethal with HR deficiency and the use of PARP inhibitors (PARPi) has significantly improved the outcome of patients with HGSOC with a greater benefit in patients with BRCA1/2 deficient tumors. However, intrinsic or acquired resistance to PARPi inevitably occurs in most HGSOC patients. Distinct heterogeneous mechanisms underlying the resistance to PARPi have been described, including a decrease in intracellular drug levels due to upregulation of multidrug efflux pumps, loss of expression/inactivating mutations in the PARP1 protein, restoration of HR and the protection of the replicative fork. Deciphering the molecular mechanisms of resistance to PARPi is of paramount importance towards the development of new treatment strategies and/or novel pharmacological agents to overcome this chemoresistance and optimize the treatment regimen for individual HGSOC patients. The current review summarizes the mechanisms underlying the resistance to PARPi, the available preclinical and clinical data on new combination treatment strategies (with chemotherapy, anti-angiogenic agents and immune checkpoint inhibitors) as well as agents under investigation which target the DNA damage response.

Keywords:  Ovarian cancer; PARPi resistance

DOI:  https://doi.org/10.1016/j.drup.2021.100744
DNA Repair (Amst). 2021 Jan 29. pii: S1568-7864(21)00002-1. [Epub ahead of print]100 103048

The DNA ligands Arg47 and Arg76 are crucial for catalysis by human PrimPol.

Elizaveta O Boldinova, Аnna А Manukyan, Аlena V Makarova.

  Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.

Keywords:  Active site; DNA polymerase; PirmPol; Primase

DOI:  https://doi.org/10.1016/j.dnarep.2021.103048
Int J Mol Sci. 2021 Jan 30. pii: 1391. [Epub ahead of print]22(3):

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells.

Andrey Kropotov, Veronika Kulikova, Kirill Nerinovski, Alexander Yakimov, Maria Svetlova, Ljudmila Solovjeva, Julia Sudnitsyna, Marie E Migaud, Mikhail Khodorkovskiy, Mathias Ziegler, Andrey Nikiforov.

  Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Keywords:  NAD+ metabolism; concentrative nucleoside transporters; equilibrative nucleoside transporters; human cells; nicotinamide riboside; nicotinic acid riboside

DOI:  https://doi.org/10.3390/ijms22031391
Nat Rev Mol Cell Biol. 2021 Feb 09.

Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.

Ci Ji Lim, Thomas R Cech.

The regulation of telomere length in mammals is crucial for chromosome end-capping and thus for maintaining genome stability and cellular lifespan. This process requires coordination between telomeric protein complexes and the ribonucleoprotein telomerase, which extends the telomeric DNA. Telomeric proteins modulate telomere architecture, recruit telomerase to accessible telomeres and orchestrate the conversion of the newly synthesized telomeric single-stranded DNA tail into double-stranded DNA. Dysfunctional telomere maintenance leads to telomere shortening, which causes human diseases including bone marrow failure, premature ageing and cancer. Recent studies provide new insights into telomerase-related interactions (the 'telomere replisome') and reveal new challenges for future telomere structural biology endeavours owing to the dynamic nature of telomere architecture and the great number of structures that telomeres form. In this Review, we discuss recently determined structures of the shelterin and CTC1-STN1-TEN1 (CST) complexes, how they may participate in the regulation of telomere replication and chromosome end-capping, and how disease-causing mutations in their encoding genes may affect specific functions. Major outstanding questions in the field include how all of the telomere components assemble relative to each other and how the switching between different telomere structures is achieved.

DOI: https://doi.org/10.1038/s41580-021-00328-y
Cell Death Dis. 2021 Feb 08. 12(2): 165

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.

Juri Na, Joseph A Newman, Chee Kin Then, Junetha Syed, Iolanda Vendrell, Ignacio Torrecilla, Sophie Ellermann, Kristijan Ramadan, Roman Fischer, Anne E Kiltie.

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

DOI: https://doi.org/10.1038/s41419-021-03437-w
J Biol Chem. 2021 Feb 08. pii: S0021-9258(21)00173-3. [Epub ahead of print] 100401

Tyrosine kinase inhibitors protect the salivary gland from radiation damage by increasing DNA double strand break repair.

Trisiani Affandi, Angela M Ohm, Dany Gaillard, Ami Haas, Mary E Reyland.

  We have previously shown that the tyrosine kinase inhibitors (TKIs) dasatinib and imatinib can protect salivary glands from irradiation (IR) damage without impacting tumor therapy. However, how they induce this protection not unknown. Here we show that TKIs mediate radioprotection by increasing the repair of DNA double stranded breaks. DNA repair in IR-treated parotid cells, but not oral cancer cells, occurs more rapidly following pretreatment with imatinib or dasatinib, and is accompanied by faster formation of DNA damage-induced foci. Similar results were observed in the parotid glands of mice pretreated with imatinib prior to IR, suggesting that TKIs "prime" cells for DNA repair. Mechanistically, we observed that TKIs increased IR-induced activation of DNA-PK, but not ATM. Pretreatment of parotid cells with the DNA-PK inhibitor NU7441 reversed the increase in DNA repair induced by TKIs. Reporter assays specific for homologous recombination (HR) or non-homologous end joining (NHEJ) verified TKIs functionally regulate both DNA repair pathways. Moreover, TKIs also increased basal and IR-induced expression of genes associated with NHEJ (DNA ligase 4, Artemis, XLF) and HR (Rad50, Rad51 and BRCA1); depletion of DNA ligase 4 or BRCA1 reversed the increase in DNA repair mediated by TKIs. In addition, TKIs increased activation of the ERK survival pathway in parotid cells, and ERK was required for the increased survival of TKI treated cells. Our studies demonstrate a dual mechanism by which TKIs provide radioprotection of salivary gland tissues and support exploration of TKIs clinically in head and neck cancer patients undergoing IR therapy.

Keywords:  DNA damage; DNA repair; Radioprotection; protein kinase C-δ; salivary gland; tyrosine kinase inhibitor

DOI:  https://doi.org/10.1016/j.jbc.2021.100401
J Biol Chem. 2021 Feb 07. pii: S0021-9258(21)00168-X. [Epub ahead of print] 100396

USP11 mediates repair of DNA-protein crosslinks by deubiquitinating SPRTN metalloprotease.

Megan Perry, Meghan Biegert, Sai Sundeep Kollala, Halle Mallard, Grace Su, Manohar Kodavati, Natasha Kreiling, Alexander Holbrook, Gargi Ghosal.

  DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as a SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.

Keywords:  DPC; DPC repair; SPRTN; TOP1-ccs; USP11; deubiquitination; monoubiquitinated SPRTN

DOI:  https://doi.org/10.1016/j.jbc.2021.100396
Biochim Biophys Acta Rev Cancer. 2021 Feb 05. pii: S0304-419X(21)00020-2. [Epub ahead of print]1875(2): 188521

DNA damage response inhibitors: An avenue for TNBC treatment.

Juan Jin, Zhonghua Tao, Jun Cao, Ting Li, Xichun Hu.

The DNA damage response (DDR) is critical for the maintenance of genomic stability by sensing DNA damage, regulating cell cycle and initiating DNA repair. Drugs targeting DDR pathways have been increasingly exploited in treating various tumors. Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive tumor with constitutive activation of oncogenes, inducing replication stress and DNA damage, which require the DDR for survival. In addition, emerging studies have demonstrated that TNBC harbors aberrant genetic alterations in DDR pathways, such as a high frequency of p53 dysfunction and BRCA1/2 mutations. DDR alterations force TNBC to rely on the existing DDR pathways for survival, and make TNBC particularly sensitive to specific DDR inhibitors, such as high sensitivity of TNBC with BRCA1/2 mutations to PARP inhibitors. This review first and comprehensively covers the current status of the development of DDR inhibitors and discusses the mechanism of targeting the DDR in TNBC. Preclinical and clinical studies on inhibitors of the ATR-CHK1-WEE1 pathway and PARP inhibitors, the most studied inhibitors, and some other DDR inhibitors as monotherapy or combination therapy in TNBC are summarized. We also highlight the possible predictive biomarkers for these DDR inhibitors and their potential combination strategies with chemotherapy, radiotherapy or other targeted agents to optimize the efficacy of DDR inhibitors in TNBC treatment. In conclusion, this review discussed the recent considerations related to the use of DDR inhibitors for TNBC and provides a perspective to address future directions and potential therapeutic strategies for patients with TNBC.

DOI: https://doi.org/10.1016/j.bbcan.2021.188521
Genome Res. 2021 Feb 09.

Rtt109 slows replication speed by histone N-terminus acetylation.

Nelly Frenkel, Felix Jonas, Miri Carmi, Gilad Yaakov, Naama Barkai.

The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses ∼3-5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.

DOI: https://doi.org/10.1101/gr.266510.120
Mol Cell Oncol. 2020 Nov 20. 8(1): 1848233

Inducing DNA damage through R-loops to kill cancer cells.

Fred C Lam, Yi Wen Kong, Michael B Yaffe.

  R-loops are intermediate structures of transcription that can accumulate when transcriptional elongation is blocked by inhibiting BRD4. In normal cells, R-loop persistence suppresses firing of adjacent replication origins. This control is lost in a subset of cancer cells, where BRD4 inhibition results in R-loop accumulation, leading to transcription-replication collisions and DNA double-strand breaks during S-phase, followed by cell death. This finding sheds new light on the mechanisms by which BRD4 inhibitors function as cancer therapies, and indicates that targeting other cellular events to cause R-loop accumulation may be useful for cancer treatment.

Keywords:  BRD4; DNA damage; R-loops; bromodomain proteins; replication stress; transcription-replication conflicts

DOI:  https://doi.org/10.1080/23723556.2020.1848233
Annu Rev Biochem. 2021 Feb 08.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway.

Benjamin M Stinson, Joseph J Loparo.

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-biochem-080320-110356
Nat Rev Cancer. 2021 Feb 10.

Solid tumours hijack the histone variant network.

Flávia G Ghiraldini, Dan Filipescu, Emily Bernstein.

Cancer is a complex disease characterized by loss of cellular homeostasis through genetic and epigenetic alterations. Emerging evidence highlights a role for histone variants and their dedicated chaperones in cancer initiation and progression. Histone variants are involved in processes as diverse as maintenance of genome integrity, nuclear architecture and cell identity. On a molecular level, histone variants add a layer of complexity to the dynamic regulation of transcription, DNA replication and repair, and mitotic chromosome segregation. Because these functions are critical to ensure normal proliferation and maintenance of cellular fate, cancer cells are defined by their capacity to subvert them. Hijacking histone variants and their chaperones is emerging as a common means to disrupt homeostasis across a wide range of cancers, particularly solid tumours. Here we discuss histone variants and histone chaperones as tumour-promoting or tumour-suppressive players in the pathogenesis of cancer.

DOI: https://doi.org/10.1038/s41568-020-00330-0
Biochem Pharmacol. 2021 Feb 08. pii: S0006-2952(21)00046-0. [Epub ahead of print] 114450

Pharmacological targeting of differential DNA repair, radio-sensitizes WRN-deficient cancer cells in vitro and in vivo.

Pooja Gupta, Bhaskar Saha, Subrata Chattopadhyay, Birija Sankar Patro.

  Werner (WRN) expression is epigenetically downregulated in various tumors. It is imperative to understand differential repair process in WRN-proficient and WRN-deficient cancers to find pharmacological targets for radio-sensitization of WRN-deficient cancer. In the current investigation, we showed that pharmacological inhibition of CHK1 mediated homologous recombination repair (HRR), but not non-homologous end joining (NHEJ) repair, can causes hyper-radiosensitization of WRN-deficient cancers. This was confirmed in cancer cell lines of different tissue origin (osteosarcoma, colon adenocarcinoma and melanoma) with WRN silencing and overexpression. We established that WRN-depleted cells are dependent on a critical but compromised CHK1-mediated HRR-pathway for repairing ionizing radiation (IR) induced DSBs for their survival. Mechanistically, we unraveled a new finding that the MRE11, CTIP and WRN proteins are largely responsible for resections of late and persistent DSBs. In response to IR-treatment, MRE11 and CTIP-positively and WRN-negatively regulate p38-MAPK reactivation, in a CHK1-dependent manner. A degradation resistant WRN protein, mutated at serine 1141, abrogates p38-MAPK activation. We also showed that CHK1-p38-MAPK axis plays important role in RAD51 mediated HRR in WRN-silenced cells. Like CHK1 inhibition, pharmacological-inhibition of p38-MAPK also hyper-radiosensitizes WRN-depleted cells by targeting HR-pathway. Combination treatment of CHK1-inhibitor (currently under various clinical trials) and IR exhibited a strong synergy against WRN-deficient melanoma tumor in vivo. Taken together, our findings suggest that pharmacological targeting of CHK1-p38-MAPK mediated HRR is an attractive strategy for enhancing therapeutic response of radiation treatment of cancer.

Keywords:  CHK1 inhibitor; WRN; checkpoint; p38-MAPK inhibitor; radio-sensitization

DOI:  https://doi.org/10.1016/j.bcp.2021.114450
Cell Biosci. 2021 Feb 08. 11(1): 34

DDB2 regulates DNA replication through PCNA-independent degradation of CDT2.

Xiaojun Wu, Min Yu, Zhuxia Zhang, Feng Leng, Yue Ma, Ni Xie, Fei Lu.

   BACKGROUND: Targeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy. CRLCDT2 and CRLDDB2 are two key E3 ubiquitin ligases involved in DNA replication and DNA damage repair. But CDT2 and DDB2 are opposite prognostic factors in kinds of cancers, and the underlining mechanism needs to be elucidated.
METHODS: Small interfering RNAs were used to determine the function of target genes. Co-immunoprecipitation (Co-IP) was performed to detect the interaction between DDB2 and CDT2. Immunofluorescence assays and fluorescence activating cell sorting (FACS) were used to measure the change of DNA content. In vivo ubiquitination assay was carried out to clarify the ubiquitination of CDT2 mediated by DDB2. Cell synchronization was performed to arrest cells at G1/S and S phase. The mechanism involved in DDB2-mediated CDT2 degradation was investigated by constructing plasmids with mutant variants and measured by Western blot. Immunohistochemistry was performed to determine the relationship between DDB2 and CDT2. Paired two-side Student's t-test was used to measure the significance of the difference between control group and experimental group.
RESULTS: Knockdown of DDB2 stabilized CDT2, while over-expression of DDB2 enhanced ubiquitination of CDT2, and subsequentially degradation of CDT2. Although both DDB2 and CDT2 contain PIP (PCNA-interacting protein) box, PIP box is dispensable for DDB2-mediated CDT2 degradation. Knockdown of PCNA had negligible effects on the stability of CDT2, but promoted accumulation of CDT1, p21 and SET8. Silencing of DDB2 arrested cell cycle in G1 phase, destabilized CDT1 and reduced the chromatin loading of MCMs, thereby blocked the formation of polyploidy induced by ablation of CDT2. In breast cancer and ovarian teratoma tissues, high level of DDB2 was along with lower level of CDT2.
CONCLUSIONS: We found that CRL4DDB2 is the novel E3 ubiquitin ligases of CDT2, and DDB2 regulates DNA replication through indirectly regulates CDT1 protein stability by degrading CDT2 and promotes the assembly of pre-replication complex. Our results broaden the horizon for understanding the opposite function of CDT2 and DDB2 in tumorigenesis, and may provide clues for drug discovery in cancer therapy.

Keywords:  CDT2; Cancer; DDB2; DNA replication; Protein degradation; Ubiquitin ligase

DOI:  https://doi.org/10.1186/s13578-021-00540-5
Mol Cell Oncol. 2021 ;8(1): 1838863

Targeting drug transporters to prevent chemotherapy-induced peripheral neuropathy.

Kevin M Huang, Shuiying Hu.

  Chemotherapy-induced peripheral neuropathy is a debilitating toxicity that adversely affects patient quality and course of treatment. Recent findings have demonstrated that the etiology of peripheral neuropathy is dependent on transporter-mediated accumulation in dorsal root ganglia, and targeting this mechanism can afford neurological protection without compromising therapeutic efficacy.

Keywords:  Chemotherapy; oxaliplatin; paclitaxel; peripheral neuropathy; solute carriers

DOI:  https://doi.org/10.1080/23723556.2020.1838863
Mol Cell Oncol. 2020 Dec 07. 8(1): 1843951

Topoisomerase I prevents transcription-replication conflicts at transcription termination sites.

Yaqun Liu, Yea-Lih Lin, Philippe Pasero, Chun-Long Chen.

  R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops, we mapped RNA:DNA hybrids, markers of replication fork stalling and DNA double-strand breaks along the human genome. This analysis indicates that transient replication fork pausing occurs at the transcription termination sites of highly expressed genes enriched in R-loops and prevents head-on conflicts with transcription, in a topoisomerase I-dependent manner.

Keywords:  R-loops; Transcription-replication conflict; chromosome breaks; topoisomerase I

DOI:  https://doi.org/10.1080/23723556.2020.1843951
J Biol Chem. 2021 Feb 07. pii: S0021-9258(21)00160-5. [Epub ahead of print] 100388

Regulation of ATR-dependent DNA damage response by nitric oxide.

Chay Teng Yeo, Jennifer S Stancill, Bryndon J Oleson, Jamie K Schnuck, Joshua D Stafford, Aaron Naatz, Polly A Hansen, John A Corbett.

We have shown that nitric oxide limits ataxia-telangiectasia mutated (ATM) signaling by inhibiting mitochondrial oxidative metabolism in a β-cell selective manner. In this study, we examined the actions of nitric oxide on a second DNA damage response (DDR) transducer kinase, ataxia-telangiectasia and Rad3-related protein (ATR). In β-cells and non-β-cells, nitric oxide activates ATR signaling by inhibiting ribonucleotide reductase (RNR); however, when produced at iNOS-derived (low μM) levels, nitric oxide impairs ATR signaling in a β-cell selective manner. The inhibitory actions of nitric oxide are associated with impaired mitochondrial oxidative metabolism and lack of glycolytic compensation that results in a decrease in β-cell ATP. Like nitric oxide, inhibitors of mitochondrial respiration reduce ATP levels and limit ATR signaling in a β-cell selective manner. When non-β-cells are forced to utilize mitochondrial oxidative metabolism for ATP generation their response is more like β-cells, as nitric oxide and inhibitors of mitochondrial respiration attenuate ATR signaling. These studies support a dual role for nitric oxide in regulating ATR signaling. Nitric oxide activates ATR in all cell types examined by inhibiting RNR, and in a β-cell selective manner, iNOS-derived levels of nitric oxide limit ATR signaling by attenuating mitochondrial oxidative metabolism and depleting ATP.

DOI: https://doi.org/10.1016/j.jbc.2021.100388
NAR Cancer. 2020 Sep;2(3): zcaa019

The uracil-DNA glycosylase UNG protects the fitness of normal and cancer B cells expressing AID.

Shiva Safavi, Ariane Larouche, Astrid Zahn, Anne-Marie Patenaude, Diana Domanska, Kiersten Dionne, Torbjørn Rognes, Felix Dingler, Seong-Kwi Kang, Yan Liu, Nathalie Johnson, Josée Hébert, Ramiro E Verdun, Cristina A Rada, Francisco Vega, Hilde Nilsen, Javier M Di Noia.

In B lymphocytes, the uracil N-glycosylase (UNG) excises genomic uracils made by activation-induced deaminase (AID), thus underpinning antibody gene diversification and oncogenic chromosomal translocations, but also initiating faithful DNA repair. Ung-/- mice develop B-cell lymphoma (BCL). However, since UNG has anti- and pro-oncogenic activities, its tumor suppressor relevance is unclear. Moreover, how the constant DNA damage and repair caused by the AID and UNG interplay affects B-cell fitness and thereby the dynamics of cell populations in vivo is unknown. Here, we show that UNG specifically protects the fitness of germinal center B cells, which express AID, and not of any other B-cell subset, coincident with AID-induced telomere damage activating p53-dependent checkpoints. Consistent with AID expression being detrimental in UNG-deficient B cells, Ung-/- mice develop BCL originating from activated B cells but lose AID expression in the established tumor. Accordingly, we find that UNG is rarely lost in human BCL. The fitness preservation activity of UNG contingent to AID expression was confirmed in a B-cell leukemia model. Hence, UNG, typically considered a tumor suppressor, acquires tumor-enabling activity in cancer cell populations that express AID by protecting cell fitness.

DOI: https://doi.org/10.1093/narcan/zcaa019
Int J Mol Sci. 2021 Feb 08. pii: 1701. [Epub ahead of print]22(4):

Glutamine Synthetase as a Therapeutic Target for Cancer Treatment.

Go Woon Kim, Dong Hoon Lee, Yu Hyun Jeon, Jung Yoo, So Yeon Kim, Sang Wu Lee, Ha Young Cho, So Hee Kwon.

  The significance of glutamine in cancer metabolism has been extensively studied. Cancer cells consume an excessive amount of glutamine to facilitate rapid proliferation. Thus, glutamine depletion occurs in various cancer types, especially in poorly vascularized cancers. This makes glutamine synthetase (GS), the only enzyme responsible for de novo synthesizing glutamine, essential in cancer metabolism. In cancer, GS exhibits pro-tumoral features by synthesizing glutamine, supporting nucleotide synthesis. Furthermore, GS is highly expressed in the tumor microenvironment (TME) and provides glutamine to cancer cells, allowing cancer cells to maintain sufficient glutamine level for glutamine catabolism. Glutamine catabolism, the opposite reaction of glutamine synthesis by GS, is well known for supporting cancer cell proliferation via contributing biosynthesis of various essential molecules and energy production. Either glutamine anabolism or catabolism has a critical function in cancer metabolism depending on the complex nature and microenvironment of cancers. In this review, we focus on the role of GS in a variety of cancer types and microenvironments and highlight the mechanism of GS at the transcriptional and post-translational levels. Lastly, we discuss the therapeutic implications of targeting GS in cancer.

Keywords:  anticancer effect; cancer metabolism; glutamine metabolism; glutamine synthetase

DOI:  https://doi.org/10.3390/ijms22041701
Redox Biol. 2021 Jan 28. pii: S2213-2317(21)00020-3. [Epub ahead of print] 101872

Biomarkers of nucleic acid oxidation - A summary state-of-the-art.

Mu-Rong Chao, Mark D Evans, Chiung-Wen Hu, Yunhee Ji, Peter Møller, Pavel Rossner, Marcus S Cooke.

  Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.

Keywords:  Biomarkers; DNA; DNA repair; Nucleotide pool; Oxidative stress; RNA

DOI:  https://doi.org/10.1016/j.redox.2021.101872
J Thorac Dis. 2021 Jan;13(1): 189-201

SAMHD1 can suppress lung adenocarcinoma progression through the negative regulation of STING.

Yun Wu, Yuxu Niu, Yue Wu, Xiaoyu Chen, Xiaoyong Shen, Wen Gao.

   Background: The sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) has been specifically linked to lung cancer. However, the underlying mechanisms in regulating lung adenocarcinoma (LAC) are unclear. The aim of this study was to assess the specific regulation between SAMHD1 and LAC.
Methods: We retrospectively reviewed 238 patients who underwent surgery for LAC between January 2018 and December 2019. The expression of SAMHD1 was detected by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in tumors and paired adjacent tissues. A lentivirus was used to overexpress SAMHD1 and stimulator of interferon genes (STING) in A549 cells; and RT-qPCR and western blot analysis were performed to verify their levels. Cell proliferation was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Celigo imaging cytometry. Cell apoptosis was detected by Annexin V staining. Overexpressed SAMHD1 suppressed LAC progression in a xenograft model. The DNA damage response inhibitor (DDRi) was used to assess the cell proliferation and apoptosis rate in SAMHD1-overexpressing A549 cells and the control group. A rescue experiment was carried out to evaluate the potential influence of SAMHD1 and STING.
Results: A low expression of SAMHD1 was associated with advanced disease. Overexpression of SAMHD1 decreased cell proliferation and invasion in A549 cells, and the apoptosis rate was significantly higher in the overexpressed SAMHD1 cells than those in the control group. The overexpression of SAMHD1 inhibited tumor progression in the xenograft model. The expression of STING was lower in SAMHD1-overexpressing A549 cells than those in the wild-type group. Furthermore, the inhibited cellular behaviors of LAC cells resulting from the stable SAMHD1 expression were partially reversed after STING overexpression. Treatment with DDRi could inhibit cancer cell progression.
Conclusions: Upregulation of SAMHD1 could suppress the progression of LAC in vivo and in vitro through the negative regulation of STING.

Keywords:  Lung adenocarcinoma (LAC); progression; sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1); stimulator of interferon genes (STING)

DOI:  https://doi.org/10.21037/jtd-20-1889
Cell Death Discov. 2020 May 04. 6(1): 34

Deubiquitinase USP47-stabilized splicing factor IK regulates the splicing of ATM pre-mRNA.

Hye In Ka, Sunyi Lee, Sora Han, Ae Lee Jeong, Ji Young Park, Hyun Jeong Joo, Su Jung Soh, Doyeon Park, Young Yang.

IK depletion leads to an aberrant mitotic entry because of chromosomal misalignment through the enhancement of Aurora B activity at the interphase. Here, we demonstrate that IK, a spliceosomal component, plays a crucial role in the proper splicing of the ATM pre-mRNA among other genes related with the DNA Damage Response (DDR). Intron 1 in the ATM pre-mRNA, having lengths <200 bp, was not spliced in the IK-depleted cells and led to a deficiency of the ATM protein. Subsequently, the IK depletion-induced ATM protein deficiency impaired the ability to repair the damaged DNA. Because the absence of SMU1 results in IK degradation, the mechanism underlying IK degradation was exploited. IK was ubiquitinated in the absence of SMU1 and then subjected to proteolysis through the 26S proteasome. To prevent the proteolytic degradation of IK, a deubiquitinating enzyme, USP47, directly interacted with IK and stabilized it through deubiquitination. Collectively, our results suggest that IK is required for proper splicing of the ATM pre-mRNA and USP47 contributes toward the stabilization of IK.

DOI: https://doi.org/10.1038/s41420-020-0268-1
Nucleic Acids Res. 2021 Feb 08. pii: gkab041. [Epub ahead of print]

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

Amit Ketkar, Lane Smith, Callie Johnson, Alyssa Richey, Makayla Berry, Jessica H Hartman, Leena Maddukuri, Megan R Reed, Julie E C Gunderson, Justin W C Leung, Robert L Eoff.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

DOI: https://doi.org/10.1093/nar/gkab041
Purinergic Signal. 2021 Feb 12.

Targeting the purinergic pathway in breast cancer and its therapeutic applications.

Julia Beatrice de Araújo, Vanessa Vitória Kerkhoff, Sarah Franco Vieira de Oliveira Maciel, Débora Tavares de Resende E Silva.

  Breast cancer (BC) is the most frequent cause of death among women, representing a global public health problem. Here, we aimed to discuss the correlation between the purinergic system and BC, recognizing therapeutic targets. For this, we analyzed the interaction of extracellular nucleotides and nucleosides with the purinergic receptors P1 and P2, as well as the influence of ectonucleotidase enzymes (CD39 and CD73) on tumor progression. A comprehensive bibliographic search was carried out. The relevant articles for this review were found in the PubMed, Scielo, Lilacs, and ScienceDirect databases. It was observed that among the P1 receptors, the A1, A2A, and A2B receptors are involved in the proliferation and invasion of BC, while the A3 receptor is related to the inhibition of tumor growth. Among the P2 receptors, the P2X7 has a dual function. When activated for a short time, it promotes metastasis, but when activated for long periods, it is related to BC cell death. P2Y2 and P2Y6 receptors are related to BC proliferation and invasiveness. Also, the high expression of CD39 and CD73 in BC is strongly related to a worse prognosis. The receptors and ectonucleotidases involved with BC become possible therapeutic targets. Several purinergic pathways have been found to be involved in BC cell survival and progression. In this review, in addition to analyzing the pathways involved, we reviewed the therapeutic interventions already studied for BC related to the purinergic system, as well as to other possible therapeutic targets.

Keywords:  Breast tumors; Ectonucleotidases; Purinergic receptors; Therapeutic possibilities; Tumor progression

DOI:  https://doi.org/10.1007/s11302-020-09760-9