bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–03–27
35 papers selected by
Sean Rudd, Karolinska Institutet
  1. Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations.
  2. Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.
  3. The Purinosome: A Case Study for a Mammalian Metabolon.
  4. Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis.
  5. SUMO-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells.
  6. Histone H2A variants: Diversifying chromatin to ensure genome integrity.
  7. BMI-1 regulates DNA end resection and homologous recombination repair.
  8. DNA repair defects in cancer and therapeutic opportunities.
  9. Expression of BRCA1, BRCA2, RAD51, and other DSB repair factors is regulated by CRL4WDR70.
  10. PARP inhibition impedes the maturation of nascent DNA strands during DNA replication.
  11. Recombination Mediator Proteins: Misnomers That Are Key to Understanding the Genomic Instabilities in Cancer.
  12. The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ.
  13. Targeting DNA Damage Response and Immune Checkpoint for Anticancer Therapy.
  14. RNA polymerase pausing, stalling and bypass during transcription of damaged DNA: from molecular basis to functional consequences.
  15. Mobile origin-licensing factors confer resistance to conflicts with RNA polymerase.
  16. Phosphorylation of MAD2 at Ser195 Promotes Spindle Checkpoint Defects and Sensitizes Cancer Cells to Radiotherapy in ATM Deficient Cells.
  17. The Initiation of Eukaryotic DNA Replication.
  18. Interactions between miRNAs and Double-Strand Breaks DNA Repair Genes, Pursuing a Fine-Tuning of Repair.
  19. Role of Base Excision Repair in Innate Immune Cells and Its Relevance for Cancer Therapy.
  20. SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones.
  21. Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster.
  22. Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90.
  23. Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.
  24. Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.
  25. MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.
  26. Measuring S-Phase Duration from Asynchronous Cells Using Dual EdU-BrdU Pulse-Chase Labeling Flow Cytometry.
  27. Prospectively defined patterns of APOBEC3A mutagenesis are prevalent in human cancers.
  28. Immunotherapy for a POLE Mutation Advanced Non-Small-Cell Lung Cancer Patient.
  29. Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.
  30. The mycobacterial guaB1 gene encodes a guanosine 5´-monophosphate reductase with a cystathionine-β-synthase domain.
  31. Adenosine Monophosphate Activated Protein Kinase (AMPK) enhances chemotherapy response in Acute Myeloid Leukemia (AML).
  32. Thymine-starvation-induced chromosomal fragmentation is not required for thymineless death in Escherichia coli.
  33. Nucleotide pyrophosphatase/phosphodiesterases (NPPs) including NPP1 and NPP2/ ATX as important drug targets: A patent review (2015-2020).
  34. Recent Development of Prodrugs of Gemcitabine.
  35. Polymerase Epsilon-Associated Ultramutagenesis in Cancer.
  1. Exp Hematol. 2022 Mar 16. pii: S0301-472X(22)00125-4. [Epub ahead of print]
    Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations.
    Daniil E Shabashvili, Yang Feng, Prabhjot Kaur, Kartika Venugopal, Olga A Guryanova.
      Cytarabine and other chain-terminating nucleoside analogs that damage replication forks in rapidly proliferating cells are a cornerstone of leukemia chemotherapy, yet the outcomes remain unsatisfactory due to resistance and toxicity. Better understanding of DNA damage repair and downstream effector mechanisms in different disease subtypes can guide combination strategies that sensitize leukemia cells to cytarabine without increasing side effects. We have previously found that mutations in DNMT3A, one of the most commonly mutated genes in acute myeloid leukemia and associated with poor prognosis, predisposed cells to DNA damage and cell killing by cytarabine, cladribine, and other nucleoside analogs, which coincided with PARP1 dysfunction and DNA repair defect (Venugopal et al, 2021). In this perspective piece, we first overview DNA repair mechanisms that remove aberrant chain-terminating nucleotides as determinants of sensitivity or resistance to cytarabine and other nucleoside analogs. Next, we discuss PARP inhibition as a rational strategy to increase cytarabine efficacy in cells without DNMT3A mutations, while considering the implications of PARP inhibitor resistance for promoting clonal hematopoiesis. Finally, we examine the utility of p53 potentiators to boost leukemia cell killing by cytarabine in the context of mutant DNMT3A. Systematic profiling of DNA damage repair proficiency has the potential to uncover subtype-specific therapeutic dependencies in AML.
    Keywords:  DNA damage repair; DNMT3A; MDM2 inhibitors; PARP inhibitors; acute myeloid leukemia (AML); cytarabine; p53 potentiators; replication fork stalling
    DOI:  https://doi.org/10.1016/j.exphem.2022.03.008
  2. J Biol Chem. 2022 Mar 21. pii: S0021-9258(22)00293-9. [Epub ahead of print] 101853
    Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.
    Jingxuan He, Ling-Nan Zou, Vidhi Pareek, Stephen J Benkovic.
      There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein-protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells; and co-immunoprecipitation using StrepTag-labelled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase (PPAT), the first enzyme of the DNPB pathway, we discovered PAICS interaction with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C-terminus disrupts these interactions, and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate (IMP) into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impacts metabolite flux through this important pathway.
    Keywords:  bimolecular fluorescence; co-immunoprecipitation; complementation; de novo purine biosynthesis; metabolic channeling; protein complex; purinosome
    DOI:  https://doi.org/10.1016/j.jbc.2022.101853
  3. Annu Rev Biochem. 2022 Feb 23.
    The Purinosome: A Case Study for a Mammalian Metabolon.
    Anthony M Pedley, Vidhi Pareek, Stephen J Benkovic.
      Over the past fifteen years, we have unveiled a new mechanism by which cells achieve greater efficiency in de novo purine biosynthesis. This mechanism relies on the compartmentalization of de novo purine biosynthetic enzymes into a dynamic complex called the purinosome. In this review, we highlight our current understanding of the purinosome with emphasis on its biophysical properties and function and on the cellular mechanisms that regulate its assembly. We propose a model for functional purinosomes in which they consist of at least ten enzymes that localize near mitochondria and carry out de novo purine biosynthesis by metabolic channeling. We conclude by discussing challenges and opportunities associated with studying the purinosome and analogous metabolons. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-032620-105728
  4. Metabolites. 2022 Mar 13. pii: 241. [Epub ahead of print]12(3):
    Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis.
    Lucie Mádrová, Olga Součková, Radana Brumarová, Dana Dobešová, Jan Václavík, Štěpán Kouřil, Julie de Sousa, Jaroslava Friedecká, David Friedecký, Veronika Barešová, Marie Zikánová, Tomáš Adam.
      Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.
    Keywords:  HeLa cells; mass spectrometry; metabolomics; purine de novo synthesis; rare metabolic disorders
    DOI:  https://doi.org/10.3390/metabo12030241
  5. PLoS Genet. 2022 Mar 25. 18(3): e1009860
    SUMO-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells.
    Hugo Dorison, Ibtissam Talhaoui, Gerard Mazón.
      The post-translational modification of DNA damage response proteins with SUMO is an important mechanism to orchestrate a timely and orderly recruitment of repair factors to damage sites. After DNA replication stress and double-strand break formation, a number of repair factors are SUMOylated and interact with other SUMOylated factors, including the Yen1 nuclease. Yen1 plays a critical role in ensuring genome stability and unperturbed chromosome segregation by removing covalently linked DNA intermediates between sister chromatids that are formed by homologous recombination. Here we show how this important role of Yen1 depends on interactions mediated by non-covalent binding to SUMOylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 impair its ability to resolve DNA intermediates and result in chromosome mis-segregation and increased genome instability.
    DOI:  https://doi.org/10.1371/journal.pgen.1009860
  6. Semin Cell Dev Biol. 2022 Mar 21. pii: S1084-9521(22)00078-7. [Epub ahead of print]
    Histone H2A variants: Diversifying chromatin to ensure genome integrity.
    Philipp Oberdoerffer, Kyle M Miller.
      Histone variants represent chromatin components that diversify the structure and function of the genome. The variants of H2A, primarily H2A.X, H2A.Z and macroH2A, are well-established participants in DNA damage response (DDR) pathways, which function to protect the integrity of the genome. Through their deposition, post-translational modifications and unique protein interaction networks, these variants guard DNA from endogenous threats including replication stress and genome fragility as well as from DNA lesions inflicted by exogenous sources. A growing body of work is now providing a clearer picture on the involvement and mechanistic basis of H2A variant contribution to genome integrity. Beyond their well-documented role in gene regulation, we review here how histone H2A variants promote genome stability and how alterations in these pathways contribute to human diseases including cancer.
    Keywords:  DNA repair; Genome integrity; H2A variants; H2AX; Histones; MacroH2A
    DOI:  https://doi.org/10.1016/j.semcdb.2022.03.011
  7. Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00277-7. [Epub ahead of print]38(12): 110536
    BMI-1 regulates DNA end resection and homologous recombination repair.
    Amira Fitieh, Andrew J Locke, Fatemeh Mashayekhi, Fajr Khaliqdina, Ajit K Sharma, Ismail Hassan Ismail.
      BMI-1 is an essential regulator of transcriptional silencing during development. Recently, the role of BMI-1 in the DNA damage response has gained much attention, but the exact mechanism of how BMI-1 participates in the process is unclear. Here, we establish a role for BMI-1 in the repair of DNA double-strand breaks by homologous recombination (HR), where it promotes DNA end resection. Mechanistically, BMI-1 mediates DNA end resection by facilitating the recruitment of CtIP, thus allowing RPA and RAD51 accumulation at DNA damage sites. Interestingly, treatment with transcription inhibitors rescues the DNA end resection defects of BMI-1-depleted cells, suggesting BMI-1-dependent transcriptional silencing mediates DNA end resection. Moreover, we find that H2A ubiquitylation at K119 (H2AK119ub) promotes end resection. Taken together, our results identify BMI-1-mediated transcriptional silencing and promotion of H2AK119ub deposition as essential regulators of DNA end resection and thus the progression of HR.
    Keywords:  BMI-1; BMI1; CP: Molecular Biology; CtIP; DNA end resection; DNA repair; PRC1; PcG; Polycomb; homologous recombination; transcriptional silencing
    DOI:  https://doi.org/10.1016/j.celrep.2022.110536
  8. Genes Dev. 2022 Mar 01. 36(5-6): 278-293
    DNA repair defects in cancer and therapeutic opportunities.
    Jessica L Hopkins, Li Lan, Lee Zou.
      DNA repair and DNA damage signaling pathways are critical for the maintenance of genomic stability. Defects of DNA repair and damage signaling contribute to tumorigenesis, but also render cancer cells vulnerable to DNA damage and reliant on remaining repair and signaling activities. Here, we review the major classes of DNA repair and damage signaling defects in cancer, the genomic instability that they give rise to, and therapeutic strategies to exploit the resulting vulnerabilities. Furthermore, we discuss the impacts of DNA repair defects on both targeted therapy and immunotherapy, and highlight emerging principles for targeting DNA repair defects in cancer therapy.
    Keywords:  ATM; ATR; BRCA; DNA damage; DNA repair; PARP; cancer; genomic instability; immunotherapy; targeted therapy
    DOI:  https://doi.org/10.1101/gad.349431.122
  9. DNA Repair (Amst). 2022 Mar 15. pii: S1568-7864(22)00049-0. [Epub ahead of print]113 103320
    Expression of BRCA1, BRCA2, RAD51, and other DSB repair factors is regulated by CRL4WDR70.
    Zachary Mirman, Keshav Sharma, Thomas S Carroll, Titia de Lange.
      Double-strand break (DSB) repair relies on DNA damage response (DDR) factors including BRCA1, BRCA2, and RAD51, which promote homology-directed repair (HDR); 53BP1, which affects single-stranded DNA formation; and proteins that mediate end-joining. Here we show that the CRL4/DDB1/WDR70 complex (CRL4WDR70) controls the expression of DDR factors. Auxin-mediated degradation of WDR70 led to reduced expression of BRCA1, BRCA2, RAD51, and other HDR factors; 53BP1 and its downstream effectors; and other DDR factors. In contrast, cNHEJ factors were generally unaffected. WDR70 loss abrogated the localization of HDR factors to DSBs and elicited hallmarks of genomic instability, although 53BP1/RIF1 foci still formed. Mutation of the DDB1-binding WD40 motif, disruption of DDB1, or inhibition of cullins phenocopied WDR70 loss, consistent with CRL4, DDB1, and WDR70 functioning as a complex. RNA-sequencing revealed that WDR70 degradation affects the mRNA levels of DDR and many other factors. The data indicate that CRL4WDR70 is critical for expression of myriad genes including BRCA1, BRCA2, and RAD51.
    Keywords:  53BP1; 5′ End resection; BRCA1; CRL4/DDB1; DSB repair; Homology-directed repair; RAD51; WDR70
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103320
  10. Nat Struct Mol Biol. 2022 Mar 24.
    PARP inhibition impedes the maturation of nascent DNA strands during DNA replication.
    Alina Vaitsiankova, Kamila Burdova, Margarita Sobol, Amit Gautam, Oldrich Benada, Hana Hanzlikova, Keith W Caldecott.
      Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.
    DOI:  https://doi.org/10.1038/s41594-022-00747-1
  11. Genes (Basel). 2022 Feb 27. pii: 437. [Epub ahead of print]13(3):
    Recombination Mediator Proteins: Misnomers That Are Key to Understanding the Genomic Instabilities in Cancer.
    Justin Courcelle, Travis K Worley, Charmain T Courcelle.
      Recombination mediator proteins have come into focus as promising targets for cancer therapy, with synthetic lethal approaches now clinically validated by the efficacy of PARP inhibitors in treating BRCA2 cancers and RECQ inhibitors in treating cancers with microsatellite instabilities. Thus, understanding the cellular role of recombination mediators is critically important, both to improve current therapies and develop new ones that target these pathways. Our mechanistic understanding of BRCA2 and RECQ began in Escherichia coli. Here, we review the cellular roles of RecF and RecQ, often considered functional homologs of these proteins in bacteria. Although these proteins were originally isolated as genes that were required during replication in sexual cell cycles that produce recombinant products, we now know that their function is similarly required during replication in asexual or mitotic-like cell cycles, where recombination is detrimental and generally not observed. Cells mutated in these gene products are unable to protect and process replication forks blocked at DNA damage, resulting in high rates of cell lethality and recombination events that compromise genome integrity during replication.
    Keywords:  RecF; RecJ; RecO; RecQ; RecR; nucleotide excision repair; recombination; translesion synthesis
    DOI:  https://doi.org/10.3390/genes13030437
  12. Sci Rep. 2022 Mar 25. 12(1): 5163
    The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ.
    Robert Toth, Miklos Halmai, Zsuzsanna Gyorfy, Eva Balint, Ildiko Unk.
      PCNA is a central orchestrator of cellular processes linked to DNA metabolism. It is a binding platform for a plethora of proteins and coordinates and regulates the activity of several pathways. The outer side of PCNA comprises most of the known interacting and regulatory surfaces, whereas the residues at the inner side constitute the sliding surface facing the DNA double helix. Here, by investigating the L154A mutation found at the inner side, we show that the inner surface mediates protein interactions essential for genome stability. It forms part of the binding site of Rad18, a key regulator of DNA damage tolerance, and is required for PCNA sumoylation which prevents unscheduled recombination during replication. In addition, the L154 residue is necessary for stable complex formation between PCNA and the replicative DNA polymerase δ. Hence, its absence increases the mutation burden of yeast cells due to faulty replication. In summary, the essential role of the L154 of PCNA in guarding and maintaining stable replication and promoting DNA damage tolerance reveals a new connection between these processes and assigns a new coordinating function to the central channel of PCNA.
    DOI:  https://doi.org/10.1038/s41598-022-09208-7
  13. Int J Mol Sci. 2022 Mar 17. pii: 3238. [Epub ahead of print]23(6):
    Targeting DNA Damage Response and Immune Checkpoint for Anticancer Therapy.
    Jau-Ling Huang, Yu-Tzu Chang, Zhen-Yang Hong, Chang-Shen Lin.
      Deficiency in DNA damage response (DDR) genes leads to impaired DNA repair functions that will induce genomic instability and facilitate cancer development. However, alterations of DDR genes can serve as biomarkers for the selection of suitable patients to receive specific therapeutics, such as immune checkpoint blockade (ICB) therapy. In addition, certain altered DDR genes can be ideal therapeutic targets through adapting the mechanism of synthetic lethality. Recent studies indicate that targeting DDR can improve cancer immunotherapy by modulating the immune response mediated by cGAS-STING-interferon signaling. Investigations of the interplay of DDR-targeting and ICB therapies provide more effective treatment options for cancer patients. This review introduces the mechanisms of DDR and discusses their crucial roles in cancer therapy based on the concepts of synthetic lethality and ICB. The contemporary clinical trials of DDR-targeting and ICB therapies in breast, colorectal, and pancreatic cancers are included.
    Keywords:  DNA damage response; PARP; cGAS-STING; cancer immunotherapy; clinical trial; immune checkpoint; synthetic lethality
    DOI:  https://doi.org/10.3390/ijms23063238
  14. Nucleic Acids Res. 2022 Mar 22. pii: gkac174. [Epub ahead of print]
    RNA polymerase pausing, stalling and bypass during transcription of damaged DNA: from molecular basis to functional consequences.
    Aleksei Agapov, Anna Olina, Andrey Kulbachinskiy.
      Cellular DNA is continuously transcribed into RNA by multisubunit RNA polymerases (RNAPs). The continuity of transcription can be disrupted by DNA lesions that arise from the activities of cellular enzymes, reactions with endogenous and exogenous chemicals or irradiation. Here, we review available data on translesion RNA synthesis by multisubunit RNAPs from various domains of life, define common principles and variations in DNA damage sensing by RNAP, and consider existing controversies in the field of translesion transcription. Depending on the type of DNA lesion, it may be correctly bypassed by RNAP, or lead to transcriptional mutagenesis, or result in transcription stalling. Various lesions can affect the loading of the templating base into the active site of RNAP, or interfere with nucleotide binding and incorporation into RNA, or impair RNAP translocation. Stalled RNAP acts as a sensor of DNA damage during transcription-coupled repair. The outcome of DNA lesion recognition by RNAP depends on the interplay between multiple transcription and repair factors, which can stimulate RNAP bypass or increase RNAP stalling, and plays the central role in maintaining the DNA integrity. Unveiling the mechanisms of translesion transcription in various systems is thus instrumental for understanding molecular pathways underlying gene regulation and genome stability.
    DOI:  https://doi.org/10.1093/nar/gkac174
  15. Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00272-8. [Epub ahead of print]38(12): 110531
    Mobile origin-licensing factors confer resistance to conflicts with RNA polymerase.
    Matthias J Scherr, Syafiq Abd Wahab, Dirk Remus, Karl E Duderstadt.
      Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during the G1 phase and where synthesis begins in S phase. However, the temporal delay between phases exposes the replisome assembly pathway to potential disruption prior to replication. Using multicolor, single-molecule imaging, we systematically study the consequences of encounters between actively transcribing RNA polymerases (RNAPs) and replication initiation intermediates in the context of chromatin. We demonstrate that RNAP can push multiple licensed MCM helicases over long distances with nucleosomes ejected or displaced. Unexpectedly, we observe that MCM helicase loading intermediates also can be repositioned by RNAP and continue origin licensing after encounters with RNAP, providing a web of alternative origin specification pathways. Taken together, our observations reveal a surprising mobility in origin-licensing factors that confers resistance to the complex challenges posed by diverse obstacles encountered on chromosomes.
    Keywords:  CP; CP: Molecular biology; DNA replication; MCM2-7; Molecular biology; ORC; RNA polymerase; TIRF; chromatin; origin licensing; single molecule; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2022.110531
  16. Front Cell Dev Biol. 2022 ;10 817831
    Phosphorylation of MAD2 at Ser195 Promotes Spindle Checkpoint Defects and Sensitizes Cancer Cells to Radiotherapy in ATM Deficient Cells.
    Yang Wang, Tianyu Yu, Yi Han, Yazhi He, Yiran Song, Leiming Guo, Liwei An, Chunying Yang, Feng Wang.
      The spindle assembly checkpoint (SAC) is a critical monitoring device in mitosis for the maintenance of genomic stability. Specifically, the SAC complex comprises several factors, including Mad1, Mad2, and Bub1. Ataxia-telangiectasia mutated (ATM) kinase, the crucial regulator in DNA damage response (DDR), also plays a critical role in mitosis by regulating Mad1 dimerization and SAC. Here, we further demonstrated that ATM negatively regulates the phosphorylation of Mad2, another critical component of the SAC, which is also involved in DDR. Mechanistically, we found that phosphorylation of Mad2 is aberrantly increased in ATM-deficient cells. Point-mutation analysis further revealed that Serine 195 mainly mediated Mad2 phosphorylation upon ATM ablation. Functionally, the phosphorylation of Mad2 causes decreased DNA damage repair capacity and is related to the resistance to cancer cell radiotherapy. Altogether, this study unveils the key regulatory role of Mad2 phosphorylation in checkpoint defects and DNA damage repair in ATM-deficient cells.
    Keywords:  ATM kinase; DNA damage repair; checkpoint defect; mad2; phosphorylation
    DOI:  https://doi.org/10.3389/fcell.2022.817831
  17. Annu Rev Biochem. 2022 Feb 23.
    The Initiation of Eukaryotic DNA Replication.
    Alessandro Costa, John F X Diffley.
      DNA replication in eukaryotic cells initiates from large numbers of sites called replication origins. Initiation of replication from these origins must be tightly controlled to ensure the entire genome is precisely duplicated in each cell cycle. This is accomplished through the regulation of the first two steps in replication: loading and activation of the replicative DNA helicase. Here we describe what is known about the mechanism and regulation of these two reactions from a genetic, biochemical, and structural perspective, focusing on recent progress using proteins from budding yeast. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-072321-110228
  18. Int J Mol Sci. 2022 Mar 17. pii: 3231. [Epub ahead of print]23(6):
    Interactions between miRNAs and Double-Strand Breaks DNA Repair Genes, Pursuing a Fine-Tuning of Repair.
    Ricardo I Peraza-Vega, Mahara Valverde, Emilio Rojas.
      The repair of DNA damage is a crucial process for the correct maintenance of genetic information, thus, allowing the proper functioning of cells. Among the different types of lesions occurring in DNA, double-strand breaks (DSBs) are considered the most harmful type of lesion, which can result in significant loss of genetic information, leading to diseases, such as cancer. DSB repair occurs through two main mechanisms, called non-homologous end joining (NHEJ) and homologous recombination repair (HRR). There is evidence showing that miRNAs play an important role in the regulation of genes acting in NHEJ and HRR mechanisms, either through direct complementary binding to mRNA targets, thus, repressing translation, or by targeting other genes involved in the transcription and activity of DSB repair genes. Therefore, alteration of miRNA expression has an impact on the ability of cells to repair DSBs, which, in turn, affects cancer therapy sensitivity. This latter gives account of the importance of miRNAs as regulators of NHEJ and HRR and places them as a promising target to improve cancer therapy. Here, we review recent reports demonstrating an association between miRNAs and genes involved in NHEJ and HRR. We employed the Web of Science search query TS ("gene official symbol/gene aliases*" AND "miRNA/microRNA/miR-") and focused on articles published in the last decade, between 2010 and 2021. We also performed a data analysis to represent miRNA-mRNA validated interactions from TarBase v.8, in order to offer an updated overview about the role of miRNAs as regulators of DSB repair.
    Keywords:  DNA repair; double-strand breaks; homologous recombination repair; miRNAs; non-homologous end joining
    DOI:  https://doi.org/10.3390/ijms23063231
  19. Biomedicines. 2022 Feb 26. pii: 557. [Epub ahead of print]10(3):
    Role of Base Excision Repair in Innate Immune Cells and Its Relevance for Cancer Therapy.
    Shengyuan Zhao, Samy L Habib, Alireza G Senejani, Manu Sebastian, Dawit Kidane.
      Innate immunity is critical for immediate recognition and elimination of invading pathogens or defense against cancer cell growth. Dysregulation of innate immune systems is associated with the pathogenesis of different types of inflammatory diseases, including cancer. In addition, the maintenance of innate immune cells' genomic integrity is crucial for the survival of all organisms. Oxidative stress generated from innate immune cells may cause self-inflicted DNA base lesions as well as DNA damage on others neighboring cells, including cancer cells. Oxidative DNA base damage is predominantly repaired by base excision repair (BER). BER process different types of DNA base lesions that are presented in cancer and innate immune cells to maintain genomic integrity. However, mutations in BER genes lead to impaired DNA repair function and cause insufficient genomic integrity. Moreover, several studies have implicated that accumulation of DNA damage leads to chromosomal instability that likely activates the innate immune signaling. Furthermore, dysregulation of BER factors in cancer cells modulate the infiltration of innate immune cells to the tumor microenvironment. In the current review, the role of BER in cancer and innate immune cells and its impact on innate immune signaling within the tumor microenvironment is summarized. This is a special issue that focuses on DNA damage and cancer therapy to demonstrate how BER inhibitor or aberrant repair modulates innate inflammatory response and impact immunotherapy approaches. Overall, the review provides substantial evidence to understand the impact of BER in innate immune response dynamics within the current immune-based therapeutic strategy.
    Keywords:  base excision repair; immunotherapy; innate immune cells; innate inflammatory signaling
    DOI:  https://doi.org/10.3390/biomedicines10030557
  20. Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00282-0. [Epub ahead of print]38(12): 110541
    SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones.
    Tadashi Nakagawa, Akane Morohoshi, Yuko Nagasawa, Makiko Nakagawa, Masaki Hosogane, Yasuhiro Noda, Toru Hosoi, Keiko Nakayama.
      The histone chaperone complex FACT comprises SPT16 and SSRP1 and contributes to DNA replication, transcription, and repair, but how it plays such various roles is unclear. Here, we show that human SPT16 is ubiquitylated at lysine-674 (K674) by the DCAF14-CRL4 ubiquitin ligase. K674 is located in the middle domain of SPT16, and the corresponding residue of the yeast ortholog is critical for binding to histone H3.1-H4. We show that the middle domain of human SPT16 binds to histone H3.1-H4 and that this binding is inhibited by K674 ubiquitylation. Cells with heterozygous knockin of a K674R mutant of SPT16 manifest reduction of both SPT16 ubiquitylation and H3.1 in chromatin, a reduced population in mid S phase, impaired proliferation, and increased susceptibility to S phase stress. Our data thus indicate that SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones and may thereby control DNA replication-coupled histone incorporation into chromatin.
    Keywords:  CP: Molecular Biology; CRL4; DCAF14; DNA replication; FACT; chromatin; histone chaperone; histone incorporation; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.celrep.2022.110541
  21. Genes (Basel). 2022 Mar 08. pii: 474. [Epub ahead of print]13(3):
    Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster.
    Adam Thomas, Julie Cox, Kelly B Wolfe, Carrie Hui Mingalone, Haleigh R Yaspan, Mitch McVey.
      Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3' end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in Drosophila melanogaster lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the blm helq and blm fancm double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.
    Keywords:  D-loop; double-strand break; end joining; mutagenesis; transposon
    DOI:  https://doi.org/10.3390/genes13030474
  22. J Biol Chem. 2022 Mar 17. pii: S0021-9258(22)00285-X. [Epub ahead of print] 101845
    Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90.
    Anthony M Pedley, Jack P Boylan, Chung Yu Chan, Erin L Kennedy, Minjoung Kyoung, Stephen J Benkovic.
      Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase (FGAMS) granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid-liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Lastly, we demonstrate that the heat shock protein HSP90 helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of HSP90 activity induced the transformation of FGAMS clusters into more irregularly-shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells.
    Keywords:  liquid condensate; liquid-liquid phase separation; metabolism; protein complex; purine biosynthesis
    DOI:  https://doi.org/10.1016/j.jbc.2022.101845
  23. Nat Commun. 2022 Mar 24. 13(1): 1240
    Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.
    Martin Peterka, Nina Akrap, Songyuan Li, Sandra Wimberger, Pei-Pei Hsieh, Dmitrii Degtev, Burcu Bestas, Jack Barr, Stijn van de Plassche, Patricia Mendoza-Garcia, Saša Šviković, Grzegorz Sienski, Mike Firth, Marcello Maresca.
      Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.
    DOI:  https://doi.org/10.1038/s41467-022-28771-1
  24. Cancers (Basel). 2022 Mar 11. pii: 1446. [Epub ahead of print]14(6):
    Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.
    Dinh Hoa Hoang, Corey Morales, Ivan Rodriguez Rodriguez, Melissa Valerio, Jiamin Guo, Min-Hsuan Chen, Xiwei Wu, David Horne, Varsha Gandhi, Lisa S Chen, Bin Zhang, Vinod Pullarkat, Steven T Rosen, Guido Marcucci, Ralf Buettner, Le Xuan Truong Nguyen.
      It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.
    Keywords:  8-chloro-adenosine; acute myeloid leukemia; metabolism; rRNA synthesis; venetoclax
    DOI:  https://doi.org/10.3390/cancers14061446
  25. Oncotarget. 2022 ;13 534-550
    MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.
    Pratima Nangia-Makker, Malathy P V Shekhar, Victor Hogan, Vitaly Balan, Avraham Raz.
      The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.
    Keywords:  DNA synthesis; MYH9; dNTP; deoxyribose-5-phosphate; myosin II
    DOI:  https://doi.org/10.18632/oncotarget.28219
  26. Genes (Basel). 2022 Feb 24. pii: 408. [Epub ahead of print]13(3):
    Measuring S-Phase Duration from Asynchronous Cells Using Dual EdU-BrdU Pulse-Chase Labeling Flow Cytometry.
    Marta Bialic, Baraah Al Ahmad Nachar, Maria Koźlak, Vincent Coulon, Etienne Schwob.
      Eukaryotes duplicate their chromosomes during the cell cycle S phase using thousands of initiation sites, tunable fork speed and megabase-long spatio-temporal replication programs. The duration of S phase is fairly constant within a given cell type, but remarkably plastic during development, cell differentiation or various stresses. Characterizing the dynamics of S phase is important as replication defects are associated with genome instability, cancer and ageing. Methods to measure S-phase duration are so far indirect, and rely on mathematical modelling or require cell synchronization. We describe here a simple and robust method to measure S-phase duration in cell cultures using a dual EdU-BrdU pulse-labeling regimen with incremental thymidine chases, and quantification by flow cytometry of cells entering and exiting S phase. Importantly, the method requires neither cell synchronization nor genome engineering, thus avoiding possible artifacts. It measures the duration of unperturbed S phases, but also the effect of drugs or mutations on it. We show that this method can be used for both adherent and suspension cells, cell lines and primary cells of different types from human, mouse and Drosophila. Interestingly, the method revealed that several commonly-used cancer cell lines have a longer S phase compared to untransformed cells.
    Keywords:  DNA replication; EdU-BrdU pulse chase; S phase; cell lines; cytometry
    DOI:  https://doi.org/10.3390/genes13030408
  27. Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00299-6. [Epub ahead of print]38(12): 110555
    Prospectively defined patterns of APOBEC3A mutagenesis are prevalent in human cancers.
    Rachel A DeWeerd, Eszter Németh, Ádám Póti, Nataliya Petryk, Chun-Long Chen, Olivier Hyrien, Dávid Szüts, Abby M Green.
      Mutational signatures defined by single base substitution (SBS) patterns in cancer have elucidated potential mutagenic processes that contribute to malignancy. Two prevalent mutational patterns in human cancers are attributed to the APOBEC3 cytidine deaminase enzymes. Among the seven human APOBEC3 proteins, APOBEC3A is a potent deaminase and proposed driver of cancer mutagenesis. In this study, we prospectively examine genome-wide aberrations by expressing human APOBEC3A in avian DT40 cells. From whole-genome sequencing, we detect hundreds to thousands of base substitutions per genome. The APOBEC3A signature includes widespread cytidine mutations and a unique insertion-deletion (indel) signature consisting largely of cytidine deletions. This multi-dimensional APOBEC3A signature is prevalent in human cancer genomes. Our data further reveal replication-associated mutations, the rate of stem-loop and clustered mutations, and deamination of methylated cytidines. This comprehensive signature of APOBEC3A mutagenesis is a tool for future studies and a potential biomarker for APOBEC3 activity in cancer.
    Keywords:  APOBEC3; CP:Cancer; cancer genome; cytidine deaminase; indel; methylcytidine; mutagenesis; mutational signature
    DOI:  https://doi.org/10.1016/j.celrep.2022.110555
  28. Front Pharmacol. 2022 ;13 817265
    Immunotherapy for a POLE Mutation Advanced Non-Small-Cell Lung Cancer Patient.
    Yang Fu, Yue Zheng, Pei-Pei Wang, Yue-Yun Chen, Zhen-Yu Ding.
      Currently, the predictive role of POLE mutations for immunotherapy is under intense investigation. The POLE gene encodes one of the four subunits of DNA polymerase important for DNA replication and repair. POLE mutations are related to other favorable predicative factors such as high expression of PD-L1, high TMB, and infiltration of CD8+ cells in the tumor microenvironment. No formal clinical trials studied the efficacy of immunotherapy in lung patients harboring POLE mutation, and only few cases were mentioned in the literature. Moreover, lung cancer patients are prone to brain metastasis, which is notorious for the unresponsiveness to chemotherapy. The efficacy of immunotherapy for brain metastasis is still controversial. Here, we described a case of a POLEmt non-small-cell lung cancer (NSCLC) patient with brain metastasis who was treated with immunotherapy. His brain lesions disappeared after treatment. Our report strongly supported the benefit of immune-combined therapy for advanced NSCLC patients with POLE mutation, even with brain metastasis.
    Keywords:  CR; PD-L1; POLE mutation; brain metastases; lung adenocarcinoma
    DOI:  https://doi.org/10.3389/fphar.2022.817265
  29. Cancer Discov. 2022 Mar 23. pii: candisc.1117.2021. [Epub ahead of print]
    Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.
    Mihir Rajurkar, Aparna R Parikh, Alexander Solovyov, Eunae You, Anupriya S Kulkarni, Chong Chu, Katherine H Xu, Christopher Jaicks, Martin S Taylor, Connie Wu, Katherine A Alexander, Charly R Good, Annamaria Szabolcs, Stefanie Gerstberger, Antuan V Tran, Nova Xu, Richard Y Ebright, Emily E Van Seventer, Kevin D Vo, Eric C Tai, Chenyue Lu, Jasmin Joseph-Chazan, Michael J Raabe, Linda T Nieman, Niyati Desai, Kshitij S Arora, Matteo Ligorio, Vishal Thapar, Limor Cohen, Padric M Garden, Yasmeen Senussi, Hui Zheng, Jill N Allen, Lawrence S Blaszkowsky, Jeffrey W Clark, Lipika Goyal, Jennifer Y Wo, David P Ryan, Ryan B Corcoran, Vikram Deshpande, Miguel N Rivera, Martin J Aryee, Theodore S Hong, Shelley L Berger, David R Walt, Kathleen H Burns, Peter J Park, Benjamin D Greenbaum, David T Ting.
      Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer (CRC) implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in CRC pre-clinical models with a preferential effect in P53 mutant cell lines linked with direct binding of P53 to repeat elements. We translate these findings to a human Phase 2 trial of single agent 3TC treatment in metastatic CRC with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on CRC tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in CRC by targeting the viral mimicry of repeat elements.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-1117
  30. FEBS J. 2022 Mar 26.
    The mycobacterial guaB1 gene encodes a guanosine 5´-monophosphate reductase with a cystathionine-β-synthase domain.
    Zdeněk Knejzlík, Michal Doležal, Klára Herkommerová, Kamila Clarova, Martin Klíma, Matteo Dedola, Eva Zborníková, Dominik Rejman, Iva Pichová.
      Mycobacteria express enzymes from both the de novo and purine-salvage pathways. However, the regulation of these processes and the roles of individual metabolic enzymes have not been sufficiently detailed. Both Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm) possess three guaB genes, but information is only available on guaB2, which encodes an essential inosine 5´-monophosphate dehydrogenase (IMPDH) involved in de novo purine biosynthesis. This paper shows that guaB1, annotated in databases as a putative IMPDH, encodes a guanosine 5´-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine-salvage pathway and contains a cystathionine β-synthase domain (CBS), which is essential for enzyme activity. GMPR activity is allosterically regulated by the ATP/GTP ratio in a pH-dependent manner. Bioinformatic analysis has indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.
    Keywords:   Mycobacterium smegmatis ; Mycobacterium tuberculosis ; guaB1 ; CBS domain; GMPR; activity regulation; purine biosynthesis
    DOI:  https://doi.org/10.1111/febs.16448
  31. Cancer Lett. 2022 Mar 20. pii: S0304-3835(22)00142-2. [Epub ahead of print]535 215659
    Adenosine Monophosphate Activated Protein Kinase (AMPK) enhances chemotherapy response in Acute Myeloid Leukemia (AML).
    Lais Ghiraldeli, Rebecca Anderson, Kristin Pladna, Timothy S Pardee.
      Adenosine monophosphate activated protein kinase (AMPK) is a master regulator of cell metabolism and is involved in cancer as both a tumor suppressor and a source of resistance to metabolic stress. The role of AMPK in response to chemotherapy has been examined in solid tumor models but remains unclear in acute myeloid leukemia (AML). To determine the role of AMPK in chemotherapy response, AML cell lines were generated lacking AMPK activity. AMPK knock out cells demonstrated significant resistance to cytarabine and doxorubicin both in vitro and in vivo. Mitochondrial mass and function were unchanged in AMPK knockout cells. Mechanistically, AMPK knock out cells demonstrated a diminished DNA damage response with significantly lower γH2AX foci, p53 and p21 induction as well as decreased apoptosis following chemotherapy exposure. Most importantly, TCGA datasets revealed that patients expressing low levels of the PRKAA1 subunit of AMPK had significantly shorter survival. Finally, AML cells were sensitized to chemotherapy with the addition of the AMPK activator AICAR. These data demonstrate that AMPK sensitizes AML cells to chemotherapy and suggests a contribution of the cellular metabolic state to cell fate decisions ultimately affecting therapy response.
    DOI:  https://doi.org/10.1016/j.canlet.2022.215659
  32. Mol Microbiol. 2022 Mar 24.
    Thymine-starvation-induced chromosomal fragmentation is not required for thymineless death in Escherichia coli.
    Sharik R Khan, Andrei Kuzminov.
      Thymine or thymidine starvation induces robust chromosomal fragmentation in E. coli thyA deoCABD mutants, and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stays unchanged in hyper-TLD-exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication-dependent, but undetectable by pulsed field gel, double-strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. Expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine-to-thymine interconversion during T-starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T-starvation, is either dispensable or redundant for the overall TLD pathology, including hyper-TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide minor contribution to TLD, but only in the repair-deficient thyA deoCABD recBCD mutant.
    Keywords:  Double-strand breaks; Nucleotide salvage pathway; Replication forks; deoCABD; recBCD
    DOI:  https://doi.org/10.1111/mmi.14897
  33. Expert Opin Ther Pat. 2022 Mar 25.
    Nucleotide pyrophosphatase/phosphodiesterases (NPPs) including NPP1 and NPP2/ ATX as important drug targets: A patent review (2015-2020).
    Sehrish Bano, Mariya Al-Rashida, Rima D Alharthy, Imtiaz Ali Khan, Jamshed Iqbal.
       INTRODUCTION: Nucleoside triphosphate diphosphohydrolases (NTPDases), alkaline phosphatases (APs), and ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) are nucleotidases found on the cell surface. It is a promising therapeutic target for a range of disorders, including fibrosis, tumour metastasis, pruritus, inflammation, multiple sclerosis, and autoimmune diseases. As a result, therapeutic intervention including selective inhibitors of NPPs is required.
    AREA COVERED: Many chemical substances, including pyrazole, pyridine and bicyclic compounds have demonstrated promising inhibitory potential for ecto-nucleotide pyrophosphatase/phosphodiesterases. The chemistry and clinical applications of NPPs inhibitors patented between 2015 and 2020 are discussed in this review.
    EXPERT OPINION: : In recent years, there has been a lot of effort put into finding effective and selective inhibitors of NPPs. Despite the fact that a variety of synthetic inhibitors have been created, only a few investigations on their in vivo activity have been published. In addition to IOA-289 which has passed Phase Ia clinical trials; potent ATX inhibitor compounds such as BLD-0409, IPF and BBT-877 have been placed in phase I clinical studies. Some of the most promising ATX inhibitors in recent years are closely related analogs of previously known inhibitors, such as PF-8380. Knowledge of the structure activity relationship of such promising inhibitors can potentially translate into the discovery of more potent and effective inhibitors of NPP with a variety of structural characteristics and favourable therapeutic activities.
    Keywords:  cancer; ecto-nucleotide pyrophosphatases/phosphodiesterases; idiopathic pulmonary fibrosis; inflammation; inhibitors
    DOI:  https://doi.org/10.1080/13543776.2022.2058874
  34. Genes (Basel). 2022 Mar 05. pii: 466. [Epub ahead of print]13(3):
    Recent Development of Prodrugs of Gemcitabine.
    Bhoomika Pandit, Maksim Royzen.
      Gemcitabine is a nucleoside analog that has been used widely as an anticancer drug for the treatment of a variety of conditions, including ovarian, bladder, non-small-cell lung, pancreatic, and breast cancer. However, enzymatic deamination, fast systemic clearance, and the emergence of chemoresistance have limited its efficacy. Different prodrug strategies have been explored in recent years, seeking to obtain better pharmacokinetic properties, efficacy, and safety. Different drug delivery strategies have also been employed, seeking to transform gemcitabine into a targeted medicine. This review will provide an overview of the recent developments in gemcitabine prodrugs and their effectiveness in treating cancerous tumors.
    Keywords:  anticancer agent; chemotherapy; drug delivery; gemcitabine; prodrug
    DOI:  https://doi.org/10.3390/genes13030466
  35. Cancers (Basel). 2022 Mar 12. pii: 1467. [Epub ahead of print]14(6):
    Polymerase Epsilon-Associated Ultramutagenesis in Cancer.
    XuanXuan Xing, Ning Jin, Jing Wang.
      With advances in next generation sequencing (NGS) technologies, efforts have been made to develop personalized medicine, targeting the specific genetic makeup of an individual. Somatic or germline DNA Polymerase epsilon (PolE) mutations cause ultramutated (>100 mutations/Mb) cancer. In contrast to mismatch repair-deficient hypermutated (>10 mutations/Mb) cancer, PolE-associated cancer is primarily microsatellite stable (MSS) In this article, we provide a comprehensive review of this PolE-associated ultramutated tumor. We describe its molecular characteristics, including the mutation sites and mutation signature of this type of tumor and the mechanism of its ultramutagenesis. We discuss its good clinical prognosis and elucidate the mechanism for enhanced immunogenicity with a high tumor mutation burden, increased neoantigen load, and enriched tumor-infiltrating lymphocytes. We also provide the rationale for immune checkpoint inhibitors in PolE-mutated tumors.
    Keywords:  cancer; immunotherapy; polymerase epsilon; prognosis; ultramutagenesis
    DOI:  https://doi.org/10.3390/cancers14061467
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