bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–05–08
28 papers selected by
Sean Rudd, Karolinska Institutet
  1. Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia.
  2. CDC7-independent G1/S transition revealed by targeted protein degradation.
  3. Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.
  4. The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells.
  5. Walking a tightrope: The complex balancing act of R-loops in genome stability.
  6. Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes.
  7. Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.
  8. The Nuclear Proteins TP73 and CUL4A Confer Resistance to Cytarabine by Induction of Translesion DNA Synthesis via Mono-ubiquitination of PCNA.
  9. Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia.
  10. A rethink about enzymes that drive DNA replication.
  11. H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication.
  12. Structural Dynamics of a Common Mutagenic Oxidative DNA Lesion in Duplex DNA and during DNA Replication.
  13. Mre11-Rad50 oligomerization promotes DNA double-strand break repair.
  14. Molecular basis for the initiation of DNA primer synthesis.
  15. DNA repair and damage pathways in mesothelioma development and therapy.
  16. The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII.
  17. Delineation of a minimal topoisomerase II binding protein 1 (TOPBP1) for regulated activation of ATR at DNA double-strand breaks.
  18. METTL3 promotes homologous recombination repair and modulates chemotherapeutic response in breast cancer by regulating the EGF/Rad51 axis.
  19. Catastrophic DNA replication in unscheduled tetraploid cells.
  20. High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control.
  21. Coordination between phospholipid pools and DNA damage sensing.
  22. FORK-seq: Single-Molecule Profiling of DNA Replication.
  23. SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.
  24. Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells.
  25. Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops.
  26. Alterations in homologous recombination repair genes in prostate cancer brain metastases.
  27. Rare and misincorporated DNA N6-methyladenine is a hallmark of cytotoxic stresses for selectively stimulating the stemness and proliferation of glioblastoma cells.
  28. CCNE1 Amplification and PKMYT1 Inhibition Are Synthetic Lethal.
  1. EMBO Mol Med. 2022 May 06. e15203
    Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia.
    Joan So, Alexander C Lewis, Lorey K Smith, Kym Stanley, Rheana Franich, David Yoannidis, Lizzy Pijpers, Pilar Dominguez, Simon J Hogg, Stephin J Vervoort, Fiona C Brown, Ricky W Johnstone, Gabrielle McDonald, Danielle B Ulanet, Josh Murtie, Emily Gruber, Lev M Kats.
      The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.
    Keywords:  DHODH; acute myeloid leukemia; leukemic stem cells; protein translation
    DOI:  https://doi.org/10.15252/emmm.202115203
  2. Nature. 2022 May 04.
    CDC7-independent G1/S transition revealed by targeted protein degradation.
    Jan M Suski, Nalin Ratnayeke, Marcin Braun, Tian Zhang, Vladislav Strmiska, Wojciech Michowski, Geylani Can, Antoine Simoneau, Konrad Snioch, Mikolaj Cup, Caitlin M Sullivan, Xiaoji Wu, Joanna Nowacka, Timothy B Branigan, Lindsey R Pack, James A DeCaprio, Yan Geng, Lee Zou, Steven P Gygi, Johannes C Walter, Tobias Meyer, Piotr Sicinski.
      The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division1. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication.
    DOI:  https://doi.org/10.1038/s41586-022-04698-x
  3. Nat Commun. 2022 May 05. 13(1): 2480
    Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.
    Chinnu Rose Joseph, Sabrina Dusi, Michele Giannattasio, Dana Branzei.
      DNA damage tolerance (DDT), activated by replication stress during genome replication, is mediated by translesion synthesis and homologous recombination (HR). Here we uncover that DDK kinase, essential for replication initiation, is critical for replication-associated recombination-mediated DDT. DDK relies on its multi-monoSUMOylation to facilitate HR-mediated DDT and optimal retention of Rad51 recombinase at replication damage sites. Impairment of DDK kinase activity, reduced monoSUMOylation and mutations in the putative SUMO Interacting Motifs (SIMs) of Rad51 impair replication-associated recombination and cause fork uncoupling with accumulation of large single-stranded DNA regions at fork branching points. Notably, genetic activation of salvage recombination rescues the uncoupled fork phenotype but not the recombination-dependent gap-filling defect of DDK mutants, revealing that the salvage recombination pathway operates preferentially proximal to fork junctions at stalled replication forks. Overall, we uncover that monoSUMOylated DDK acts with Rad51 in an axis that prevents replication fork uncoupling and mediates recombination-dependent gap-filling.
    DOI:  https://doi.org/10.1038/s41467-022-30215-9
  4. Cell Cycle. 2022 May 04.
    The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells.
    Seok-Won Jang, Jung Min Kim.
      The Fanconi anemia (FA) DNA repair pathway is required for DNA inter-strand crosslink (ICL) repair. Besides its role in ICL repair, FA proteins play a central role in stabilizing stalled replication forks, thereby ensuring genome integrity. We previously demonstrated that depletion of replication protein A (RPA) induces the activation of FA pathway leading to FANCD2 monoubiquitination and FANCD2 foci formation. Thus, we speculated that FA-deficient cells would be more sensitive to RPA inhibition compared to FA-proficient cells. Following treatment with RPA inhibitor HAMNO, we observed significant induction in FANCD2 monoubiquitination and foci formation as observed in RPA depletion. In addition, HAMNO treatment caused increased levels of ϒ-H2AX and S-phase accumulation in FA-deficient cells. Importantly, FA-deficient cells showed more increased sensitivity to HAMNO than FA-proficient cells. Moreover, in combination with cisplatin, HAMNO further enhanced the cytotoxicity of cisplatin in FA-deficient cells, while being less toxic against FA-proficient cells. This result suggests that RPA inhibition might be a potential therapeutic candidate for the treatment of FA pathway-deficient tumors.
    Keywords:  Chemosensitization; FANCD2; Fanconi anemia pathway; HAMNO; Replication Protein A; Replication stress
    DOI:  https://doi.org/10.1080/15384101.2022.2074200
  5. Mol Cell. 2022 Apr 29. pii: S1097-2765(22)00325-2. [Epub ahead of print]
    Walking a tightrope: The complex balancing act of R-loops in genome stability.
    Joshua R Brickner, Jada L Garzon, Karlene A Cimprich.
      Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic.
    Keywords:  DNA damage; R-loop pathology; R-loops; RNA-DNA hybrid; double-strand break repair; genome stability; replication stress
    DOI:  https://doi.org/10.1016/j.molcel.2022.04.014
  6. Toxicol Appl Pharmacol. 2022 May 02. pii: S0041-008X(22)00187-9. [Epub ahead of print] 116042
    Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes.
    Alexandra N Nail, Lakynkalina M McCaffrey, Mayukh Banerjee, Ana P Ferragut Cardoso, J Christopher States.
      An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure.
    Keywords:  ATM; Arsenic; DNA damage response; Keratinocytes; MRN; Phosphatases
    DOI:  https://doi.org/10.1016/j.taap.2022.116042
  7. Nucleic Acids Res. 2022 May 07. pii: gkac299. [Epub ahead of print]
    Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.
    Bhushan L Thakur, Adrian M Baris, Haiqing Fu, Christophe E Redon, Lorinc S Pongor, Sara Mosavarpour, Jacob M Gross, Sang-Min Jang, Robin Sebastian, Koichi Utani, Lisa M Jenkins, Fred E Indig, Mirit I Aladjem.
      During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.
    DOI:  https://doi.org/10.1093/nar/gkac299
  8. Hemasphere. 2022 May;6(5): e0708
    The Nuclear Proteins TP73 and CUL4A Confer Resistance to Cytarabine by Induction of Translesion DNA Synthesis via Mono-ubiquitination of PCNA.
    Miriam Rehberger, Jonas A Schäfer, Anna-Maria Krampitz, Anne Catherine Bretz, Lukas Jost, Torsten Haferlach, Thorsten Stiewe, Andreas Neubauer.
      Resistance to cytarabine is a key problem in the treatment of acute myeloid leukemia (AML). To understand the molecular biology of resistance to cytarabine, a viability-based chemosensitizer screen was utilized. We screened synthetic lethal targets using 437 different small interfering RNAs (siRNAs) directed against factors involved in DNA repair mechanisms and cytarabine as the chemical compound. Three hits were identified: CUL4A, TP73, and RFC2. We show here that the ubiquitin ligase CULLIN 4A (CUL4A) and the tumor-suppressive transcription factor p73 contribute to drug resistance by modulating DNA damage response. P73 confers resistance to cytarabine therapy by transactivation of REV3L, encoding the catalytic subunit of translesion DNA polymerase ζ, and CUL4A probably by influencing proliferating cell nuclear antigen (PCNA) and the polymerase switch towards error-prone translesion DNA polymerases. Abrogation of the polymerase ζ by siRNA causes identical effects as siRNAs against CUL4A or TP73 and resensitizes cells towards cytarabine therapy in vitro. As CUL4A needs to be activated by neddylation to facilitate the degradation of several proteins including PCNA, we propose a novel explanation for the synergism between cytarabine and the neddylation inhibitor pevonedistat by inhibition of translesion synthesis. In keeping with this, in AML patients treated with cytarabine, we found high expression of CUL4A and TP73 to be associated with poor prognosis.
    DOI:  https://doi.org/10.1097/HS9.0000000000000708
  9. J Mol Biol. 2022 Apr 29. pii: S0022-2836(22)00197-8. [Epub ahead of print] 167617
    Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia.
    Dipayan Ghosh, Susmita Kumari, Sathees C Raghavan.
      Ribonuclease H2 (RNase H2) is a member of the ribonuclease H family of enzymes involved in removal of RNA from RNA-DNA hybrids as well as ribonucleotides which get misincorporated into the genomic DNA. Recent studies have shown that RNase H2 function is also needed for successful DNA repair through NHEJ events where DNA pol µ uses ribonucleotides during the gap filling stage. Mammalian RNase H2 is composed of three subunits, RNASEH2A, RNASEH2B and RNASEH2C. There have been studies suggesting changes in expression of these genes in various cancers of breast, prostate, colon, liver, and kidney. In this study, we have investigated the functional role of RNASEH2A and RNASEH2B in leukemic T-cells, MOLT4 and Jurkat. shRNA mediated knockdown of RNASEH2A/ RNASEH2B expression led to reduced cell survival and increase in apoptotic cell population. Importantly, knockdown of RNASEH2A or RNASEH2B, led to cell cycle arrest at S phase and increased number of 53BP1 foci due to abrogation of NHEJ. Interestingly, RNASEH2A or RNASEH2B depleted cells showed significantly retarded DSB repair kinetics compared to scrambled shRNA control, when exposed to ionizing radiation suggesting that NHEJ is abrogated due to loss of RNASEH2 activity in T-ALL cells. Thus, we uncover the importance of RNase H2 function in leukemic cells and suggest that it can be targeted for cancer therapy.
    Keywords:  DSB repair; Double-strand break; End joining; Error-prone polymerase; Genomic instability; NHEJ; Pol X family; Pol mu
    DOI:  https://doi.org/10.1016/j.jmb.2022.167617
  10. Nature. 2022 May 04.
    A rethink about enzymes that drive DNA replication.
    Masato T Kanemaki.
      
    Keywords:  Cancer; Cell biology
    DOI:  https://doi.org/10.1038/d41586-022-01128-w
  11. Sci Adv. 2022 May 06. 8(18): eabm6246
    H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication.
    Albert Serra-Cardona, Shoufu Duan, Chuanhe Yu, Zhiguo Zhang.
      During DNA replication, parental H3-H4 marked by H3K4me3 are transferred almost equally onto leading and lagging strands of DNA replication forks. Mutations in replicative helicase subunit, Mcm2 (Mcm2-3A), and leading strand DNA polymerase subunit, Dpb3 (dpb3∆), result in asymmetric distributions of H3K4me3 at replicating DNA strands immediately following DNA replication. Here, we show that mcm2-3A and dpb3∆ mutant cells markedly reduce the asymmetric distribution of H3K4me3 during cell cycle progression before mitosis. Furthermore, the restoration of a more symmetric distribution of H3K4me3 at replicating DNA strands in these mutant cells is driven by methylating nucleosomes without H3K4me3 by the H3K4 methyltransferase complex, COMPASS. Last, both gene transcription machinery and the binding of parental H3K4me3 by Spp1 subunit of the COMPASS complex help recruit the enzyme to chromatin for the restoration of the H3K4me3-marked state following DNA replication, shedding light on inheritance of this mark following DNA replication.
    DOI:  https://doi.org/10.1126/sciadv.abm6246
  12. J Am Chem Soc. 2022 May 02.
    Structural Dynamics of a Common Mutagenic Oxidative DNA Lesion in Duplex DNA and during DNA Replication.
    Benjamin J Ryan, Haozhe Yang, Jan Henric T Bacurio, Mallory R Smith, Ashis K Basu, Marc M Greenberg, Bret D Freudenthal.
      N6-(2-Deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy•dG) is a prevalent form of genomic DNA damage. Fapy•dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo). Fapy•dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy•dG is facile epimerization, but prior works with Fapy•dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy•dG in duplex DNA and as the template base for DNA polymerase β (Pol β). Fapy•dG adopts the β-anomer when base paired with cytosine but exists as a mixture of α- and β-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy•dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy•dG mutagenicity in wild type and Pol β knockdown HEK 293T cells indicates that Pol β contributes to G → T transversions but also suppresses G → A transitions. Complementary kinetic studies have determined that Fapy•dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy•dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the β-anomer is ∼90 times faster than the α-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy•dG and potentially the repair of this structurally unusual lesion.
    DOI:  https://doi.org/10.1021/jacs.2c00193
  13. Nat Commun. 2022 May 02. 13(1): 2374
    Mre11-Rad50 oligomerization promotes DNA double-strand break repair.
    Vera M Kissling, Giordano Reginato, Eliana Bianco, Kristina Kasaciunaite, Janny Tilma, Gea Cereghetti, Natalie Schindler, Sung Sik Lee, Raphaël Guérois, Brian Luke, Ralf Seidel, Petr Cejka, Matthias Peter.
      The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.
    DOI:  https://doi.org/10.1038/s41467-022-29841-0
  14. Nature. 2022 May 04.
    Molecular basis for the initiation of DNA primer synthesis.
    Arthur W H Li, Katerina Zabrady, Lewis J Bainbridge, Matej Zabrady, Sehr Naseem-Khan, Madison B Berger, Peter Kolesar, G Andrés Cisneros, Aidan J Doherty.
      During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
    DOI:  https://doi.org/10.1038/s41586-022-04695-0
  15. Cancer Cell Int. 2022 May 02. 22(1): 176
    DNA repair and damage pathways in mesothelioma development and therapy.
    Faezeh Malakoti, Niloufar Targhazeh, Erfan Abadifard, Reza Zarezadeh, Sahar Samemaleki, Zatollah Asemi, Simin Younesi, Reza Mohammadnejad, Seyed Hadi Hossini, Ansar Karimian, Forough Alemi, Bahman Yousefi.
      Malignant mesothelioma (MMe) is an aggressive neoplasm that occurs through the transformation of mesothelial cells. Asbestos exposure is the main risk factor for MMe carcinogenesis. Other important etiologies for MMe development include DNA damage, over-activation of survival signaling pathways, and failure of DNA damage response (DDR). In this review article, first, we will describe the most important signaling pathways that contribute to MMe development and their interaction with DDR. Then, the contribution of DDR failure in MMe progression will be discussed. Finally, we will review the latest MMe therapeutic strategies that target the DDR pathway.
    Keywords:  BAP1; BRCA1 associated protein 1; DNA damage repair; Malignant mesothelioma; Malignant peritoneal mesothelioma; Signaling pathways
    DOI:  https://doi.org/10.1186/s12935-022-02597-9
  16. PLoS One. 2022 ;17(5): e0267615
    The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII.
    Barbara N Borsos, Vasiliki Pantazi, Zoltán G Páhi, Hajnalka Majoros, Zsuzsanna Ujfaludi, Ivett Berzsenyi, Tibor Pankotai.
      DNA double-strand breaks are one of the most deleterious lesions for the cells, therefore understanding the macromolecular interactions of the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. We previously demonstrated that following transcription block, p53 plays a pivotal role in transcription elongation by interacting with S2P RNAPII. In the current study, we reveal that p53 is involved in the fine-tune regulation of S2P RNAPII ubiquitylation. Furthermore, we emphasize the potential role of p53 in delaying the premature ubiquitylation and the subsequent chromatin removal of S2P RNAPII as a response to transcription block.
    DOI:  https://doi.org/10.1371/journal.pone.0267615
  17. J Biol Chem. 2022 Apr 28. pii: S0021-9258(22)00432-X. [Epub ahead of print] 101992
    Delineation of a minimal topoisomerase II binding protein 1 (TOPBP1) for regulated activation of ATR at DNA double-strand breaks.
    Kenna Ruis, Oanh Huynh, Katrina Montales, Nina A Barr, W Matthew Michael.
      Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangeictasia and Rad3-Related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal Repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-Interacting Protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks (DSBs) in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DSBs and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1, and provides insight into the mechanism of ATR activation.
    Keywords:  ATR; BRCT; CHK1; DNA damage; DNA double-strand break; DNA repair; TOPBP1; cell biology; checkpoint control; protein kinase
    DOI:  https://doi.org/10.1016/j.jbc.2022.101992
  18. Elife. 2022 May 03. pii: e75231. [Epub ahead of print]11
    METTL3 promotes homologous recombination repair and modulates chemotherapeutic response in breast cancer by regulating the EGF/Rad51 axis.
    Enjie Li, Mingyue Xia, Yu Du, Kaili Long, Feng Ji, Feiyan Pan, Lingfeng He, Zhigang Hu, Zhigang Guo.
      METTL3 and N6-methyladenosine (m6A) are involved in many types of biological and pathological processes, including DNA repair. However, the function and mechanism of METTL3 in DNA repair and chemotherapeutic response remain largely unknown. In present study, we identified that METTL3 participates in the regulation of homologous recombination repair (HR), which further influences chemotherapeutic response in both MCF-7 and MDA-MB-231 breast cancer (BC) cells. Knockdown of METTL3 sensitized these BC cells to Adriamycin (ADR; also named as doxorubicin) treatment and increased accumulation of DNA damage. Mechanically, we demonstrated that inhibition of METTL3 impaired HR efficiency and increased ADR-induced DNA damage by regulating m6A modification of EGF/RAD51 axis. METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. We further demonstrated that the m6A 'reader', YTHDC1, bound to the m6A modified EGF transcript and promoted EGF synthesis, which enhanced HR and cell survival during ADR treatment in breast cancer cells. Our findings reveal a pivotal mechanism of METTL3-mediated HR and chemotherapeutic drug response, which may contribute to cancer therapy.
    Keywords:  biochemistry; cell biology; chemical biology; mouse
    DOI:  https://doi.org/10.7554/eLife.75231
  19. Trends Genet. 2022 Apr 27. pii: S0168-9525(22)00085-3. [Epub ahead of print]
    Catastrophic DNA replication in unscheduled tetraploid cells.
    Gwenola Manic, Lorenzo Galluzzi, Ilio Vitale.
      Unscheduled tetraploidy is a metastable state that rapidly evolves into aneuploidy. Recent findings reported by Gemble et al. demonstrate that freshly formed tetraploid cells fail to accumulate the required amounts of DNA replication factors during the first G1 phase after whole-genome duplication (WGD), culminating in genetic instability in the subsequent S phase and extensive karyotypic alterations.
    Keywords:  cell cycle checkpoint; chromosomal instability; mitotic spindle; regulated cell death; replication stress
    DOI:  https://doi.org/10.1016/j.tig.2022.04.005
  20. Nat Commun. 2022 May 03. 13(1): 2402
    High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control.
    Dashiell J Massey, Amnon Koren.
      DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.
    DOI:  https://doi.org/10.1038/s41467-022-30212-y
  21. Biol Cell. 2022 May 07.
    Coordination between phospholipid pools and DNA damage sensing.
    Sara Ovejero, Caroline Soulet, Sylvain Kumanski, María Moriel-Carretero.
      Both phospholipid synthesis and the detection of DNA damage are coupled to cell cycle progression, yet whether these two aspects crosstalk to each other remains unassessed. We postulate here that shortage of phospholipids, which negatively affects proliferation, may reduce the need for checkpoint activation in response to DNA damage. By exploring the DDR activation in response to seven different genotoxins, in three distinct cell types, and manipulating phospholipid synthesis both pharmacologically and genetically, we point at the DNA damage response kinase ATR as responsible for the coordination between phospholipid levels and DNA damage sensing. Further, our analysis reveals the functional significance of this crosstalk to keep genome homeostasis. This article is protected by copyright. All rights reserved.
    Keywords:  ATR; Chk1; DNA Damage Response; membranes; phospholipids
    DOI:  https://doi.org/10.1111/boc.202200007
  22. Methods Mol Biol. 2022 ;2477 107-128
    FORK-seq: Single-Molecule Profiling of DNA Replication.
    Magali Hennion, Bertrand Theulot, Jean-Michel Arbona, Benjamin Audit, Olivier Hyrien.
      Most genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution using a nanopore current interpretation tool allowing the quantification of BrdU incorporation. Along pulse-chased replication intermediates from Saccharomyces cerevisiae, we can orient replication tracks and reproduce population-based replication directionality profiles. Additionally, we can map individual initiation and termination events. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.
    Keywords:  Convolutional neural network; DNA replication; Nanopore sequencing; Replication fork direction; Replication origins; Single-molecule analysis; Termination sites; Whole-genome
    DOI:  https://doi.org/10.1007/978-1-0716-2257-5_8
  23. Nucleic Acids Res. 2022 Apr 30. pii: gkac304. [Epub ahead of print]
    SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.
    Yongjuan Guan, Huijuan Lin, N Adrian Leu, Gordon Ruthel, Serge Y Fuchs, Luca Busino, Mengcheng Luo, P Jeremy Wang.
      Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.
    DOI:  https://doi.org/10.1093/nar/gkac304
  24. Cancer Lett. 2022 Apr 28. pii: S0304-3835(22)00160-4. [Epub ahead of print] 215677
    Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells.
    Jun Wang, Aftab Amin, Man Hei Cheung, Linjing Shi, Chun Liang.
      MicroRNAs are noncoding RNAs with a typical length of 22 nucleotides that post-transcriptionally suppress gene expression by inducing target mRNA degradation and/or impairing translation in eukaryotes. Thousands of miRNA genes in the human genome are involved in various physiological and pathological processes. Each miRNA targets many different mRNAs, while each mRNA may be targeted by various miRNAs. Mini-chromosome maintenance (MCM2-7) protein complex functions as essential components of the pre-replicative complex (pre-RC) and forms a helicase together with other proteins to unwind the DNA duplex in S phase. MCM proteins are overexpressed in all cancer cells, while they are strictly regulated in normal cells, with no expression in non-proliferating normal cells. Here we report that miRNA-214-3p (miR-214) targets both MCM5 and MCM7. The level of miR-214 is lower in HepG2 and Hep3B hepatocellular carcinoma cells than the L-02 normal liver cells. Introduction of miRNA-214 mimic into HepG2 and Hep3B cells reduced the mRNA and protein levels of MCM5/7 and inhibited DNA replication, cell cycle progression, cell proliferation and colony formation. Comparatively, miRNA-214 mimic had little effect in L-02 cells. Importantly, miR-214 mimic can also inhibit the growth of HepG2 xenografts in nude mice. Our data suggest that miRNA-214 regulates DNA replication by targeting MCM5/7 and has the potential to be developed into a liver cancer drug. IMPLICATIONS: This study supports the notion that DNA replication-initiation proteins (DRIPs), including MCM2-7 proteins, are attractive anticancer targets. Furthermore, the potential of miR-214 as an anticancer agent, with activity against liver cancer cells but not normal livre cells, may be of high significance.
    DOI:  https://doi.org/10.1016/j.canlet.2022.215677
  25. Nucleic Acids Res. 2022 May 07. pii: gkac296. [Epub ahead of print]
    Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops.
    Ramin Sakhtemani, Madusha L W Perera, Daniel Hübschmann, Reiner Siebert, Michael S Lawrence, Ashok S Bhagwat.
      Activation-induced deaminase (AID) is a DNA-cytosine deaminase that mediates maturation of antibodies through somatic hypermutation and class-switch recombination. While it causes mutations in immunoglobulin heavy and light chain genes and strand breaks in the switch regions of the immunoglobulin heavy chain gene, it largely avoids causing such damage in the rest of the genome. To help understand targeting by human AID, we expressed it in repair-deficient Escherichia coli and mapped the created uracils in the genomic DNA using uracil pull-down and sequencing, UPD-seq. We found that both AID and the human APOBEC3A preferentially target tRNA genes and transcription start sites, but do not show preference for highly transcribed genes. Unlike A3A, AID did not show a strong replicative strand bias or a preference for hairpin loops. Overlapping uracilation peaks between these enzymes contained binding sites for a protein, FIS, that helps create topological domains in the E. coli genome. To confirm whether these findings were relevant to B cells, we examined mutations from lymphoma and leukemia genomes within AID-preferred sequences. These mutations also lacked replicative strand bias or a hairpin loop preference. We propose here a model for how AID avoids causing mutations in the single-stranded DNA found within replication forks.
    DOI:  https://doi.org/10.1093/nar/gkac296
  26. Nat Commun. 2022 May 03. 13(1): 2400
    Alterations in homologous recombination repair genes in prostate cancer brain metastases.
    Antonio Rodriguez-Calero, John Gallon, Dilara Akhoundova, Sina Maletti, Alison Ferguson, Joanna Cyrta, Ursula Amstutz, Andrea Garofoli, Viola Paradiso, Scott A Tomlins, Ekkehard Hewer, Vera Genitsch, Achim Fleischmann, Erik Vassella, Elisabeth J Rushing, Rainer Grobholz, Ingeborg Fischer, Wolfram Jochum, Gieri Cathomas, Adeboye O Osunkoya, Lukas Bubendorf, Holger Moch, George Thalmann, Charlotte K Y Ng, Silke Gillessen, Salvatore Piscuoglio, Mark A Rubin.
      Improved survival rates for prostate cancer through more effective therapies have also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. Here we investigate the repertoire of somatic genetic alterations present in brain metastases from 51 patients with prostate cancer brain metastases (PCBM). We highlight the clonal evolution occurring in PCBM and demonstrate an increased mutational burden, concomitant with an enrichment of the homologous recombination deficiency mutational signature in PCBM compared to non-brain metastases. Focusing on known pathogenic alterations within homologous recombination repair genes, we find 10 patients (19.6%) fulfilling the inclusion criteria used in the PROfound clinical trial, which assessed the efficacy of PARP inhibitors (PARPi) in homologous recombination deficient prostate cancer. Eight (15.7%) patients show biallelic loss of one of the 15 genes included in the trial, while 5 patients (9.8%) harbor pathogenic alterations in BRCA1/2 specifically. Uncovering these molecular features of PCBM may have therapeutic implications, suggesting the need of clinical trial enrollment of PCBM patients when evaluating potential benefit from PARPi.
    DOI:  https://doi.org/10.1038/s41467-022-30003-5
  27. Cell Discov. 2022 Apr 30. 8(1): 39
    Rare and misincorporated DNA N6-methyladenine is a hallmark of cytotoxic stresses for selectively stimulating the stemness and proliferation of glioblastoma cells.
    Cong Lyu, Yamei Niu, Weiyi Lai, Yu Wang, Yaning Wang, Peibin Dai, Chunhui Ma, Shaokun Chen, Yao Li, Guibin Jiang, Zhiyong Liang, Wenbin Ma, Zhengliang Gao, Wei-Min Tong, Hailin Wang.
      The entity of DNA N6-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N6-methyladenine (m6A) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of m6A nucleoside. The released m6A nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.
    DOI:  https://doi.org/10.1038/s41421-022-00399-x
  28. Cancer Discov. 2022 May 06. OF1
    CCNE1 Amplification and PKMYT1 Inhibition Are Synthetic Lethal.
      Using a genome-wide screen, PKMYT1 inhibition was found to be synthetic lethal with CCNE1 amplification.
    DOI:  https://doi.org/10.1158/2159-8290.CD-RW2022-080
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