bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–05–15
48 papers selected by
Sean Rudd, Karolinska Institutet
  1. Resistance to DNA repair inhibitors in cancer.
  2. Translesion DNA synthesis mediates acquired resistance to olaparib plus temozolomide in small cell lung cancer.
  3. Unique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions.
  4. Study of Interaction of the PARP Family DNA-Dependent Proteins with Nucleosomes Containing DNA Intermediates of the Initial Stages of BER Process.
  5. Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair.
  6. DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders.
  7. DNA Replication Checkpoint Activates Respiration in Budding Yeast.
  8. Novel Therapeutic Approaches with DNA Damage Response Inhibitors for Melanoma Treatment.
  9. PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.
  10. MicroRNA-145 Impairs Classical Non-Homologous End-Joining in Response to Ionizing Radiation-Induced DNA Double-Strand Breaks via Targeting DNA-PKcs.
  11. Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice.
  12. A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single-molecule experiments.
  13. Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair.
  14. Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.
  15. The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.
  16. Kub5-Hera Deficiency Promotes R-Loop-Induced Genomic Instability and Carcinogenesis Following Whole-Body Exposure to Ionizing Radiation.
  17. Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma.
  18. Joining the PARty: PARP Regulation of KDM5A during DNA Repair (and Transcription?).
  19. G9a Responds to KRAS-Mediated Replication Stress by Increased Origin Licensing.
  20. Transcriptional regulation via strand displacement DNA repair in G-quadruplexes.
  21. H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication.
  22. Targeting Keratin 17 in Pancreatic Cancer: A Novel Rewired Pathway of Nucleotide Metabolism that Drives Chemoresistance.
  23. Development of New Assays for the Simultaneous Measurement of DNA Double-Strand Break Repair by Multiple Pathways.
  24. Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.
  25. Determinants of RPA megafoci localization to the nuclear periphery in response to replication stress.
  26. Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks.
  27. cMET inhibition potentiates the tumor-selective damaging effects of NQO1-bioactivatable agents by compromising DNA repair.
  28. Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome.
  29. Too much and never enough: Synthetic excess and metabolic inefficiency of aneuploidy in tumorigenesis.
  30. MRNIP condensates promote DNA double-strand break sensing and end resection.
  31. ARL2 is required for homologous recombination repair and colon cancer stem cell survival.
  32. Differential Expression of Nuclear Body Component, Zc3h8, Disrupts DNA Double Strand Break Repair.
  33. Detection of Multi-Protein Complexes Containing PCNA Using Fluorescence Anisotropy and Hormetic Modeling.
  34. Single-Molecule Analysis of in vivo DNA Replication Origin Licensing Stoichiometry.
  35. Nucleic Acid Sensing Pathways in DNA Repair Targeted Cancer Therapy.
  36. Purine biosynthesis and related enzymes play the oncogenic function in FGFR3 mutant urothelial carcinoma.
  37. Combined Inhibition of the Ataxia-telangiectasia mutated and Rad3-related Pathway and the G9a Methyltransferase Synergize to Reduce Pancreatic Cancer Cell Growth.
  38. Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR.
  39. Does Intracellular Metabolism Render Gemcitabine Uptake Undetectable in Mass Spectrometry?
  40. Alternative Lengthening of Telomeres and Mediated Telomere Synthesis.
  41. DNA Damage Response Inhibitors in Cholangiocarcinoma: Current Progress and Perspectives.
  42. Effect of Uracil DNA Glycosylase Activity on the Efficacy of Thymidylate Synthase Inhibitor/HDAC Inhibitor Combination Therapies in Colon Cancer.
  43. Genome maintenance in retinoblastoma: Implications for therapeutic vulnerabilities.
  44. Human TREX1 Repairs 3'-End DNA Lesions in Vitro.
  45. A Collapsed Fingers Subdomain is the Basis for DNA Polymerase β I260M Mutator Activity.
  46. LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.
  47. Inhibition of SUMO-mediated Protein-Protein Interactions in DNA Damage Repair.
  48. Biochemical Characterization of a Human Ribonucleotide Reductase (HsRNR) Variant Lacking Activity Regulation.
  1. Mol Oncol. 2022 May 14.
    Resistance to DNA repair inhibitors in cancer.
    Joseph S Baxter, Diana Zatreanu, Stephen J Pettitt, Christopher J Lord.
      The DNA damage response (DDR) represents a complex network of proteins which detect and repair DNA damage, thereby maintaining the integrity of the genome and preventing the transmission of mutations and rearranged chromosomes to daughter cells. Faults in the DDR are a known driver and hallmark of cancer. Furthermore, inhibition of DDR enzymes can be used to treat the disease. This is exemplified by PARP inhibitors (PARPi) used to treat cancers with defects in the homologous recombination DDR pathway. A series of novel DDR targets are now also under pre-clinical or clinical investigation, including inhibitors of ATR kinase, WRN helicase or the DNA polymerase/helicase Polθ (Pol-Theta). Drug resistance is a common phenomenon that impairs the overall effectiveness of cancer treatments and there is already some understanding of how resistance to PARPi occurs. Here, we discuss how an understanding of PARPi resistance could inform how resistance to new drugs targeting the DDR emerges. We also discuss potential strategies that could limit the impact of these therapy resistance mechanisms in cancer.
    Keywords:  ATR; Cancer; DDR; PARP; PolQ; WRN
    DOI:  https://doi.org/10.1002/1878-0261.13224
  2. Sci Adv. 2022 May 13. 8(19): eabn1229
    Translesion DNA synthesis mediates acquired resistance to olaparib plus temozolomide in small cell lung cancer.
    Marcello Stanzione, Jun Zhong, Edmond Wong, Thomas J LaSalle, Jillian F Wise, Antoine Simoneau, David T Myers, Sarah Phat, Moshe Sade-Feldman, Michael S Lawrence, M Kyle Hadden, Lee Zou, Anna F Farago, Nicholas J Dyson, Benjamin J Drapkin.
      In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.
    DOI:  https://doi.org/10.1126/sciadv.abn1229
  3. FASEB J. 2022 May;36 Suppl 1
    Unique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions.
    Shilpi Paul, Paolo Cifani, Darryl Pappin, Thomas Spratt.
      Translesion DNA synthesis (TLS) polymerases evolved to tolerate DNA damage that bypasses DNA lesions, thus preventing genomic instability. Multiple TLS polymerases exist with different damage tolerance capabilities, since they have low fidelity their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein-protein interactions are used to regulate TLS polymerase activity. A major knowledge-gap in elucidating the roles of these polymerases is that it is difficult to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2 -benzyl- 2'-deoxyguanosine (EBndG) that is highly selective toward DNA polymerase kappa (Pol κ), a Y family TLS polymerase. Pol k can bypass bulky lesions in the minor groove generated by a metabolite benzo[a]pyrene diolepoxide (BPDE), an environmental carcinogen. Although Pol κ has been identified to have multiple cellular roles, the mechanisms regulating its different cellular activities are unknown. To interrogate the identity of proteins surrounding the Pol κ active sites, we performed an extensive study using modified iPOND (isolation of proteins on nascent DNA), called iPoKD (isolation of proteins on Pol kappa synthesized DNA). Human cell lines were treated with BPDE, subsequently 5-ethynyl-2'-deoxyuridine (EdU) or EBndG was added and proteins bound to the DNA containing EdU and EBndG were analyzed by mass spectrometry. In addition, we performed quantitative analysis of the Pol κ active sites interactome using iPoKD followed by iTRAQ (isobaric tags for relative and absolute quantitation). Our data identified DNA replication and repair proteins previously identified with EdU pull-downs; and interestingly enrichment of RNA binding, ribosome biogenesis, nucleolar proteins and transcriptional repressive complex(es) associated with EBndG pull-downs. Chromatin modifiers, histone chaperones and histone variants are identified suggesting changes in chromatin structure that facilitates Pol κ-mediated DNA lesion bypass, repair and restorative process. identification of unique proteins associated with EBndG-containing DNA, suggests novel roles for Pol κ's activity in the cell. Using super-resolution confocal microscopy, Pol κ activity is identified in the nucleolus after BPDE damage. EBndG is observed in the nucleolar DNA and Pol κ 's activity regulated by the canonical polycomb-complex recruited by the PARylation of PARP1. BPDE lead to transcriptional stress and repression that is gradually recovered. We are investigating whether Pol κ maintains ribosomal DNA integrity after BPDE damage and is required for TLS DNA synthesis or repair. Pol κ active site associated candidate proteins are being validated using CRISPR-Cas9 knockout or siRNA knockdown strategies. These data will provide first insight into Pol κ's core interactome, it's regulation, chromatin surrounding Pol κ active sites and its novel cellular roles.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R478
  4. Biochemistry (Mosc). 2022 Apr;87(4): 331-345
    Study of Interaction of the PARP Family DNA-Dependent Proteins with Nucleosomes Containing DNA Intermediates of the Initial Stages of BER Process.
    Alexander A Ukraintsev, Ekaterina A Belousova, Mikhail M Kutuzov, Olga I Lavrik.
      Reaction of (ADP-ribosyl)ation catalyzed by DNA-dependent proteins of the poly(ADP-ribose)polymerase (PARP) family, PARP1, PARP2, and PARP3, comprises the cellular response to DNA damage. These proteins are involved in the base excision repair (BER) process. Despite the extensive research, it remains unknown how PARPs are involved in the regulation of the BER process and how the roles are distributed between the DNA-dependent members of the PARP family. Here, we investigated the interaction of the PARP's family DNA-dependent proteins with nucleosome core particles containing DNA intermediates of the initial stages of BER. To do that, the nucleosomes containing damage in the vicinity of one of the DNA duplex blunt ends were reconstituted based on the Widom's Clone 603 DNA sequence. Dissociation constants of the PARP complexes with nucleosomes bearing DNA contained uracil (Native), apurine/apyrimidine site (AP site), or a single-nucleotide gap with 5'-dRp fragment (Gap) were determined. It was shown that the affinity of the proteins for the nucleosomes increased in the row: PARP3<<PARP2<PARP1; whereas the affinity of each protein for the certain damage type increased in the row: Native = AP site < Gap for PARP1 and PARP2, Gap<<<Native = AP site for PARP3. The interaction regions of each PARP protein with nucleosome were also determined by sodium borohydride cross-linking and footprinting assay. Based on the obtained and published data, the involvement pattern of the PARP1, PARP2, and PARP3 into the interaction with nucleosome particles containing DNA intermediates of the BER process was discussed.
    Keywords:  BER; PARP1; PARP2; PARP3; nucleosome
    DOI:  https://doi.org/10.1134/S0006297922040034
  5. FASEB J. 2022 May;36 Suppl 1
    Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair.
    Melike Caglayan.
      DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the covalent LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R459
  6. Int J Mol Sci. 2022 Apr 22. pii: 4653. [Epub ahead of print]23(9):
    DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders.
    Vincent E Provasek, Joy Mitra, Vikas H Malojirao, Muralidhar L Hegde.
      The damage and repair of DNA is a continuous process required to maintain genomic integrity. DNA double-strand breaks (DSBs) are the most lethal type of DNA damage and require timely repair by dedicated machinery. DSB repair is uniquely important to nondividing, post-mitotic cells of the central nervous system (CNS). These long-lived cells must rely on the intact genome for a lifetime while maintaining high metabolic activity. When these mechanisms fail, the loss of certain neuronal populations upset delicate neural networks required for higher cognition and disrupt vital motor functions. Mammalian cells engage with several different strategies to recognize and repair chromosomal DSBs based on the cellular context and cell cycle phase, including homologous recombination (HR)/homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the classic non-homologous end-joining (NHEJ). In addition to these repair pathways, a growing body of evidence has emphasized the importance of DNA damage response (DDR) signaling, and the involvement of heterogeneous nuclear ribonucleoprotein (hnRNP) family proteins in the repair of neuronal DSBs, many of which are linked to age-associated neurological disorders. In this review, we describe contemporary research characterizing the mechanistic roles of these non-canonical proteins in neuronal DSB repair, as well as their contributions to the etiopathogenesis of selected common neurological diseases.
    Keywords:  DNA damage response; DNA double-strand break repair; TDP-43; dementia; hnRNPs; neurodegeneration
    DOI:  https://doi.org/10.3390/ijms23094653
  7. FASEB J. 2022 May;36 Suppl 1
    DNA Replication Checkpoint Activates Respiration in Budding Yeast.
    Shreya Nagar, Riddhi Mehta, Pritpal Kaur, Ales Vancura.
      In addition to DNA damage checkpoint (DDC), eukaryotic cells have also DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. We have showed previously that DDC activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage-induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain (ETC), and oxidative phosphorylation. Here we show that similarly to DDC, activation of DRC also induces respiration. However, unlike DDC, DRC does not affect histone gene expression and chromatin structure. DRC induces respiration by inducing transcription of RNR1-4, upregulating synthesis of dNTPs, and elevating mtDNA copy number. Fitness of rrm3∆ and sgs1∆, mutants with defects in DNA replication, requires checkpoint kinase Dun1, but does not require ETC and oxidative metabolism. However, in the absence of Dun1, ETC is required for viability of rrm3Δ and sgs1Δ cells. In addition, inactivation of ETC in dun1Δ cells results in a synthetic growth defect. Together, our data show that when Dun1p function is compromised, rrm3Δand sgs1Δ cells depend on oxidative metabolism.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3529
  8. Cells. 2022 Apr 26. pii: 1466. [Epub ahead of print]11(9):
    Novel Therapeutic Approaches with DNA Damage Response Inhibitors for Melanoma Treatment.
    Luisa Maresca, Barbara Stecca, Laura Carrassa.
      Targeted therapies against components of the mitogen-activated protein kinase (MAPK) pathway and immunotherapies, which block immune checkpoints, have shown important clinical benefits in melanoma patients. However, most patients develop resistance, with consequent disease relapse. Therefore, there is a need to identify novel therapeutic approaches for patients who are resistant or do not respond to the current targeted and immune therapies. Melanoma is characterized by homologous recombination (HR) and DNA damage response (DDR) gene mutations and by high replicative stress, which increase the endogenous DNA damage, leading to the activation of DDR. In this review, we will discuss the current experimental evidence on how DDR can be exploited therapeutically in melanoma. Specifically, we will focus on PARP, ATM, CHK1, WEE1 and ATR inhibitors, for which preclinical data as single agents, taking advantage of synthetic lethal interactions, and in combination with chemo-targeted-immunotherapy, have been growing in melanoma, encouraging the ongoing clinical trials. The overviewed data are suggestive of considering DDR inhibitors as a valid therapeutic approach, which may positively impact the future of melanoma treatment.
    Keywords:  ATM; ATR; CHK1; DNA damage response; PARP; WEE1; combined therapy; inhibitors; melanoma
    DOI:  https://doi.org/10.3390/cells11091466
  9. Development. 2022 May 12. pii: dev.200351. [Epub ahead of print]
    PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.
    Ming-Zhe Dong, Ying-Chun Ouyang, Shi-Cai Gao, Xue-Shan Ma, Yi Hou, Heide Schatten, Zhen-Bo Wang, Qing-Yuan Sun.
      Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here we showed that protein phosphatase 4 catalytic subunit (PPP4C) deletion caused severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibit DNA lesions which inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of essential HR factor RAD51. On the other hand, if inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods coupling the checkpoint to HR.
    Keywords:  CDK1; DNA damage; Embryo; Homologous recombination; PLK1; PPP4C
    DOI:  https://doi.org/10.1242/dev.200351
  10. Cells. 2022 Apr 30. pii: 1509. [Epub ahead of print]11(9):
    MicroRNA-145 Impairs Classical Non-Homologous End-Joining in Response to Ionizing Radiation-Induced DNA Double-Strand Breaks via Targeting DNA-PKcs.
    Muddenahalli Srinivasa Sudhanva, Gurusamy Hariharasudhan, Semo Jun, Gwanwoo Seo, Radhakrishnan Kamalakannan, Hyun Hee Kim, Jung-Hee Lee.
      DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3'-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.
    Keywords:  DNA-PKcs; DSBs; classical non-homologous end-joining pathway (c-NHEJ); microRNA
    DOI:  https://doi.org/10.3390/cells11091509
  11. DNA Repair (Amst). 2022 Apr 28. pii: S1568-7864(22)00065-9. [Epub ahead of print]115 103332
    Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice.
    Aditya Mojumdar, Nancy Adam, Jennifer A Cobb.
      A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5' end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5' resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330-338) is important for two events that drive NHEJ as it stimulates ligation and inhibits resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly by inhibiting Dna2-mediated resection.
    Keywords:  5' resection; DSB repair; Dna2; Nej1; Non-homologous end-joining (NHEJ); Repair pathway choice
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103332
  12. FASEB J. 2022 May;36 Suppl 1
    A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single-molecule experiments.
    Jason Lin, Marco Simonetta, Evan Gates, Shiba Dandpat, Mattijs de Groot, Bas Groen, Trey Simpson.
      Fundamental biological processes such as DNA replication, DNA repair, and chromatin organization are precisely regulated in space and time by sophisticated multiprotein molecular machinery. The high spatiotemporal resolution of optical tweezers combined with single molecule fluorescence microscopy enables real-time visualization of these processes while simultaneously measuring their enzymatic activity via detection of mechanical manipulation of the DNA substrates. Our biochemistry platform provides a set of tools to incorporate replication forks, DNA lesions, and specific sequences (e.g. nucleosome arrays) in DNA molecules tethered between two beads of the optical tweezers. Importantly, our tools also allow labeling of exact positions within these DNA substrates with fluorophores. This enables precise localization of the imaged proteins in the DNA sequence during processes including DNA scanning, as well as recognition and enzymatic manipulation of specific DNA structures or lesions. Our next developments aim to combine multiple DNA features in a single DNA molecule, such as a replication fork in the presence of DNA lesions or in the context of a nucleosome array. These advances will allow more complex experimental set ups and increase the capability to mimic cellular biological processes at a single molecule level.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6134
  13. FASEB J. 2022 May;36 Suppl 1
    Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair.
    Armand M Berry.
      DNA is under constant attack by exogenous and endogenous factors. Endogenous sources of DNA lesions include free radicals such as reactive oxygen and nitrogen species (RONS), as well as many naturally occurring enzymatic and chemical processes. To prevent mutations and possibly cell death these lesions must be repaired prior to DNA replication. There are five major pathways that are utilized for repair depending on the type of DNA damage. Eukaryotes utilize two pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ), for repair of DNA double-strand breaks (DSBs). In addition, the cells utilize base excision repair (BER) for repair of damaged or missing bases. Previous work in the lab has shown that HR-deficient yeast mutant cell cultures exhibit high levels of G2 phase cells during normal growth, without exposure to exogenous DNA damaging agents. Inactivation of DNA damage checkpoint genes, but not spindle checkpoint genes, abolished the high G2 cell phenotype, indicating that the damage checkpoint is persistently activated in the cells. In a screen of yeast mutant library strains deficient in each of the major repair pathways, the high G2 phenotype was observed in HR- and BER-deficient cells, but not in mutants specifically defective in nucleotide excision repair (NER), mismatch repair (MMR) or NHEJ. Experiments performed for the current project have assessed the consequences of inactivation of the HR and BER pathways on yeast cells in more detail. The high G2 cell phenotype was heightened in both diploid HR (rad52) and BER (apn1, ogg1) mutants relative to their haploid counterparts. Expression of DIN1 (RNR3), a DNA damage-inducible gene, was found to be constitutively induced in the HR mutant cells. Preliminary tests of the role of free radicals in generation of endogenous signaling were performed by overexpression of Yap1p, a transcriptional activator of antioxidant enzyme genes. The high G2 cell phenotype was abolished in ogg1 mutants and strongly reduced in rad52 cells when Yap1p was overexpressed. Current work is focused on (a) analysis of DIN1 promoter activation in BER mutants, (b) assessment of the impact of neutralization of free radicals using chemical antioxidants, and (c) measurement of phosphorylated Rad53 checkpoint protein levels using Western blots to characterize the DNA damage checkpoint signaling response.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3195
  14. Cancer Sci. 2022 May 09.
    Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.
    Caroline Moyret-Lalle, Mélanie K Prodhomme, Delphine Burlet, Ayaka Kashiwagi, Virginie Petrilli, Alain Puisieux, Hiroyuki Seimiya, Agnès Tissier.
      Numerous epithelial-mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties and therapy-associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that the EMT-associated transcription factors (EMT-TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT-TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor-specific molecular vulnerabilities, which results in selective destruction of cancer cells. Here we review recent advances providing novel insights into the role of EMT in the DDR and repair pathways with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.
    Keywords:  DNA damage response; DNA repair; ZEB1; epithelial-mesenchymal transition; synthetic lethality
    DOI:  https://doi.org/10.1111/cas.15389
  15. Nat Struct Mol Biol. 2022 May 09.
    The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.
    Daniel R Semlow, Victoria A MacKrell, Johannes C Walter.
      The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated during replication-coupled DNA interstrand cross-link repair. We show that HMCES cross-links form on DNA after the replicative CDC45-MCM2-7-GINS (CMG) helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses double-strand break formation, slows translesion synthesis past the AP site and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data demonstrate that HMCES-DPCs form as intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.
    DOI:  https://doi.org/10.1038/s41594-022-00764-0
  16. FASEB J. 2022 May;36 Suppl 1
    Kub5-Hera Deficiency Promotes R-Loop-Induced Genomic Instability and Carcinogenesis Following Whole-Body Exposure to Ionizing Radiation.
    Edward Motea, Julio Morales, S L Pay, Genevieve Crane.
      Formation of persistent R-loops (a structure containing an RNA:DNA hybrid and a displaced single-stranded DNA) during transcription are now emerging as a direct threat to genomic stability with links to cancer and various neurodegenerative, autoimmune, and developmental disorders. However, the molecular mechanisms by which R-loops are resolved or aberrantly formed to induce DNA damage and genomic instability remains unclear. Aberrant accumulation of these nucleic acid structures through compromised transcription and exposure to genotoxic agents (e.g., ionizing radiation, IR) imposes a roadblock to ongoing replication and transcription causing genomic instability by generating double-stranded DNA breaks (DSBs). Kub5-Hera (Ku70-binding protein 5-Hera, K-H; also known as RPRD1B, Regulation of nuclear pre-mRNA-domain-containing protein 1B) is a novel factor discovered in our lab that is involved in transcription termination and DNA repair. We found that loss of K-H increases R-loop-induced DSBs that activates the error-prone alternative non-homologous end-joining (alt-EJ or back-up NHEJ) due to defects in classical NHEJ (c-NHEJ) and homologous recombination (HR). Using our transgenic mouse model, we discovered that a complete knockout of K-H is embryonic lethal suggesting a critical role for this protein in cellular development and proliferation. Interestingly, deletion of one K-H allele in mice caused rapid development of cancer-related deaths over time after whole-body exposure to IR in a dose-dependent manner compared to their wild-type littermates. Using targeted gene silencing, western blot, proximity ligation and immunofluorescent assays in our cell line models and mouse tissues, we showed that depletion of K-H increases the formation of R-loop-induced double-stranded breaks (DSBs) and PARP1 activity due to a compromised homologous recombination (HR). Loss of K-H in cells increases the error-prone activity of PARP1 in alt-EJ to possibly repair harmful R-loop-induced single-stranded breaks (SSBs) and DSBs due to HR and c-NHEJ deficiency, which could provide the mechanism of carcinogenesis in mice after exposure to IR. Overall, our studies offer new mechanistic insights into carcinogenesis caused by K-H genetic alterations and persistent R-loops, as well as identify novel strategies to leverage Kub5-Hera expression or the formation of lethal R-loop-induced-DSBs for innovative therapeutic approaches.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L6387
  17. J Biomed Sci. 2022 May 12. 29(1): 32
    Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma.
    Yongsheng Xie, Yingcong Wang, Zhijian Xu, Yumeng Lu, Dongliang Song, Lu Gao, Dandan Yu, Bo Li, Gege Chen, Hui Zhang, Qilin Feng, Yong Zhang, Ke Hu, Cheng Huang, Yu Peng, Xiaosong Wu, Zhiyong Mao, Jimin Shao, Weiliang Zhu, Jumei Shi.
       BACKGROUND: Aberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM.
    METHODS: We applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy.
    RESULTS: HDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM.
    CONCLUSIONS: Our study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM.
    TRIAL REGISTRATION: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018.
    Keywords:  4-Hydroxysalicylanilid; DNA damage repair; Deoxyribonucleotides; Multiple myeloma; Ribonucleotide reductase
    DOI:  https://doi.org/10.1186/s12929-022-00813-2
  18. Bioessays. 2022 May 09. e2200015
    Joining the PARty: PARP Regulation of KDM5A during DNA Repair (and Transcription?).
    Anthony Sanchez, Bethany A Buck-Koehntop, Kyle M Miller.
      The lysine demethylase KDM5A collaborates with PARP1 and the histone variant macroH2A1.2 to modulate chromatin to promote DNA repair. Indeed, KDM5A engages poly(ADP-ribose) (PAR) chains at damage sites through a previously uncharacterized coiled-coil domain, a novel binding mode for PAR interactions. While KDM5A is a well-known transcriptional regulator, its function in DNA repair is only now emerging. Here we review the molecular mechanisms that regulate this PARP1-macroH2A1.2-KDM5A axis in DNA damage and consider the potential involvement of this pathway in transcription regulation and cancer. Using KDM5A as an example, we discuss how multifunctional chromatin proteins transition between several DNA-based processes, which must be coordinated to protect the integrity of the genome and epigenome. The dysregulation of chromatin and loss of genome integrity that is prevalent in human diseases including cancer may be related and could provide opportunities to target multitasking proteins with these pathways as therapeutic strategies.
    Keywords:  DNA double-strand breaks; DNA repair; KDM5A; PARP; chromatin; histone demethylation; poly (ADP-ribose)
    DOI:  https://doi.org/10.1002/bies.202200015
  19. FASEB J. 2022 May;36 Suppl 1
    G9a Responds to KRAS-Mediated Replication Stress by Increased Origin Licensing.
    Anju Thomas, Thiago Milech de Assuncao, Gwen Lomberk.
      Pancreatic Ductal Adenocarcinoma (PDAC) remains as one of the leading causes of cancer death in the US. KRAS mutations are recognized as the leading oncogenic driver of PDAC growth. Activation of KRAS causes increased proliferation that requires a level of adaptation during processes, such as DNA replication. This rapid duplication of the genome and epigenome generates havoc in the timing and coordination of replication origin firing, triggering a response known as replication stress (RS). The epigenomic regulator G9a is responsible for catalyzing histone H3 lysine 9 mono- and di-methylation (H3K9me1 and H3K9me2). G9a is abundantly present in many forms of cancer, including PDAC, and because of its interaction with replication proteins, such as RPA and PCNA, it is known to have an important role during DNA replication. DNA replication happens at the S phase of the cell cycle, which requires that replication origins are recognized at late G1 phase by the origin recognition complex (ORC) followed by assembly of the pre-replication complex (pre-RC), comprised of CDC6, cdt1 and mini chromosome maintenance (MCM) proteins, to allow origin licensing and subsequent firing. We have shown that the G9a complex increases in response to KRAS activation and is critical to the growth-promoting effects of this oncogene in PDAC. Though it is known that KRAS reduces distances between replication origins, there has been a paucity of knowledge regarding the role of G9a during replication origin licensing and whether it is directly involved with the pre-RC. In the current study, we aim to better understand whether G9a interacts with the replication origins and the pre-RC to increase origin licensing under conditions of KRAS-mediated RS. To simulate the early events in oncogene-induced RS to study compensatory mechanisms, we generated an inducible KRAS model (iKRAS-HPNE) in an hTERT-immortalized human non-cancerous pancreatic ductal cell line, hTERT-HPNE E6/E7. We found that KRAS induction for 48h in this iKRAS-HPNE model activated the ATR RS-response pathway, indicated by increased levels of P-T1989-ATR, P-S345-CHK1 and P-S33-RPA32 by western blot and immunofluorescence-based microscopy. Concurrently, members of the G9a epigenomic complex (G9a, GLP and WIZ) significantly increased along with two subunits of the ORC (ORC1 and ORC2) and several other pre-RC proteins (Cdt1, MCM2, MCM5 and MCM7). Co-immunoprecipitation of G9a in the iKRAS-HPNE cells revealed that G9a interacts with ORC2 protein under conditions of oncogene-induced RS. Pharmacological inhibition of G9a with UNC0642 during KRAS-induced RS resulted in a decrease of chromatin-bound ORC1 and ORC2 as well as pre-RC proteins in subcellular fractionation experiments, suggesting that functional G9a is critical for enhanced origin licensing. In summary, our results demonstrate that G9a directly interacts with replication origins and the pre-RC during origin licensing under conditions of RS. Our study reveals one potential mechanism by which cancerous cells with KRAS mutation meet the demands for rapid cellular proliferation during RS and reinforces the role of G9a as a promising therapeutic target for PDAC.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5141
  20. FASEB J. 2022 May;36 Suppl 1
    Transcriptional regulation via strand displacement DNA repair in G-quadruplexes.
    Amy Whitaker, Bret Freudenthal.
      Environmental exposures and lifestyle choices can result in cellular oxidative stress, characterized by the generation of an abundance of reactive oxygen species (ROS). ROS wreak havoc on the structure of DNA bases, with guanine modification yielding the lesion 8-oxo-7,8- dihydroguanine (8oxoG) being particularly prevalent. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote human disease. Guanine-rich G- quadruplex (G4) forming sequences are enriched at promoter proximal regions of the genome, making these regions hot spots for 8oxoG lesions. The cells primary defense against 8oxoG is base excision repair (BER). Recently, it was shown that the repair of 8oxoG by the BER enzymes OGG1 and APE1 perturbs the structural equilibrium of the VEGFpromoter DNA sequence between duplex and G4 conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details regulating this equilibrium remain somewhat enigmatic, including the activity and coordination of BER enzymes on an 8oxoG containing G4 promoter. To address this, we determined the requirement and efficiency of each BER factor on G4 substates (including OGG1, APE1, Polβ, and DNA ligase 1) by employing a combination of pre-steady state kinetics assays and in vitroBER reconstitution assays with G4 DNA substrates. Surprisingly, we observe OGG1 is capable of initiating BER on oxidized G4 VEGFDNA substrates in which the G4 region is flanked by non-G4, duplex regions of DNA. Pre-steady state kinetics revealed that compared to abasic duplex DNA, APE1-mediated strand cleavage is only slightly decreased and that a majority of the reduction in its activity on G4 DNA results from slow product release. Thus, APE1 may have an opportunity to recruit transcription factors to the VEGF promoter during this slow product release step of its reaction. Interestingly, Polβ performs multiple insertions on G4 substates via strand displacement DNA synthesis, with 3 - 5 insertions depending on the position of damage. In contrast, there is only a single insertion by Polβ with corresponding duplex DNA substates and no insertion when the G4 structure lacks an opposing strand with flanking duplex regions. Ligase I does not efficiently ligate G4 substrates, and hence cannot complete the BER repair cycle. Instead, proper repair requires the long-patch BER enzyme flap-endonuclease activity of FEN1 in response to the multiple insertions by Polβ prior to ligation.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R292
  21. 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
  22. FASEB J. 2022 May;36 Suppl 1
    Targeting Keratin 17 in Pancreatic Cancer: A Novel Rewired Pathway of Nucleotide Metabolism that Drives Chemoresistance.
    Chun-Hao Pan, Robert Tseng, Katie Donnelly, Cindy V Leiton, Simon J Hogg, Natalia Marchenko, Pankaj K Singh, Kenneth R Shroyer, Luisa F Escobar-Hoyos.
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by two molecular subtypes, of which the basal-like subtype is associated with the worst survival and is highly resistant to the first-line chemotherapy. We previously reported that keratin 17 (K17), a signature gene in basal-like subtype, is a novel negative-prognostic and predictive biomarker, whose overexpression results in resistance to Gemcitabine (Gem) and 5-fluorouracil, the major chemotherapeutic agents in standard-of-care treatments, and leads to shortened patient survival. Here, we set out to uncover the mechanisms of chemoresistance and explore targeted therapies for K17-expressing PDAC. We hypothesized that K17 reprograms cancer metabolism and leads to therapeutic resistance. We manipulated the expression of K17 in multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells and orthotopic xenografts, for drug-testing, metabolomic and mechanistic studies. To uncover the mechanisms associated with K17-induced chemoresistance, we performed unbiased metabolomic studies in isogenic PDAC cell lines and found that compared to control cells, K17 increases intracellular levels of deoxycytidine (dC) by four-fold that promote Gem (dC analogue) resistance. Interestingly, K17-expressing cells are more sensitive to a compound that targets de novo pyrimidine biosynthesis. Based on previous findings that K17 enters nucleus to regulate gene expression, weexplored whether K17 triggers metabolic reprogramming at the transcriptional level and found that enzymes involved in pyrimidine biosynthesis are positively correlated with K17 expression in PDAC cells. Given that it is still poorly understood how K17 regulates gene expression, we performed domain-prediction analyses. We discovered and validated a novel chromatin remodeling domain in K17 that is required for metabolic reprogramming. Importantly, mice bearing tumors with the deletion of the chromatin remodeling domain in K17 survived significantly longer than those with tumors expressing wild type K17. We are now performing ATAC-seq, ChIP-Seq and RNA-Seq to understand how this domain alters pyrimidine biosynthesis. In summary, we identified a novel pathway of chemoresistance that could result in developing a biomarker-based personalized therapy.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3889
  23. FASEB J. 2022 May;36 Suppl 1
    Development of New Assays for the Simultaneous Measurement of DNA Double-Strand Break Repair by Multiple Pathways.
    Diego F Valdez-Oranday, L K Lewis.
      Cellular DNA is damaged by both endogenous and exogenous sources. Among the different types of DNA lesions that are formed, double-strand breaks (DSBs) are considered to be the most consequential lesions. They can cause genomic instability and cellular death if not repaired via one of two pathways: nonhomologous end-joining (NHEJ) or homologous recombination (HR). Deficiency of DSB repair is implicated in several congenital diseases and cancer-prone disorders. In the budding yeast Saccharomyces cerevisiae NHEJ is accomplished by the actions of the Yku, Mrx, and Dnl4 protein complexes. Recent experiments in this lab have demonstrated that, in contrast to Yku and Dnl4, the Mrx complex is required for NHEJ repair of some DSB end structures but not others. The major goal of the current project is to characterize the role of Mrx and other protein complexes in repair by NHEJ and HR using newly developed plasmid and PCR fragment-based assays. Experiments performed for the project demonstrated that repair of DSBs with 3' single-stranded DNA overhangs was reduced in yku70, mre11 and dnl4 mutants; however, the defect in NHEJ was strongly suppressed when EXO1, encoding a major 5'-to-3' exonuclease, was inactivated in mre11 cells, i.e., in mre11 exo1 double mutants. By contrast, absence of the Sgs1-Dna2 nuclease-helicase complex (in mre11 sgs1 mutants) had no effect. NHEJ repair of DSBs with 5' single-stranded DNA overhangs was not changed by inactivation of EXO1 or SGS1. These findings implicate Mrx as a potential protector of DSB ends with 3' overhangs during NHEJ repair and Exo1 as the main degradative nuclease. Additionally, we have developed new plasmid- and PCR fragment-based repair assays that can analyze NHEJ and HR simultaneously using auxotrophic markers and type IIS restriction endonucleases. We anticipate that the new methods for simultaneous measurement will permit exploration of pathway choice in ways not previously possible.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3626
  24. Front Pharmacol. 2022 ;13 860682
    Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.
    Nikolaos Karantzelis, Michalis Petropoulos, Valeria De Marco, David A Egan, Alexander Fish, Evangelos Christodoulou, David W Will, Joe D Lewis, Anastassis Perrakis, Zoi Lygerou, Stavros Taraviras.
      DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.
    Keywords:  AlphaScreen; CDT1; Geminin; cancer; high-throughput screening; small molecule inhibitor
    DOI:  https://doi.org/10.3389/fphar.2022.860682
  25. G3 (Bethesda). 2022 May 14. pii: jkac116. [Epub ahead of print]
    Determinants of RPA megafoci localization to the nuclear periphery in response to replication stress.
    Seong Min Kim, Susan L Forsburg.
      Upon replication stress, ssDNA, coated by the ssDNA binding protein RPA, accumulates and generates a signal to activate the replication stress response. Severe replication stress induced by the loss of MCM helicase subunit Mcm4 in the temperature sensitive Schizosaccharomyces pombe degron mutant (mcm4-dg) results in formation of a large RPA focus that is translocated to the nuclear periphery. We show that resection and repair processes and chromatin remodeller Swr1/Ino80 are involved in the large RPA foci formation and its relocalization to nuclear periphery. This concentrated accumulation of RPA increases recruitment of Cds1 to chromatin and results in an aberrant cell cycle that lacks MBF-mediated G1/S accumulation of Tos4. These findings reveal a distinct replication stress response mediated by localized accumulation of RPA that allows evasion of cell cycle arrest.
    Keywords:  DNA repair; MCM4; RPA; Replication stress; checkpoint
    DOI:  https://doi.org/10.1093/g3journal/jkac116
  26. FASEB J. 2022 May;36 Suppl 1
    Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks.
    Jessica Packard, Jill Dembowski.
      Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that replicates the 152kbp viral genome within the nucleus of host cells. Replication of the HSV-1 genome is catalyzed by viral replication machinery consisting of a DNA polymerase (UL30), processivity factor (UL42), helicase-primase complex, origin binding protein (UL9), and a single stranded DNA binding protein. Despite encoding a viral DNA polymerase processivity factor, we found that host Proliferating Cell Nuclear Antigen (PCNA) interacts with HSV-1 DNA at replication forks and associates with viral DNA in a replication-dependent manner. PCNA is a homotrimer that provides processivity to cellular DNA polymerases and selectively recruits DNA damage response and DNA repair factors to cellular replication forks. We therefore hypothesized that PCNA associates at viral replication forks to promote HSV-1 replication while also tethering cellular DNA repair proteins to replicating viral DNA. To test this, we used two models to identify how associated factors change as a function of PCNA: a known PCNA inhibitor, PCNA-I1, and a cell line that was engineered to inducibly express a shRNA targeting PCNA. We performed a technique adapted from isolation of proteins on nascent DNA (iPOND) to isolate HSV-1 DNA from infected cells and identified associated viral and cellular proteins. Cells were infected and replicating viral DNA was selectively labeled with EdC. Labeled DNA was specifically and irreversibly tagged via the covalent attachment of biotin azide via click reaction. Biotin-tagged DNA was purified on streptavidin-coated beads and associated proteins were eluted and identified by mass spectrometry. As a complimentary approach, we also performed immunofluorescence (IF) to confirm iPOND data. We found that PCNA is associated with viral DNA despite treatment with PCNA-I1, an observation that is consistent with IF imaging data. Viral replication proteins UL30, UL42, and UL9 decreased in the presence of PCNA-I1. DNA damage response proteins such as mismatch repair proteins and RECQL both decreased up to 10-fold. Of note, however, proteins that make up the MRN complex, a class of proteins that functions in DNA break repair, restart of stalled replication forks, and viral DNA infection, increased in response to PCNA-I1 treatment. Given this data, PCNA may be involved in tethering viral replication proteins, cellular DNA repair proteins, and/or virion assembly proteins to viral replication forks.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2044
  27. FASEB J. 2022 May;36 Suppl 1
    cMET inhibition potentiates the tumor-selective damaging effects of NQO1-bioactivatable agents by compromising DNA repair.
    Snehal B Bhandare, I-Ju Yeh, Jon-Luc Poirier, Edward A Motea.
      The overall prognosis for solid tumors overexpressing mesenchymal-epithelial transition factor (cMET) receptor tyrosine kinase and NADPH:Quinone oxidoreductase 1 (NQO1) is poor, and innovative treatment strategies that selectively target these cancers are critically needed. Recent studies have implicated cMET in the activation of PARP1, a critical factor involved in DNA damage response and repair. Therefore, targeting cMET is an attractive strategy for cancer therapy. However, the overall efficacies of cMET inhibitors and NQO1 bioactivable agents are limited due to dose-limiting toxicities and lack of tumor selectivity at high concentrations as a monotherapy. Here, we report that the combination treatment with sublethal doses of cMET inhibitors (some in clinical trials and FDA-approved) and β-lapachone (β-lap, an NQO1-bioactivatable drug in clinical trials) induced synergistic lethality in cancer cells in an NQO1-dependent manner. Mechanistically, a sublethal dose of β-lap creates reactive oxygen species (ROS) that damage DNA nucleobases (e.g., 8-oxoguanine) but rapidly gets repaired by the ability of ROS-activated cMET to enhance PARP1 activity for efficient DNA damage repair in NQO1+ cells. Thus, β-lap in combination with cMET inhibition elevated DNA damage by compromising DNA repair, increased double-strand break (DSB) formation and promoted tumor selective apoptosis in NQO1+cancer cells. We further determined that the combination treatment significantly inhibit tumor growth in 3D spheroids. Our results add a new strategy for personalized therapy: the targeting of cMET in NQO1+ cancers to potentiate the toxic effects of sub-lethal doses of NQO1-bioativatable agents and cMET inhibitors.
    Keywords:  DNA repair; NQO1; PARP1; ROS; cMET; β-Lapachone
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4326
  28. FASEB J. 2022 May;36 Suppl 1
    Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome.
    Carolina de Bovi Pontes, Cody Jefferys, Muhamad Bedwan, Elena J Ciesielska, Grzegorz L Ciesielski.
      Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post-replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4339
  29. FASEB J. 2022 May;36 Suppl 1
    Too much and never enough: Synthetic excess and metabolic inefficiency of aneuploidy in tumorigenesis.
    Emma V Watson.
      Aneuploidy is a hallmark feature of cancer. Relative to normal diploid tissue, aneuploid tumors are often net copy number increased, in a range from hyper-diploid to tetraploid. This results in widespread replication stress and proteotoxic stress that affects diverse cellular complexes. Utilizing multi-omics approaches in novel models of cancer-associated copy number alterations derived from diploid human mammary epithelial cells, we uncovered an aneuploidy stress response driven in part by Myc that transcriptionally upregulates biomass production capacity and stresses metabolic capacity. Basal and peak glycolytic and oxidative rates are increased in aneuploid cells compared to diploids, however free nucleotide pools and metabolic intermediates, particularly pyrimidines, are depleted. CRISPR screens revealed a universally increased dependence on de novo pyrimidine synthesis genes in aneuploid cells with diverse CNAs, suggesting a struggle to match pyrimidine synthesis capacity with need. The biosynthetic demand and inefficiency driven by aneuploidy may partially explain the switch to aerobic glycolysis in tumors and present vulnerabilities to be therapeutically exploited.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0I210
  30. Nat Commun. 2022 May 12. 13(1): 2638
    MRNIP condensates promote DNA double-strand break sensing and end resection.
    Yun-Long Wang, Wan-Wen Zhao, Shao-Mei Bai, Li-Li Feng, Shu-Ying Bie, Li Gong, Fang Wang, Ming-Biao Wei, Wei-Xing Feng, Xiao-Lin Pang, Cao-Litao Qin, Xin-Ke Yin, Ying-Nai Wang, Weihua Zhou, Daniel R Wahl, Quentin Liu, Ming Chen, Mien-Chie Hung, Xiang-Bo Wan.
      The rapid recognition of DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex is critical for the initiation of DNA damage response and DSB end resection. Here, we show that MRN complex interacting protein (MRNIP) forms liquid-like condensates to promote homologous recombination-mediated DSB repair. The intrinsically disordered region is essential for MRNIP condensate formation. Mechanically, the MRN complex is compartmentalized and concentrated into MRNIP condensates in the nucleus. After DSB formation, MRNIP condensates move to the damaged DNA rapidly to accelerate the binding of DSB by the concentrated MRN complex, therefore inducing the autophosphorylation of ATM and subsequent activation of DNA damage response signaling. Meanwhile, MRNIP condensates-enhanced MRN complex loading further promotes DSB end resection. In addition, data from xenograft models and clinical samples confirm a correlation between MRNIP and radioresistance. Together, these results reveal an important role of MRNIP phase separation in DSB response and the MRN complex-mediated DSB end resection.
    DOI:  https://doi.org/10.1038/s41467-022-30303-w
  31. FEBS Open Bio. 2022 May 14.
    ARL2 is required for homologous recombination repair and colon cancer stem cell survival.
    Hani Lee, SeokGyeong Choi, Sojung Ha, Sukjoon Yoon, Woo-Young Kim.
      ARL2 regulates the dynamics of cytological components and is highly expressed in colon cancer tissues. Here, we report novel roles of ARL2 in the cell nucleus and colon cancer stem cells (CSCs). ARL2 is expressed at relatively low levels in K-RAS active colon cancer cells, but its expression is induced in CSC. Depletion of ARL2 results in M phase arrest exclusively in non-CSC cultured cells; in addition, DNA break stress accumulates in CSCs leading to apoptosis. ARL2 expression is positively associated with the expression of all 6 RAD51 family genes, which are essential for homologous recombination repair (HRR). Furthermore, ARL2 is required for HRR and detected within chromatin compartments. These results demonstrate the requirement of ARL2 in colon CSC maintenance, which possibly occurs through mediating double strand break DNA repair in the nucleus.
    Keywords:  ARL2; Cancer stem cells; Colon cancer; Double strand repair; Homologous recombination repair
    DOI:  https://doi.org/10.1002/2211-5463.13438
  32. FASEB J. 2022 May;36 Suppl 1
    Differential Expression of Nuclear Body Component, Zc3h8, Disrupts DNA Double Strand Break Repair.
    John A Schmidt, Rhiann Sato.
      The processes of DNA damage recognition and repair are paramount to maintaining genome integrity in the cell. Failure to do so results in genetic instability and the accumulation of mutations. Double strand breaks have the greatest potential to introduce mutations because rearrangements, non-homologous end joining, deletions, or insertions may result. Unrepaired or incorrectly repaired DNA double strand breaks can lead to cell death, apoptosis, or disease such as cancer. We previously demonstrated that the C3H1 zinc finger-containing protein Zc3h8 (also called Fliz1) promotes oncogenic phenotypes when overexpressed in cells, while oncogenic phenotypes are suppressed when expression is reduced. In mice, Zc3h8 expression affected tumorigenesis and tumor size in vivo. Higher expression of Zc3h8 led to more frequent tumors and larger tumors in mice. In humans Zc3h8 is overexpressed in 2-6% of cancers and is a statistically significant prognostic indicator for poor rates of survival. The Zc3h8 protein has been localized to nuclear bodies including promyelocytic leukemia (PML) and Cajal Bodies where key cellular processes including transcription, translation, post-translational modifications, and RNA processing are regulated. PML Bodies, in particular, have a role in DNA damage response and repair since P53 is located in PML Bodies along with DAXX, ATRX, and CK2. Since Zc3h8 co-localized with DNA damage response and repair proteins in PML bodies and promotes oncogenic properties in cells, we investigated if Zc3h8 expression alters how cells respond to DNA damage such as double strand breaks and how this damage is mitigated in cells. Differential expression of Zc3h8 affected mouse mammary tumor cell's ability to repair double strand breaks caused by the topoisomerase II inhibitor etoposide as indicated by the presence of γH2AX, a marker for unrepaired double strand breaks. Using a Comet assay, we found extensive DNA fragmentation in cells with elevated Zc3h8 expression despite high rates of proliferation and migration. Furthermore, Zc3h8 increases cellular tolerance for DNA damage and alters apoptosis. These phenotypes promote further mutation and may indicate a molecular cause for highly aggressive and progressive cancers with elevated Zc3h8 expression. These conclusions suggest a DNA damage repair and response pathway that is altered by Zc3h8 expression levels can affect multiple aspects of cellular behavior and promote oncogenic phenotypes.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.00R97
  33. FASEB J. 2022 May;36 Suppl 1
    Detection of Multi-Protein Complexes Containing PCNA Using Fluorescence Anisotropy and Hormetic Modeling.
    Sharon N Greenwood, Regina G Belz, Brian P Weiser.
      Proliferating Cell Nuclear Antigen (PCNA) is a homotrimeric protein involved in DNA replication and repair. It contains three equivalent binding sites that are known to interact with dozens of replication and repair proteins. In theory, PCNA could bind multiple proteins at the same time, but steric hindrance prevents this for some protein combinations. Here, we developed an approach to detect the formation of multi-protein complexes containing PCNA and base excision repair proteins using fluorescence anisotropy and hormetic modeling. Initially, we measured binding of fluorescent-labeled pogo-ligase peptide (PL) to PCNA and determined a Kd of 118 nM. This assay measured an increase in the fluorescence anisotropy of 50 nM PL upon PCNA binding because the fluorescent peptide becomes part of a larger macromolecular complex that slows its movement in solution (anisotropy min/max: 0.043/0.121). Next, we competed 50 nM PL from 0.25 μM PCNA using uracil DNA glycosylase (UNG2), which reduced the anisotropy from 0.086 to 0.057, and we determined an IC50 of 4 μM with a conventional sigmoidal curve. In contrast, displacement of PL from 3 μM PCNA using UNG2 resulted in a hormetic dose response that was fit using the Brain-Cousens equation for hormesis. In these experiments, the anisotropy of 50 nM PL increased from 0.095 in the presence of 3 μM PCNA to 0.127 with the addition of 5 μM UNG2, and the anisotropy then reduced to 0.086 with 50 μM UNG2. The hormetic response occurred because a ternary PCNA-PL-UNG2 complex formed at UNG2 concentrations that were sufficient to bind open PCNA sites, but were insufficient to displace PL from PCNA, and the anisotropy of PL bound to the ternary complex was higher than its anisotropy bound to PCNA alone. Additional values derived from the Brain-Cousens curve included a significant hormesis parameter f (0.024), a maximum stimulatory response of 134% of control at a dose of 4 μM UNG2, and a limited dose for stimulation at 31 μM UNG2. We also report additional datasets where UNG2 displaced 50 nM PL from other PCNA concentrations to train our hormetic modeling and optimize assay signal/noise. Continuing experiments using fluorescent-labeled UNG2 and fluorescent-labeled DNA polymerase β (POLB), in addition to DNA Ligase 1 (LIG1), will explore the simultaneous and/or sequential interactions of base excision repair proteins with PCNA.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R254
  34. FASEB J. 2022 May;36 Suppl 1
    Single-Molecule Analysis of in vivo DNA Replication Origin Licensing Stoichiometry.
    Nick Rhind, Johnson Chung, Larry Friedman, Jeff Gelles.
      The organization of DNA replication location and timing is regulated by the establishment of replication origins. Origins are established in G1 by the loading of a pair of MCMs, the catalytic core of the replicative helicase, as an inactive double-hexamer complex. Activation of MCM during S phase is the rate-limiting step for replication initiation, and therefore regulates where and when replication occurs. We have previously shown that origins at which multiple MCM complexes are loaded have a higher probability of initiation and therefore replicate earlier, on average. Bulk biochemical experiments suggest the ARS1, a early-firing budding yeast origin, loads, on average, about three MCM complexes in G1, whereas ARS316, a late-firing origin, loads less than one. However, these bulk measurements have two major drawbacks. First, the required normalization controls make our absolute stoichiometry estimates uncertain. Second, they only reveal the average numbers of MCM's loaded; they provide no insight into the distribution of MCM stoichiometry, information which could elucidate how MCM loading stoichiometry is controlled.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5092
  35. Front Cell Dev Biol. 2022 ;10 903781
    Nucleic Acid Sensing Pathways in DNA Repair Targeted Cancer Therapy.
    Bingteng Xie, Aiqin Luo.
      The repair of DNA damage is a complex process, which helps to maintain genome fidelity, and the ability of cancer cells to repair therapeutically DNA damage induced by clinical treatments will affect the therapeutic efficacy. In the past decade, great success has been achieved by targeting the DNA repair network in tumors. Recent studies suggest that DNA damage impacts cellular innate and adaptive immune responses through nucleic acid-sensing pathways, which play essential roles in the efficacy of DNA repair targeted therapy. In this review, we summarize the current understanding of the molecular mechanism of innate immune response triggered by DNA damage through nucleic acid-sensing pathways, including DNA sensing via the cyclic GMP-AMP synthase (cGAS), Toll-like receptor 9 (TLR9), absent in melanoma 2 (AIM2), DNA-dependent protein kinase (DNA-PK), and Mre11-Rad50-Nbs1 complex (MRN) complex, and RNA sensing via the TLR3/7/8 and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). Furthermore, we will focus on the recent developments in the impacts of nucleic acid-sensing pathways on the DNA damage response (DDR). Elucidating the DDR-immune response interplay will be critical to harness immunomodulatory effects to improve the efficacy of antitumor immunity therapeutic strategies and build future therapeutic approaches.
    Keywords:  DDR inhibitors; DNA damage and repair; immunotherapy; innate immunity; nucleic acid-sensing pathways
    DOI:  https://doi.org/10.3389/fcell.2022.903781
  36. FASEB J. 2022 May;36 Suppl 1
    Purine biosynthesis and related enzymes play the oncogenic function in FGFR3 mutant urothelial carcinoma.
    Yu-Chan Chang.
      Urothelial carcinoma (UC), as one of the malignant tumors, has a high incidence, mortality, and poor survival rate. FGFR3 is one of the frequency mutation genetic events of UC. Recent studies have reported that FGFR3 mutations are associated with lower aggressiveness and better prognosis of UC, and small molecule compounds with FGFR3 mutations have been developed and treated. However, they are still limited, especially for wild-type cells. We used Ingenuity Pathways Analysis (IPA) to analyze the significantly expression of 351 genes (>2-fold change cutoff value) between FGFR3 wild-type and mutation/fusion UC cells by FGFR3 inhibitors treatment. Through the predicted canonical pathways, several metabolism-related events have been regulated, including purine biosynthesis. Focusing on de novo purine biosynthesis, we found that several involved enzymes are upregulated in UC. In addition, we have also observed that several enzymes are inhibited by FGFR3 inhibitors in mutant cells, but not in wild type. We then hypothesized that purine biosynthetic events might confer drug resistance and promote UC tumorigenicity. To verify our observations, we determined the expression level of purine biosynthesis after FGFR3 classification. The results indicate a similar trend in clinical cohorts that overexpression of purine biosynthesis genes in FGFR3 wild-type. Moreover, combining these genes can be used as an independent prognostic factor (HR=1.41, p=0.026). We further established the overexpression of purine biosynthesis genes in some FGFR3 mutant UC cells. We determined that purine biosynthesis genes cause FGFR3 mutations models to be more prone to mesenchymal and metastatic than controls. Our studies also observed that FGFR3 wild-type cells promote UC cell radioresistance via purine biosynthesis and further activate the homologous recombination repair mechanism. Combining all evidence, we claim that purine biosynthesis should be focused and regarded as a novel strategy against UC.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R497
  37. FASEB J. 2022 May;36 Suppl 1
    Combined Inhibition of the Ataxia-telangiectasia mutated and Rad3-related Pathway and the G9a Methyltransferase Synergize to Reduce Pancreatic Cancer Cell Growth.
    Daniela Rodrigues de Oliveira, Thiago Milech de Assuncao, Guillermo Urrutia, Gwen Lomberk.
      Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States with a devastating overall five-year survival rate of only 10%. Consequently, there is an urgent need to identify innovative therapeutic strategies. PDAC exhibits chronic replication stress (RS), becoming reliant on a proficient Ataxia-telangiectasia mutated and Rad3-related (ATR) pathway to adapt to high RS levels in cells. This also presents an exploitable therapeutic vulnerability in tumors via strategies that enhance the endogenous RS to trigger replication catastrophe and cell death. Our laboratory is focused on utilizing epigenetic inhibitors in this context as novel therapeutic approaches. Recently, we have shown that combined inhibition of the major ATR effector, CHK1, and the G9a methyltransferase leads to replication catastrophe, providing an effective therapeutic approach for PDAC by priming replication failure through the epigenome rather than genomic damage. In the current study, we sought to investigate additional targets in the RS response pathway through direct ATR inhibition combined with G9a inhibition and perform mechanistic studies on DNA damage response checkpoints and cell cycle. We treated multiple PDAC cell lines with a combination of the ATR inhibitor, AZD6738, and either BRD4770 or UNC0642 to inhibit G9a at various concentrations to determine the effect on cell viability and cytotoxicity by live-cell analysis. We find that the AZD6738-BRD4770 and AZD6738-UNC0642 combinations display synergistic effects to disrupt the RS response and inhibit PDAC growth. Overall, our work provides quantitative insights into a novel therapeutic opportunity for PDAC and expands the universality of a synergistic interaction resulting from simultaneous targeting of the ATR-CHK1 RS pathway and the epigenomic regulator G9a.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4969
  38. Acta Pharm Sin B. 2022 Mar;12(3): 1339-1350
    Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR.
    Fangfang Wang, Sora Jin, Franklin Mayca Pozo, Danmei Tian, Xiyang Tang, Yi Dai, Xinsheng Yao, Jinshan Tang, Youwei Zhang.
      DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.
    Keywords:  ATM; ATM, ataxia telangiectasia mutated; ATR; ATR, ATM and RAD3-related; ATRIP; ATRIP, ATR-interacting protein; BAF, bafilomycin A; CHK1/2, checkpoint kinase 1/2; CIS, cisplatin; CPT, camptothecin; Chemical screen; Chemo sensitizing; DDR, DNA damage response; DNA damage Response; ETO, etoposide; GEM, gemcitabine; KAP1, KRAB-associated protein 1; Luc, Luciferase; PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffered saline; Protein degradation; RNAi, RNA interference; SKN, shikonin; Shikonin; ULK1, Unc-51-like kinase 1; Z-VAD, Z-VAD-FMK; qPCR, quantitative polymerase chain reaction
    DOI:  https://doi.org/10.1016/j.apsb.2021.08.025
  39. Int J Mol Sci. 2022 Apr 23. pii: 4690. [Epub ahead of print]23(9):
    Does Intracellular Metabolism Render Gemcitabine Uptake Undetectable in Mass Spectrometry?
    Julian Peter Müller, Dirk Gründemann.
      The ergothioneine transporter ETT (formerly OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC-MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC-MS/MS measurements when we expressed the Na+/nucleoside cotransporter CNT3 (SLC28A3). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2'-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.
    Keywords:  CNT3; SLC22A4; drug transport; ergothioneine; ergothioneine transporter; nucleoside metabolism; nucleoside transport
    DOI:  https://doi.org/10.3390/ijms23094690
  40. Cancers (Basel). 2022 Apr 27. pii: 2194. [Epub ahead of print]14(9):
    Alternative Lengthening of Telomeres and Mediated Telomere Synthesis.
    Kailong Hou, Yuyang Yu, Duda Li, Yanduo Zhang, Ke Zhang, Jinkai Tong, Kunxian Yang, Shuting Jia.
      Telomeres are DNA-protein complexes that protect eukaryotic chromosome ends from being erroneously repaired by the DNA damage repair system, and the length of telomeres indicates the replicative potential of the cell. Telomeres shorten during each division of the cell, resulting in telomeric damage and replicative senescence. Tumor cells tend to ensure cell proliferation potential and genomic stability by activating telomere maintenance mechanisms (TMMs) for telomere lengthening. The alternative lengthening of telomeres (ALT) pathway is the most frequently activated TMM in tumors of mesenchymal and neuroepithelial origin, and ALT also frequently occurs during experimental cellular immortalization of mesenchymal cells. ALT is a process that relies on homologous recombination (HR) to elongate telomeres. However, some processes in the ALT mechanism remain poorly understood. Here, we review the most recent understanding of ALT mechanisms and processes, which may help us to better understand how the ALT pathway is activated in cancer cells and determine the potential therapeutic targets in ALT pathway-stabilized tumors.
    Keywords:  alternative lengthening of telomeres; homologous recombination; telomere maintenance mechanisms
    DOI:  https://doi.org/10.3390/cancers14092194
  41. Cells. 2022 Apr 26. pii: 1463. [Epub ahead of print]11(9):
    DNA Damage Response Inhibitors in Cholangiocarcinoma: Current Progress and Perspectives.
    Öykü Gönül Geyik, Giulia Anichini, Engin Ulukaya, Fabio Marra, Chiara Raggi.
      Cholangiocarcinoma (CCA) is a poorly treatable type of cancer and its incidence is dramatically increasing. The lack of understanding of the biology of this tumor has slowed down the identification of novel targets and the development of effective treatments. Based on next generation sequencing profiling, alterations in DNA damage response (DDR)-related genes are paving the way for DDR-targeting strategies in CCA. Based on the notion of synthetic lethality, several DDR-inhibitors (DDRi) have been developed with the aim of accumulating enough DNA damage to induce cell death in tumor cells. Observing that DDRi alone could be insufficient for clinical use in CCA patients, the combination of DNA-damaging regimens with targeted approaches has started to be considered, as evidenced by many emerging clinical trials. Hence, novel therapeutic strategies combining DDRi with patient-specific targeted drugs could be the next level for treating cholangiocarcinoma.
    Keywords:  DNA damage; PARP; Wee1; biliary tract cancer; synthetic lethality; targeted therapy
    DOI:  https://doi.org/10.3390/cells11091463
  42. FASEB J. 2022 May;36 Suppl 1
    Effect of Uracil DNA Glycosylase Activity on the Efficacy of Thymidylate Synthase Inhibitor/HDAC Inhibitor Combination Therapies in Colon Cancer.
    Rashmi S Kulkarni, Brian P Weiser.
      Human uracil DNA glycosylase (UNG2) is responsible for removal of uracil bases from DNA and initiates base excision repair pathways. Accumulation of uracil or its fluorinated analogs in DNA is one of the killing mechanisms of thymidylate synthase (TS) inhibitors in cancer cells, and depletion of UNG2 often enhances the toxicity of these anticancer drugs. We used CRISPR to knockout UNG2 from HT29 colon cancer cells and confirmed the absence of protein by western blot and uracil excision assays. We tested the effect of UNG2 KO on the efficacy of multiple TS inhibitors (5-fluorouracil, fluorodeoxyuridine, pemetrexed, and raltitrexed), and we determined that only fluorodeoxyuridine and raltitrexed were significantly more potent in UNG2 KO cells compared to wild-type HT29 cells (fluorodeoxyuridine IC50: 2 mM (wt) vs. 3 nM (KO); raltitrexed IC50: 14 nM (wt) vs. 2 nM (KO)). Interestingly, UNG2 protein levels can also be depleted by the HDAC inhibitors SAHA and MS275, providing a pharmacologic strategy to reduce UNG2 activity in cells. Unexpectedly, the HDAC inhibitors synergized with 5-fluorouracil, but not fluorodeoxyuridine, in both wild-type and UNG2-knockout cells. This suggested that HDAC inhibitors sensitized cells to 5-fluorouracil through an UNG2-independent mechanism. Moreover, cell death pathways activated by fluorodeoxyuridine and regulated by UNG2 activity are not sensitized by HDAC inhibitors. Our combined genetic and pharmacologic strategies targeting UNG2 activity in cells are defining cell death mechanisms for combination therapies of TS inhibitors and HDAC inhibitors. Future work will examine these drug combinations in additional cell lines to understand optimal therapeutic combinations and to further refine mechanisms of cell death.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3096
  43. Oncol Lett. 2022 Jun;23(6): 192
    Genome maintenance in retinoblastoma: Implications for therapeutic vulnerabilities.
    Chunsik Lee, Jong Kyong Kim.
      Retinoblastoma (RB) is a pediatric ocular malignancy that is initiated mostly by biallelic inactivation of the RB transcriptional corepressor 1 (RB1) tumor suppressor gene in the developing retina. Unlike the prevailing prediction based on multiple studies involving RB1 gene disruption in experimental models, human RB tumors have been demonstrated to possess a relatively stable genome, characterized by a low mutation rate and a few recurrent chromosomal alterations related to somatic copy number changes. This suggests that RB may harbor heightened genome maintenance mechanisms to counteract or compensate for the risk of massive genome instability, which can potentially be driven by the early RB1 loss as a tumor-initiating event. Although the genome maintenance mechanisms might have been evolved to promote RB cell survival by preventing lethal genomic defects, emerging evidence suggests that the dependency of RB cells on these mechanisms also exposes their unique vulnerability to chemotherapy, particularly when the genome maintenance machineries are tumor cell-specific. This review summarizes the genome maintenance mechanisms identified in RB, including findings on the roles of chromatin regulators in DNA damage response/repair and protein factors involved in maintaining chromosome stability and promoting survival in RB. In addition, advantages and challenges for exploiting these therapeutic vulnerabilities in RB are discussed.
    Keywords:  DNA damage response; DNA repair; RB1 deficiency; chemotherapy; chromatin; eye tumor; genome stability; retinoblastoma
    DOI:  https://doi.org/10.3892/ol.2022.13312
  44. Chem Res Toxicol. 2022 May 10.
    Human TREX1 Repairs 3'-End DNA Lesions in Vitro.
    Kun Yang, Xiaoying Wei, Jennifer Le, Nestor Rodriguez.
      Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,β-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.
    DOI:  https://doi.org/10.1021/acs.chemrestox.2c00087
  45. FASEB J. 2022 May;36 Suppl 1
    A Collapsed Fingers Subdomain is the Basis for DNA Polymerase β I260M Mutator Activity.
    Cristian G Chavira, Carel Fijen, Khadijeh Alnajjar, Joann Sweasy.
      DNA Polymerase β fills single nucleotide gaps as a part of the Base Excision Repair pathway (BER); thus, deficiencies in Pol β can lead to increased mutation frequency in the cell, which can result in cancer. Our lab has previously shown that the I260M germline mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide selectivity in a sequence context dependent manner, incorporating G opposite A. To identify the molecular mechanism of the reduced fidelity of I260M, we studied polymerization using single turnover kinetics, and conformational changes using steady state fluorescence and stopped flow FRET. Our data indicate that the I260M mutation affects the fingers region of Pol β by creating a "collapsed" state in both the opened (in the absence of nucleotide) and closed (prior to chemistry) states. Importantly, we show that the incorrect incoming nucleotide binds more tightly to I260M when compared to the wild-type by a factor of 3.4x. Based on this data, we found that the collapsed fingers subdomain state of I260M may decrease nucleotide discrimination in I260M, illustrating the importance of the "fingers closing" conformational change for polymerase fidelity. A model is being developed to compare the rate of the "fingers closing" conformational change and the reverse reaction of nucleotide release between I260M and the WT.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R343
  46. FEBS Lett. 2022 May 09.
    LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.
    Noëlle Dommann, Jacopo Gavini, Daniel Sánchez-Taltavull, Felix Alexander Baier, Fabienne Birrer, Giulio Loforese, Daniel Candinas, Deborah Stroka.
      The LIM-domain protein Ajuba is associated with cell proliferation, a fundamental process of tissue regeneration and cancer. We report that in the liver, Ajuba expression is increased during regeneration and in tumor cells and tissues. Knockout of Ajuba using CRISPR/Cas9 is embryonic lethal in mice. shRNA targeting of Ajuba reduces cell proliferation, delays cell entry into S-phase, reduces cell survival and tumor growth in vivo, and increases expression of the DNA damage marker γH2AX. Ajuba binding partners include proteins involved in DNA replication and damage, such as SKP2, MCM2, MCM7 and RPA70. Taken together, our data support that Ajuba promotes liver cell proliferation associated with development, regeneration, and tumor growth and is involved in DNA replication and damage repair.
    DOI:  https://doi.org/10.1002/1873-3468.14371
  47. FASEB J. 2022 May;36 Suppl 1
    Inhibition of SUMO-mediated Protein-Protein Interactions in DNA Damage Repair.
    Emily E Jones, Yee Mon Thu.
      Maintenance of genomic integrity requires functional repair mechanisms. Some mechanisms include the use of sumoylation, a process in which proteins are post-translationally modified with a small peptide SUMO (small ubiquitin-like modifier). In response to DNA damage, multiple proteins become sumoylated. Current evidence suggests that sumoylation in this context mediates protein-protein interactions to promote DNA damage repair. SUMO-interacting motifs (SIMs) play an essential role in SUMO-mediated protein-protein interactions. The SIM on a protein is a region that allows it to recognize and bind the SUMO attached to another protein. Previous studies have shown that inhibiting this type of interaction can lead to sensitized cancer cells by impairing DNA damage repair and response. Thus, this project aims to study a similar inhibition by overexpressing SIMs naturally found in two proteins, Slx5 and Sgs1, in the eukaryotic model organism, Saccharomyces cerevisiae. Both proteins are a part of their respective protein complexes, and their SIMs serve as a functional way for the complexes to interact with sumoylated proteins during DNA repair. We hypothesize that overexpressed SIMs will disrupt endogenous SUMO-SIM interactions important for DNA damage repair and that cells with overexpression of the SIM would be sensitized to DNA damaging agents. We observed that cells overexpressing SIMs from Slx5 exhibited a growth defect compared to cells without overexpression. However, when treated with DNA damaging agents, such as methyl methanesulfonate and UV, cells with SIM overexpression exhibited little to no sensitivity compared to the wild type. To further explore the effect of SUMO-SIM interaction, we have designed another protein containing the DNA-binding (SAP) domain of Siz2, a SUMO protein ligase specific to DNA damage, and the SUMO-binding domain of Ulp1, a SUMO isopeptidase, to potentially disrupt SUMO:SIM interactions involved in DNA repair. Inclusion of the SAP domain along with a nuclear localization signal (NLS) is hypothesized to increase the nuclear localization of the protein during DNA repair, which will be observed using fluorescent microscopy of a GFP tag incorporated into the protein. With increased nuclear localization and inclusion of the SUMO-binding domain, we hypothesize that there will be disruption to the SUMO:SIM interactions involved in DNA damage repair, resulting in an increase in the cells' sensitivity to DNA damaging agents.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2544
  48. FASEB J. 2022 May;36 Suppl 1
    Biochemical Characterization of a Human Ribonucleotide Reductase (HsRNR) Variant Lacking Activity Regulation.
    Paulina Rios, Gerardo Perez Goncalves, Kelsey R Miller, Catherine Drennan.
      Ribonucleotide reductase (RNR) is responsible for the synthesis of deoxyribonucleotides used in DNA synthesis and repair. Class Ia RNRs are composed of two dimeric subunits: a catalytic subunit (α2 ) which contains the active site and two allosteric regulation sites, and radical generating (β2 ) subunit housing the diferric-tyrosyl radical cofactor. The two subunits form an α2 β2 heterotetrametric complex that allows the radical to travel from β to α subunit for catalysis. RNR activity is regulated by the binding of ATP/dATP to an N-terminal cone domain. Human RNRs (HsRNRs) are class Ia enzymes that form a homohexameric α6 in the presence of either dATP or ATP. Although, both α6 assemblies are morphologically identical, only the α6 -ATP can be disturbed by the addition of β to form the α2 β2 complex necessary for catalysis. Recent studies have shown that α subunits contact each other via their cone domains in the α6 rings, but it is unknown if the cone domains contact β2 in the α2 β2 active complex. Here, we aim to probe the role of the cone domains in catalysis through biochemical and biophysical studies of an α subunit variant lacking the cone domain. We used site-directed mutagenesis to delete residues 2-92 of α, and expressed and purified the variant a protein. The variant protein was used for HPLC-MS based activity assays of HsRNR. Preliminary results show the cone domain is important for catalysis. Understanding more about RNR's structure and activity regulation can facilitate development of new cancer therapies.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R354
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