bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020–05–31
47 papers selected by
Sean Rudd, Karolinska Institutet
  1. Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication.
  2. Biomarker-Guided Development of DNA Repair Inhibitors.
  3. DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status.
  4. CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.
  5. Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.
  6. Combined TP53 and RB1 Loss Promotes Prostate Cancer Resistance to a Spectrum of Therapeutics and Confers Vulnerability to Replication Stress.
  7. Genome Maintenance by DNA Helicase B.
  8. Preemptive Homology-Directed DNA Repair Fosters Complex Genomic Rearrangements in Hepatocellular Carcinoma.
  9. Interaction of Deubiquitinase 2A-DUB/MYSM1 with DNA Repair and Replication Factors.
  10. The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links.
  11. H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres.
  12. Gene expression regulation by CDK12: a versatile kinase in cancer with functions beyond CTD phosphorylation.
  13. HTS-Compatible CometChip Enables Genetic Screening for Modulators of Apoptosis and DNA Double-Strand Break Repair.
  14. The bromodomain containing protein BRD-9 orchestrates RAD51-RAD54 complex formation and regulates homologous recombination-mediated repair.
  15. EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair.
  16. RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia.
  17. HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response.
  18. NHJ-1 Is Required for Canonical Non-homologous End Joining in Caenorhabditis elegans.
  19. MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair.
  20. Effects of Ochratoxin A exposure on DNA damage in porcine granulosa cells in vitro.
  21. c‑Abl‑mediated tyrosine phosphorylation of DNA damage response proteins and implications in important cellular functions (Review).
  22. Ubiquitin-like proteins in the DNA damage response: the next generation.
  23. Clinical Implications of DNA Repair Defects in High-Grade Serous Ovarian Carcinomas.
  24. CD147 promotes DNA damage response and gemcitabine resistance via targeting ATM/ATR/p53 and affects prognosis in pancreatic cancer.
  25. Resolving DNA Damage: Epigenetic Regulation of DNA Repair.
  26. Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer.
  27. Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.
  28. Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network.
  29. Emerging strategies to target cancer metabolism and improve radiation therapy outcomes.
  30. Tumour predisposition and cancer syndromes as models to study gene-environment interactions.
  31. The BCL-2 pathway preserves mammalian genome integrity by eliminating recombination-defective oocytes.
  32. DNA damage invokes mitophagy through a pathway involving Spata18.
  33. Targeted Regulation of Nuclear Lamins by Ubiquitin and Ubiquitin-Like Modifiers.
  34. A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.
  35. Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis.
  36. Metabolic cofactors NADH and FAD act as non-canonical initiating substrates for a primase and affect replication primer processing in vitro.
  37. Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.
  38. Inhibition of ubiquitin-specific protease 7 sensitizes acute myeloid leukemia to chemotherapy.
  39. Freedom of assembly: metabolic enzymes come together.
  40. Molecular and structural characterization of disease-associated APE1 polymorphisms.
  41. CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes.
  42. Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes.
  43. Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates.
  44. Pemetrexed Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication through Blocking dTMP Synthesis.
  45. Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.
  46. Human equilibrative nucleoside transporter-1 (hENT1) and ribonucleotide reductase regulatory subunit M1 (RRM1) expression; do they have survival impact to pancreatic cancer?
  47. Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp.
  1. Cell Rep. 2020 May 26. pii: S2211-1247(20)30628-8. [Epub ahead of print]31(8): 107675
    Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication.
    Roberto Bellelli, Jillian Youds, Valerie Borel, Jennifer Svendsen, Visnja Pavicic-Kaltenbrunner, Simon J Boulton.
      Genome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans. Through a genome-wide synthetic lethal screen, we uncovered an essential genetic interaction between RTEL1 and DNA polymerase (Pol) epsilon. Loss of POLE4, an accessory subunit of Pol epsilon, has no overt phenotype in worms. In contrast, the combined loss of POLE-4 and RTEL-1 results in embryonic lethality, accumulation of HR intermediates, genome instability, and cessation of DNA replication. Similarly, loss of Rtel1 in Pole4-/- mouse cells inhibits cellular proliferation, which is associated with persistent HR intermediates and incomplete DNA replication. We propose that RTEL1 facilitates genome-wide fork progression through its ability to metabolize DNA secondary structures that form during DNA replication. Loss of this function becomes incompatible with cell survival under conditions of reduced origin activation, such as Pol epsilon hypomorphy.
    Keywords:  DNA polymerase epsilon; DNA replication; RTEL1; genome stability; origin activation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107675
  2. Mol Cell. 2020 May 17. pii: S1097-2765(20)30277-X. [Epub ahead of print]
    Biomarker-Guided Development of DNA Repair Inhibitors.
    James M Cleary, Andrew J Aguirre, Geoffrey I Shapiro, Alan D D'Andrea.
      Anti-cancer drugs targeting the DNA damage response (DDR) exploit genetic or functional defects in this pathway through synthetic lethal mechanisms. For example, defects in homologous recombination (HR) repair arise in cancer cells through inherited or acquired mutations in BRCA1, BRCA2, or other genes in the Fanconi anemia/BRCA pathway, and these tumors have been shown to be particularly sensitive to inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP). Recent work has identified additional genomic and functional assays of DNA repair that provide new predictive and pharmacodynamic biomarkers for these targeted therapies. Here, we examine the development of selective agents targeting DNA repair, including PARP inhibitors; inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM); and inhibitors of classical non-homologous end joining (cNHEJ) and alternative end joining (Alt EJ). We also review the biomarkers that guide the use of these agents and current clinical trials with these therapies.
    Keywords:  DNA repair; PARP inhibitor; cell-cycle kinases; polymerase theta
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.035
  3. Mol Cancer Res. 2020 May 28. pii: molcanres.1051.2019. [Epub ahead of print]
    DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status.
    Yuki Kataoka, Makoto Iimori, Ryo Fujisawa, Tomomi Morikawa-Ichinose, Shinichiro Niimi, Takeshi Wakasa, Hiroshi Saeki, Eiji Oki, Daisuke Miura, Toshiki Tsurimoto, Yoshihiko Maehara, Hiroyuki Kitao.
      DNA replication stress (DRS) is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DRS in tumor cells. However, the mechanisms underlying the anti-tumor effect of these drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases by acting both as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand, which caused DRS. In cells, FTD decreased the thymidine triphosphate level in the dNTP pool and increased the FTD triphosphate level, resulting in the activation of DRS-induced cellular responses during S phase. Additionally, replication protein A-coated single-stranded DNA associated with FancD2 and accumulated after tumor cells completed S phase. Finally, FTD activated the p53-p21 pathway and suppressed tumor cell growth by inducing cellular senescence via mitosis skipping. By contrast, tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results demonstrate that DRS induced by a nucleoside analog-type chemotherapeutic drug suppresses tumor growth irrespective of p53 status by directing tumor cell fate toward cellular senescence or apoptotic cell death according to p53 status. Implications: Chemotherapeutic drugs that increase DRS during S phase but allow tumor cells to complete S phase may have significant anti-tumor activity even when functional p53 is lost.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-19-1051
  4. Nat Commun. 2020 May 26. 11(1): 2641
    CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.
    Elaine Sanij, Katherine M Hannan, Jiachen Xuan, Shunfei Yan, Jessica E Ahern, Anna S Trigos, Natalie Brajanovski, Jinbae Son, Keefe T Chan, Olga Kondrashova, Elizabeth Lieschke, Matthew J Wakefield, Daniel Frank, Sarah Ellis, Carleen Cullinane, Jian Kang, Gretchen Poortinga, Purba Nag, Andrew J Deans, Kum Kum Khanna, Linda Mileshkin, Grant A McArthur, John Soong, Els M J J Berns, Ross D Hannan, Clare L Scott, Karen E Sheppard, Richard B Pearson.
      Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
    DOI:  https://doi.org/10.1038/s41467-020-16393-4
  5. J Exp Med. 2020 Aug 03. pii: e20191779. [Epub ahead of print]217(8):
    Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.
    Qingxin Zhou, Meihua Lin, Xing Feng, Fei Ma, Yuekun Zhu, Xing Liu, Chao Qu, Hong Sui, Bei Sun, Anlong Zhu, Heng Zhang, He Huang, Zhi Gao, Yongxiang Zhao, Jiangyun Sun, Yuxian Bai, Junfei Jin, Xuehui Hong, Chang Zou, Zhiyong Zhang.
      CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility.
    DOI:  https://doi.org/10.1084/jem.20191779
  6. Cell Rep. 2020 May 26. pii: S2211-1247(20)30622-7. [Epub ahead of print]31(8): 107669
    Combined TP53 and RB1 Loss Promotes Prostate Cancer Resistance to a Spectrum of Therapeutics and Confers Vulnerability to Replication Stress.
    Michael D Nyquist, Alexandra Corella, Ilsa Coleman, Navonil De Sarkar, Arja Kaipainen, Gavin Ha, Roman Gulati, Lisa Ang, Payel Chatterjee, Jared Lucas, Colin Pritchard, Gail Risbridger, John Isaacs, Bruce Montgomery, Colm Morrissey, Eva Corey, Peter S Nelson.
      Prostate cancers (PCs) with loss of the potent tumor suppressors TP53 and RB1 exhibit poor outcomes. TP53 and RB1 also influence cell plasticity and are frequently lost in PCs with neuroendocrine (NE) differentiation. Therapeutic strategies that address these aggressive variant PCs are urgently needed. Using deep genomic profiling of 410 metastatic biopsies, we determine the relationships between combined TP53 and RB1 loss and PC phenotypes. Notably, 40% of TP53/RB1-deficient tumors are classified as AR-active adenocarcinomas, indicating that NE differentiation is not an obligate consequence of TP53/RB1 inactivation. A gene expression signature reflecting TP53/RB1 loss is associated with diminished responses to AR antagonists and reduced survival. These tumors exhibit high proliferation rates and evidence of elevated DNA repair processes. While tumor cells lacking TP53/RB1 are highly resistant to all single-agent therapeutics tested, the combination of PARP and ATR inhibition is found to produce significant responses, reflecting a clinically exploitable vulnerability resulting from replication stress.
    Keywords:  ATR; DNA damage; PARP; RB1; TP53; androgen receptor; antiandrogen; neuroendocrine; plasticity; prostate cancer
    DOI:  https://doi.org/10.1016/j.celrep.2020.107669
  7. Genes (Basel). 2020 May 21. pii: E578. [Epub ahead of print]11(5):
    Genome Maintenance by DNA Helicase B.
    Lindsey Hazeslip, Maroof Khan Zafar, Muhammad Zain Chauhan, Alicia K Byrd.
      DNA Helicase B (HELB) is a conserved helicase in higher eukaryotes with roles in the initiation of DNA replication and in the DNA damage and replication stress responses. HELB is a predominately nuclear protein in G1 phase where it is involved in initiation of DNA replication through interactions with DNA topoisomerase 2-binding protein 1 (TOPBP1), cell division control protein 45 (CDC45), and DNA polymerase α-primase. HELB also inhibits homologous recombination by reducing long-range end resection. After phosphorylation by cyclin-dependent kinase 2 (CDK2) at the G1 to S transition, HELB is predominately localized to the cytosol. However, this cytosolic localization in S phase is not exclusive. HELB has been reported to localize to chromatin in response to replication stress and to localize to the common fragile sites 16D (FRA16D) and 3B (FRA3B) and the rare fragile site XA (FRAXA) in S phase. In addition, HELB is phosphorylated in response to ionizing radiation and has been shown to localize to chromatin in response to various types of DNA damage, suggesting it has a role in the DNA damage response.
    Keywords:  DNA damage; DNA helicase; DNA repair; DNA replication; genomic stability
    DOI:  https://doi.org/10.3390/genes11050578
  8. Transl Oncol. 2020 May 22. pii: S1936-5233(20)30028-0. [Epub ahead of print]13(9): 100796
    Preemptive Homology-Directed DNA Repair Fosters Complex Genomic Rearrangements in Hepatocellular Carcinoma.
    Shirley Ming-Hui Sy, Yingying Guo, Ying Lan, Howin Ng, Michael Shing-Yan Huen.
      Degree of genomic instability closely correlates with poor prognosis, drug resistance as well as poor survival across human cancer of different origins. This study assessed the relationship between DNA damage response (DDR) and chromosome instability in hepatocellular carcinoma (HCC). We investigated DDR signaling in HCC cells by analyzing DNA damage-dependent redistribution of major DDR proteins to damaged chromatin using immunofluorescence microscopy and Western blotting experimentations. We also performed gene conversion and metaphase analyses to address whether dysregulated DDR may bear any biological significance during hepatocarcinogenesis. Accordingly, we found that HCC cell lines suffered from elevated spontaneous DNA double-strand breaks (DSBs). In addition, analyses of HCC metaphases revealed marked aneuploidy and frequent sister chromatid exchanges when compared to immortalized hepatocytes, the latter of which were further induced following camptothecin-induced DSBs. We propose that genomic instability in HCC may be caused by erroneous DNA repair in a desperate attempt to mend DSBs for cell survival and that such preemptive measures inadvertently foster chromosome instability and thus complex genomic rearrangements.
    DOI:  https://doi.org/10.1016/j.tranon.2020.100796
  9. Int J Mol Sci. 2020 May 26. pii: E3762. [Epub ahead of print]21(11):
    Interaction of Deubiquitinase 2A-DUB/MYSM1 with DNA Repair and Replication Factors.
    Carsten Kroeger, Reinhild Roesler, Sebastian Wiese, Adelheid Hainzl, Martina Vanessa Gatzka.
      The deubiquitination of histone H2A on lysine 119 by 2A-DUB/MYSM1, BAP1, USP16, and other enzymes is required for key cellular processes, including transcriptional activation, apoptosis, and cell cycle control, during normal hematopoiesis and tissue development, and in tumor cells. Based on our finding that MYSM1 colocalizes with γH2AX foci in human peripheral blood mononuclear cells, leukemia cells, and melanoma cells upon induction of DNA double-strand breaks with topoisomerase inhibitor etoposide, we applied a mass spectrometry-based proteomics approach to identify novel 2A-DUB/MYSM1 interaction partners in DNA-damage responses. Differential display of MYSM1 binding proteins significantly enriched after exposure of 293T cells to etoposide revealed an interacting network of proteins involved in DNA damage and replication, including factors associated with poor melanoma outcome. In the context of increased DNA-damage in a variety of cell types in Mysm1-deficient mice, in bone marrow cells upon aging and in UV-exposed Mysm1-deficient skin, our current mass spectrometry data provide additional evidence for an interaction between MYSM1 and key DNA replication and repair factors, and indicate a potential function of 2A-DUB/MYSM1 in DNA repair processes.
    Keywords:  DNA damage; HELLS; MYSM1; apoptosis; cancer; histone deubiquitinase; homologous recombination (HR); melanoma; p53; γH2AX
    DOI:  https://doi.org/10.3390/ijms21113762
  10. Genes (Basel). 2020 May 25. pii: E585. [Epub ahead of print]11(5):
    The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links.
    Xavier Renaudin, Filippo Rosselli.
      DNA interstrand cross-links (ICLs) represent a major barrier blocking DNA replication fork progression. ICL accumulation results in growth arrest and cell death-particularly in cell populations undergoing high replicative activity, such as cancer and leukemic cells. For this reason, agents able to induce DNA ICLs are widely used as chemotherapeutic drugs. However, ICLs are also generated in cells as byproducts of normal metabolic activities. Therefore, every cell must be capable of rescuing lCL-stalled replication forks while maintaining the genetic stability of the daughter cells in order to survive, replicate DNA and segregate chromosomes at mitosis. Inactivation of the Fanconi anemia/breast cancer-associated (FANC/BRCA) pathway by inherited mutations leads to Fanconi anemia (FA), a rare developmental, cancer-predisposing and chromosome-fragility syndrome. FANC/BRCA is the key hub for a complex and wide network of proteins that-upon rescuing ICL-stalled DNA replication forks-allows cell survival. Understanding how cells cope with ICLs is mandatory to ameliorate ICL-based anticancer therapies and provide the molecular basis to prevent or bypass cancer drug resistance. Here, we review our state-of-the-art understanding of the mechanisms involved in ICL resolution during DNA synthesis, with a major focus on how the FANC/BRCA pathway ensures DNA strand opening and prevents genomic instability.
    Keywords:  DNA repair; FANC/BRCA pathway; genomic instability; interstrand cross-link (ICL)
    DOI:  https://doi.org/10.3390/genes11050585
  11. Oncogene. 2020 May 29.
    H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres.
    Inge de Krijger, Jaco van der Torre, Marieke H Peuscher, Mathias Eder, Jacqueline J L Jacobs.
      The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.
    DOI:  https://doi.org/10.1038/s41388-020-1334-0
  12. Exp Mol Med. 2020 May 25.
    Gene expression regulation by CDK12: a versatile kinase in cancer with functions beyond CTD phosphorylation.
    Seung Hyuk Choi, Seongjae Kim, Katherine A Jones.
      Cyclin-dependent kinases (CDKs) play critical roles in cell cycle progression and gene expression regulation. In human cancer, transcription-associated CDKs can activate oncogenic gene expression programs, whereas cell cycle-regulatory CDKs mainly induce uncontrolled proliferation. Cyclin-dependent kinase 12 (CDK12) belongs to the CDK family of serine/threonine kinases and has been recently found to have multiple roles in gene expression regulation and tumorigenesis. Originally, CDK12 was thought to be one of the transcription-associated CDKs, acting with its cyclin partner Cyclin K to promote the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II and induce transcription elongation. However, recent studies have demonstrated that CDK12 also controls multiple gene expression processes, including transcription termination, mRNA splicing, and translation. Most importantly, CDK12 mutations are frequently found in human tumors. Loss of CDK12 function causes defective expression of DNA damage response (DDR) genes, which eventually results in genome instability, a hallmark of human cancer. Here, we discuss the diverse roles of CDK12 in gene expression regulation and human cancer, focusing on newly identified CDK12 kinase functions in cellular processes and highlighting CDK12 as a promising therapeutic target for human cancer treatment.
    DOI:  https://doi.org/10.1038/s12276-020-0442-9
  13. SLAS Discov. 2020 May 26. 2472555220918367
    HTS-Compatible CometChip Enables Genetic Screening for Modulators of Apoptosis and DNA Double-Strand Break Repair.
    Ian J Tay, James J H Park, Anna L Price, Bevin P Engelward, Scott R Floyd.
      Dysfunction of apoptosis and DNA damage response pathways often drive cancer, and so a better understanding of these pathways can contribute to new cancer therapeutic strategies. Diverse discovery approaches have identified many apoptosis regulators, DNA damage response, and DNA damage repair proteins; however, many of these approaches rely on indirect detection of DNA damage. Here, we describe a novel discovery platform based on the comet assay that leverages previous technical advances in assay precision by incorporating high-throughput robotics. The high-throughput screening (HTS) CometChip is the first high-throughput-compatible assay that can directly detect physical damage in DNA. We focused on DNA double-strand breaks (DSBs) and utilized our HTS CometChip technology to perform a first-of-its-kind screen using an shRNA library targeting 2564 cancer-relevant genes. Conditions of the assay enable detection of DNA fragmentation from both exogenous (ionizing radiation) and endogenous (apoptosis) sources. Using this approach, we identified LATS2 as a novel DNA repair factor as well as a modulator of apoptosis. We conclude that the HTS CometChip is an effective assay for HTS to identify modulators of physical DNA damage and repair.
    Keywords:  Comet assay; DNA double-strand breaks; Hippo pathway; LATS2; RNA interference; high-throughput screening; homologous recombination; ionizing radiation; nonhomologous end joining
    DOI:  https://doi.org/10.1177/2472555220918367
  14. Nat Commun. 2020 May 26. 11(1): 2639
    The bromodomain containing protein BRD-9 orchestrates RAD51-RAD54 complex formation and regulates homologous recombination-mediated repair.
    Qin Zhou, Jinzhou Huang, Chao Zhang, Fei Zhao, Wootae Kim, Xinyi Tu, Yong Zhang, Somaira Nowsheen, Qian Zhu, Min Deng, Yuping Chen, Bo Qin, Kuntian Luo, Baohua Liu, Zhenkun Lou, Robert W Mutter, Jian Yuan.
      Homologous recombination (HR) is important for error-free DNA double strand break repair and maintenance of genomic stability. However, upregulated HR is also used by cancer cells to promote therapeutic resistance. Therefore, inducing HR deficiency (HRD) is a viable strategy to sensitize HR proficient cancers to DNA targeted therapies in order to overcome therapeutic resistance. A bromodomain containing protein, BRD9, was previously reported to regulate chromatin remodeling and transcription. Here, we discover that following DNA damage, the bromodomain of BRD9 binds acetylated K515 on RAD54 and facilitates RAD54's interaction with RAD51, which is essential for HR. BRD9 is overexpressed in ovarian cancer and depleting BRD9 sensitizes cancer cells to olaparib and cisplatin. In addition, inhibitor of BRD9, I-BRD9, acts synergistically with olaparib in HR-proficient cancer cells. Overall, our results elucidate a role for BRD9 in HR and identify BRD9 as a potential therapeutic target to promote synthetic lethality and overcome chemoresistance.
    DOI:  https://doi.org/10.1038/s41467-020-16443-x
  15. Oncogene. 2020 May 26.
    EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair.
    Allyson E Koyen, Matthew Z Madden, Dongkyoo Park, Elizabeth V Minten, Priya Kapoor-Vazirani, Erica Werner, Neil T Pfister, Ramona Haji-Seyed-Javadi, Hui Zhang, Jie Xu, Nikita Deng, Duc M Duong, Turner J Pecen, Zoë Frazier, Zachary D Nagel, Jean-Bernard Lazaro, Kent W Mouw, Nicholas T Seyfried, Carlos S Moreno, Taofeek K Owonikoko, Xingming Deng, David S Yu.
      Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to cisplatin-based chemotherapy. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which silences transcription through trimethylation of histone H3 lysine 27 (H3K27me3) and has emerged as an important therapeutic target with inhibitors targeting its methyltransferase activity under clinical investigation. Here, we show that EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and govern cisplatin resistance in SCLC. Using a synthetic lethality screen, we identified important regulators of cisplatin resistance in SCLC cells, including EZH2. EZH2 depletion causes cellular cisplatin and UV hypersensitivity in an epistatic manner with DDB1-DDB2. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC.
    DOI:  https://doi.org/10.1038/s41388-020-1332-2
  16. PLoS Pathog. 2020 May 26. 16(5): e1008618
    RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia.
    Huijun Zhi, Xin Guo, Yik-Khuan Ho, Nagesh Pasupala, Hampus Alexander Anders Engstrom, Oliver John Semmes, Chou-Zen Giam.
      The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.
    DOI:  https://doi.org/10.1371/journal.ppat.1008618
  17. Cell Death Dis. 2020 May 26. 11(5): 400
    HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response.
    Zongpei Guo, Shaozheng Wang, Ying Xie, Yang Han, Sai Hu, Hua Guan, Dafei Xie, Chenjun Bai, Xiaodan Liu, Yongqing Gu, Ping-Kun Zhou, Teng Ma.
      DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.
    DOI:  https://doi.org/10.1038/s41419-020-2611-0
  18. Genetics. 2020 May 26. pii: genetics.303328.2020. [Epub ahead of print]
    NHJ-1 Is Required for Canonical Non-homologous End Joining in Caenorhabditis elegans.
    Aleksandar Vujin, Steven J Jones, Monique Zetka.
      DNA double-strand breaks (DSBs) are a particularly lethal form of DNA damage that must be repaired to restore genomic integrity. Canonical non-homologous end joining (NHEJ), is the widely conserved pathway that detects and directly ligates the broken ends to repair the DSB. These events globally require the two proteins that form the Ku ring complex, Ku70 and Ku80, and the terminal ligase Lig4. While the NHEJ pathway in vertebrates is elaborated by more than a dozen factors of varying conservation and is similarly complex in other eukaryotes, the entire known NHEJ toolkit in Caenorhabditis elegans consists only of the core components CKU-70, CKU-80, and LIG-4. Here, we report the discovery of new NHEJ factor in C. elegans Our analysis of the DDR in young larvae revealed that the canonical wild-type N2 strain consisted of two lines that exhibited a differential response phenotypic to ionizing radiation (IR). Following the mapping of the causative locus to a candidate on chromosome V and CRISPR-Cas9 mutagenesis, we show that disruption of the nhj-1 sequence induces IR-sensitivity in the N2 line that previously exhibited IR resistance. Using genetic and cytological analyses, we demonstrate that nhj-1 functions in the NHEJ pathway to repair DSBs. Double mutants of nhj-1 and lig-4 or cku-80 do not exhibit additive IR-sensitivity and the post-IR somatic and fertility phenotypes of nhj-1 mimic those of the other NHEJ factors. Furthermore, in com-1 mutants that permit repair of meiotic DSBs by NHEJ instead of restricting their repair to the homologous recombination (HR) pathway, loss of nhj-1 mimics the consequences of loss of lig-4. Diakinesis-stage nuclei in nhj-1; com-1 and nhj-1; lig-4 mutant germlines exhibit increased numbers of DAPI-staining bodies, consistent with increased chromosome fragmentation in the absence of NHEJ-mediated meiotic DSB repair. Finally, we show that NHJ-1 and LIG-4 localize to somatic nuclei in larvae, but are excluded from the germline progenitor cells, consistent with NHEJ being the dominant DNA repair pathway in the soma. nhj-1 shares no sequence homology with other known eukaryotic NHEJ factors and is taxonomically restricted to the Rhabditid family, underscoring the evolutionary plasticity of even highly conserved pathways.
    Keywords:  C. elegans; DNA repair; nonhomologous end joining
    DOI:  https://doi.org/10.1534/genetics.120.303328
  19. DNA Repair (Amst). 2020 May 21. pii: S1568-7864(20)30118-X. [Epub ahead of print]91-92 102870
    MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair.
    Ingrid L Berg, Jan-Olov Persson, Stefan U Åström.
      By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutSα complex, partially suppressed the MMS super-sensitivity of the rad5Δ strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2Δ, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5Δ, showing that the suppression was specific for disabling MutSα. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAG1) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutSα.
    Keywords:  DNA damage tolerance; Msh2; Postreplication repair; Rad5; Template switch
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102870
  20. Toxicol Lett. 2020 May 23. pii: S0378-4274(20)30144-2. [Epub ahead of print]
    Effects of Ochratoxin A exposure on DNA damage in porcine granulosa cells in vitro.
    Tian-Yu Zhang, Li Kong, Jia-Xing Hao, Han Wang, Zi-Hui Yan, Xiao-Feng Sun, Wei Shen.
      Ochratoxin A (OTA), a feed mycotoxin, tends to impair the reproductive performance of animals. Our previous studies have demonstrated that OTA exposure inhibits porcine ovarian granulosa cell (GC) proliferation and induces their apoptosis, but the underlying toxic mechanism is still uncertain. In this study, we explored the OTA exposure on porcine GCs in vitro and found that OTA exposure inhibited the proliferation of porcine GCs and arrested cell cycle of GCs in the G2/M phase. The results based on RNA-Seq revealed that 20 μM and 40 μM OTA exposure increase DNA damage of porcine GCs in vitro. The differentially expressed genes (DEGs) of 40 μM OTA exposure were enriched in the pathways of mismatch repair, nucleotide excision repair and homologous recombination in DNA replication compared with control group and 20 μM OTA exposure group. Meanwhile, OTA exposure increased the expression levels of DNA double-strand breaks (DSBs) gene γ-H2AX, and DNA repair related genes, such as BRCA1, XRCC1, PARP1, and RAD51. Above all, our research revealed that OTA might exert deleterious effects on porcine ovarian GCs, influencing DNA repair-related biological processes and causing DNA damage response.
    Keywords:  DNA damage; Ochratoxin A; Porcine; RNA-Seq; granulosa cells
    DOI:  https://doi.org/10.1016/j.toxlet.2020.05.011
  21. Mol Med Rep. 2020 May 18.
    c‑Abl‑mediated tyrosine phosphorylation of DNA damage response proteins and implications in important cellular functions (Review).
    Ameer Ali Bohio, Ruoxi Wang, Xianlu Zeng, Xueqing Ba.
      Tyrosine phosphorylation is an essential post‑translational protein modification catalyzed by tyrosine kinases. c‑Abl is a crucial non‑receptor tyrosine kinase, which is most commonly activated by auto‑phosphorylation, DNA damage and by interacting with other protein kinases. DNA damage response (DDR) proteins stimulated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. The fundamental roles of activated c‑Abl tyrosine kinase in cellular response pathways have been intensively and extensively investigated and in recent years, a number of c‑Abl protein binding partners have been determined; however, the functional roles of these molecules remain to be determined. The present review aimed to summarize the DDR proteins phosphorylated by c‑Abl tyrosine kinase that have been identified to date, in addition to the functional outcomes of these phosphotyrosine events. Notably, it has been discovered that c‑Abl tyrosine kinase can bind with and phosphorylate DDR proteins at different tyrosine sites, which serve distinct roles in various cellular contexts.
    DOI:  https://doi.org/10.3892/mmr.2020.11156
  22. Essays Biochem. 2020 May 26. pii: EBC20190095. [Epub ahead of print]
    Ubiquitin-like proteins in the DNA damage response: the next generation.
    Isabelle C Da Costa, Christine K Schmidt.
      DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.
    Keywords:  DNA damage response (DDR); DNA repair; FAT10; ISG15; UBL5; UFM1; Ubiquitin-like proteins (UBLs)
    DOI:  https://doi.org/10.1042/EBC20190095
  23. Cancers (Basel). 2020 May 21. pii: E1315. [Epub ahead of print]12(5):
    Clinical Implications of DNA Repair Defects in High-Grade Serous Ovarian Carcinomas.
    Michela Camilla Milanesio, Silvia Giordano, Giorgio Valabrega.
      Despite significant improvements in surgical and medical management, high grade serous ovarian cancer (HGSOC) still represents the deadliest gynecologic malignancy and the fifth most frequent cause of cancer-related mortality in women in the USA. Since DNA repair alterations are regarded as the "the Achille's heel" of HGSOC, both DNA homologous recombination and DNA mismatch repair deficiencies have been explored and targeted in epithelial ovarian cancers in the latest years. In this review, we aim at focusing on the therapeutic issues deriving from a faulty DNA repair machinery in epithelial ovarian cancers, starting from existing and well-established treatments and investigating new therapeutic approaches which could possibly improve ovarian cancer patients' survival outcomes in the near future. In particular, we concentrate on the role of both Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPis) and immune checkpoint inhibitors in HGSOC, highlighting their activity in relation to BRCA1/2 mutational status and homologous recombination deficiency (HRD). We investigate the biological rationale supporting their use in the clinical setting, pointing at tracking their route from the laboratory bench to the patient's bedside. Finally, we deal with the onset of mechanisms of primary and acquired resistance to PARPis, reporting the pioneering strategies aimed at converting homologous-recombination (HR) proficient tumors into homologous recombination (HR)-deficient HGSOC.
    Keywords:  BRCA reversion mutations; DNA homologous recombination; DNA mismatch repair; DNA repair deficiency; PARP inhibitors; epithelial ovarian cancer
    DOI:  https://doi.org/10.3390/cancers12051315
  24. Biochem Biophys Res Commun. 2020 May 23. pii: S0006-291X(20)30908-6. [Epub ahead of print]
    CD147 promotes DNA damage response and gemcitabine resistance via targeting ATM/ATR/p53 and affects prognosis in pancreatic cancer.
    Yinghui Zhou, Ming Zheng, Zhenyu Liu, Haijiao Yang, Ping Zhu, Jian-Li Jiang, Juan Tang, Zhi-Nan Chen.
      The acquisition of chemoresistance is a major clinical challenge for pancreatic cancer (PC) treatment. Chemoresistance is largely attributed to aberrant DNA damage repair. However, the underlying mechanisms of chemoresistance in pancreatic cancer remain unclear. Here, we showed that CD147 was strongly correlated to DNA damage response (DDR) indices and poor prognosis in pancreatic ductal adenocarcinoma (PDAC) patients. CD147 knockdown or monoclonal antibodies improved the killing effects of gemcitabine in gemcitabine resistant cells, exhibiting reduced activation of ATM/p53. Moreover, we found the interaction of CD147 with ATM, ATR and p53, which was augmented in gemcitabine resistant cells. High CD147/p-ATM/p-ATR/p-p53 cytoplasmic expression associated with poor survival of PC patients. Our studies thus identify CD147 as a critical player in DDR programing that affects gemcitabine therapeutic outcomes of pancreatic cancer patients.
    Keywords:  CD147 antigens; DNA damage; Drug resistance; Gemcitabine; Pancreatic ductal adenocarcinoma; Prognosis
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.005
  25. Molecules. 2020 May 27. pii: E2496. [Epub ahead of print]25(11):
    Resolving DNA Damage: Epigenetic Regulation of DNA Repair.
    Panagiotis Karakaidos, Dimitris Karagiannis, Theodoros Rampias.
      Epigenetic research has rapidly evolved into a dynamic field of genome biology. Chromatin regulation has been proved to be an essential aspect for all genomic processes, including DNA repair. Chromatin structure is modified by enzymes and factors that deposit, erase, and interact with epigenetic marks such as DNA and histone modifications, as well as by complexes that remodel nucleosomes. In this review we discuss recent advances on how the chromatin state is modulated during this multi-step process of damage recognition, signaling, and repair. Moreover, we examine how chromatin is regulated when different pathways of DNA repair are utilized. Furthermore, we review additional modes of regulation of DNA repair, such as through the role of global and localized chromatin states in maintaining expression of DNA repair genes, as well as through the activity of epigenetic enzymes on non-nucleosome substrates. Finally, we discuss current and future applications of the mechanistic interplays between chromatin regulation and DNA repair in the context cancer treatment.
    Keywords:  DNA damage; DNA repair; chromatin remodeling; epigenomics
    DOI:  https://doi.org/10.3390/molecules25112496
  26. Nat Commun. 2020 May 29. 11(1): 2662
    Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer.
    Neha Chopra, Holly Tovey, Alex Pearson, Ros Cutts, Christy Toms, Paula Proszek, Michael Hubank, Mitch Dowsett, Andrew Dodson, Frances Daley, Divya Kriplani, Heidi Gevensleben, Helen Ruth Davies, Andrea Degasperi, Rebecca Roylance, Stephen Chan, Andrew Tutt, Anthony Skene, Abigail Evans, Judith M Bliss, Serena Nik-Zainal, Nicholas C Turner.
      Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.
    DOI:  https://doi.org/10.1038/s41467-020-16142-7
  27. PLoS Genet. 2020 May 29. 16(5): e1008816
    Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.
    Erin K Schwartz, Shih-Hsun Hung, Damon Meyer, Aurèle Piazza, Kevin Yan, Becky Xu Hua Fu, Wolf-Dietrich Heyer.
      Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.
    DOI:  https://doi.org/10.1371/journal.pgen.1008816
  28. Cell Death Dis. 2020 May 26. 11(5): 398
    Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network.
    Jeong-Hwa Baek, Hong Shik Yun, Ju-Young Kim, Janet Lee, Yeon-Joo Lee, Chang-Woo Lee, Jie-Young Song, Jiyeon Ahn, Jong Kuk Park, Jae-Sung Kim, Kee-Ho Lee, Eun Ho Kim, Sang-Gu Hwang.
      The poor therapeutic efficacy of non-small cell lung cancer (NSCLC) is partly attributed to the acquisition of chemoresistance. To investigate the mechanism underlying this resistance, we examined the potential link between kinesin light chain 4 (KLC4), which we have previously reported to be associated with radioresistance in NSCLC, and sensitivity to chemotherapy in human lung cancer cell lines. KLC4 protein levels in lung cancer cells correlated with the degree of chemoresistance to cisplatin treatment. Furthermore, KLC4 silencing enhanced the cytotoxic effect of cisplatin by promoting DNA double-strand breaks and apoptosis. These effects were mediated by interaction with the checkpoint kinase CHK2, as KLC4 knockdown increased CHK2 activation, which was further enhanced in combination with cisplatin treatment. In addition, KLC4 and CHEK2 expression levels showed negative correlation in lung tumor samples from patients, and KLC4 overexpression correlated negatively with survival. Our results indicate a novel link between the KLC4 and CHK2 pathways regulating DNA damage response in chemoresistance, and highlight KLC4 as a candidate for developing lung cancer-specific drugs and customized targeted molecular therapy.
    DOI:  https://doi.org/10.1038/s41419-020-2592-z
  29. Br J Radiol. 2020 May 28. 20200067
    Emerging strategies to target cancer metabolism and improve radiation therapy outcomes.
    Martin Kery, Ioanna Papandreou.
      Cancer-specific metabolic changes support the anabolic needs of the rapidly growing tumor, maintain a favorable redox balance and help cells adapt to microenvironmental stresses like hypoxia and nutrient deprivation. Radiation is extensively applied in a large number of cancer treatment protocols but despite its curative potential, radiation resistance and treatment failures pose a serious problem. Metabolic control of DNA integrity and genomic stability can occur through multiple processes, encompassing cell cycle regulation, nucleotide synthesis, epigenetic regulation of gene activity and antioxidant defenses. Given the important role of metabolic pathways in oxidative damage responses, it is necessary to assess the potential for tumor-specific radiosensitization by novel metabolism-targeted therapies. Additionally, there are opportunities to identify molecular and functional biomarkers of vulnerabilities to combination treatments, which could then inform clinical decisions. Here, we present a curated list of metabolic pathways in the context of ionizing radiation responses. Glutamine metabolism influences DNA damage responses by mechanisms such as synthesis of nucleotides for DNA repair or of glutathione for ROS detoxification. Repurposed oxygen consumption inhibitors have shown promising radiosensitizing activity against murine model tumors and are now in clinical trials. Production of 2-hydroxy glutarate by Isocitrate Dehydrogenase1/2 neomorphic oncogenic mutants interferes with the function of alpha-ketoglutarate-dependent enzymes and modulates ATM signaling and glutathione pools. Radiation-induced oxidative damage to membrane phospholipids promotes ferroptotic cell loss and cooperates with immunotherapies to improve tumor control. In summary, there are opportunities to enhance the efficacy of radiotherapy by exploiting cell-inherent vulnerabilities and dynamic microenvironmental components of the tumor.
    DOI:  https://doi.org/10.1259/bjr.20200067
  30. Nat Rev Cancer. 2020 May 29.
    Tumour predisposition and cancer syndromes as models to study gene-environment interactions.
    Michele Carbone, Sarah T Arron, Bruce Beutler, Angela Bononi, Webster Cavenee, James E Cleaver, Carlo M Croce, Alan D'Andrea, William D Foulkes, Giovanni Gaudino, Joanna L Groden, Elizabeth P Henske, Ian D Hickson, Paul M Hwang, Richard D Kolodner, Tak W Mak, David Malkin, Raymond J Monnat, Flavia Novelli, Harvey I Pass, John H Petrini, Laura S Schmidt, Haining Yang.
      Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.
    DOI:  https://doi.org/10.1038/s41568-020-0265-y
  31. Nat Commun. 2020 May 25. 11(1): 2598
    The BCL-2 pathway preserves mammalian genome integrity by eliminating recombination-defective oocytes.
    Elias ElInati, Agata P Zielinska, Afshan McCarthy, Nada Kubikova, Valdone Maciulyte, Shantha Mahadevaiah, Mahesh N Sangrithi, Obah Ojarikre, Dagan Wells, Kathy K Niakan, Melina Schuh, James M A Turner.
      DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
    DOI:  https://doi.org/10.1038/s41467-020-16441-z
  32. Nucleic Acids Res. 2020 May 26. pii: gkaa393. [Epub ahead of print]
    DNA damage invokes mitophagy through a pathway involving Spata18.
    Xiuli Dan, Mansi Babbar, Anthony Moore, Noah Wechter, Jingyan Tian, Joy G Mohanty, Deborah L Croteau, Vilhelm A Bohr.
      Mitochondria are vital for cellular energy supply and intracellular signaling after stress. Here, we aimed to investigate how mitochondria respond to acute DNA damage with respect to mitophagy, which is an important mitochondrial quality control process. Our results show that mitophagy increases after DNA damage in primary fibroblasts, murine neurons and Caenorhabditis elegans neurons. Our results indicate that modulation of mitophagy after DNA damage is independent of the type of DNA damage stimuli used and that the protein Spata18 is an important player in this process. Knockdown of Spata18 suppresses mitophagy, disturbs mitochondrial Ca2+ homeostasis, affects ATP production, and attenuates DNA repair. Importantly, mitophagy after DNA damage is a vital cellular response to maintain mitochondrial functions and DNA repair.
    DOI:  https://doi.org/10.1093/nar/gkaa393
  33. Cells. 2020 May 27. pii: E1340. [Epub ahead of print]9(6):
    Targeted Regulation of Nuclear Lamins by Ubiquitin and Ubiquitin-Like Modifiers.
    Michael Blank.
      Nuclear lamins (NLs) are essential components of the animal cell nucleus involved in the regulation of a plethora of molecular and cellular processes. These include the nuclear envelope assembly and stability, mechanotransduction and chromatin organization, transcription, DNA replication, damage repair, and genomic integrity maintenance. Mutations in NLs can lead to the development of a wide range of distinct disease phenotypes, laminopathies, consisting of cardiac, neuromuscular, metabolic and premature aging syndromes. In addition, alterations in the expression of nuclear lamins were associated with different types of neoplastic diseases. Despite the importance and critical roles that NLs play in the diverse cellular activities, we only recently started to uncover the complexity of regulatory mechanisms governing their expression, localization and functions. This integrative review summarizes and discusses the recent findings on the emerging roles of ubiquitin and ubiquitin-like modifiers (ULMs) in the regulation of NLs, highlighting the intriguing molecular associations and cross-talks occurring between NLs and these regulatory molecules under physiological conditions and in the disease states.
    Keywords:  autophagy; laminopathies; nuclear lamins; proteasome; ubiquitin; ubiquitin-like modifiers
    DOI:  https://doi.org/10.3390/cells9061340
  34. Mol Cell. 2020 May 19. pii: S1097-2765(20)30279-3. [Epub ahead of print]
    A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.
    Enrico Monachino, Slobodan Jergic, Jacob S Lewis, Zhi-Qiang Xu, Allen T Y Lo, Valerie L O'Shea, James M Berger, Nicholas E Dixon, Antoine M van Oijen.
      Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
    Keywords:  DNA replication; DnaB helicase; DnaG primase; clamp loader-helicase interaction; conformational switch; polymerase turnover
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.037
  35. Crit Rev Biochem Mol Biol. 2020 May 24. 1-33
    Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis.
    Huidong Zhang.
      Environmental mutagens lead to mutagenesis. However, the mechanisms are very complicated and not fully understood. Environmental mutagens produce various DNA lesions, including base-damaged or sugar-modified DNA lesions, as well as epigenetically modified DNA. DNA polymerases produce mutation spectra in translesion DNA synthesis (TLS) through misincorporation of incorrect nucleotides, frameshift deletions, blockage of DNA replication, imbalance of leading- and lagging-strand DNA synthesis, and genome instability. Motif or subunit in DNA polymerases further affects the mutations in TLS. Moreover, protein interactions and accessory proteins in DNA replisome also alter mutations in TLS, demonstrated by several representative DNA replisomes. Finally, in cells, multiple DNA polymerases or cellular proteins collaborate in TLS and reduce in vivo mutagenesis. Summaries and perspectives were listed. This review shows mechanisms of mutagenesis induced by DNA lesions and the effects of multiple factors on mutations in TLS in vitro and in vivo.
    Keywords:  DNA lesion or damage; DNA polymerase and DNA replisome; Environmental mutagens; in vivo mutagensis; translesion DNA synthesis
    DOI:  https://doi.org/10.1080/10409238.2020.1768205
  36. Nucleic Acids Res. 2020 May 28. pii: gkaa447. [Epub ahead of print]
    Metabolic cofactors NADH and FAD act as non-canonical initiating substrates for a primase and affect replication primer processing in vitro.
    Christina Julius, Paula S Salgado, Yulia Yuzenkova.
      To initiate replication on a double-stranded DNA de novo, all organisms require primase, an RNA polymerase making short RNA primers which are then extended by DNA polymerases. Here, we show that primase can use metabolic cofactors as initiating substrates, instead of its canonical substrate ATP. DnaG primase of Escherichia coli initiates synthesis of RNA with NADH (the reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) in vitro. These cofactors consist of an ADP core covalently bound to extra moieties. The ADP component of these metabolites base-pairs with the DNA template and provides a 3'-OH group for RNA extension. The additional cofactors moieties apparently contact the 'basic ridge' domain of DnaG, but not the DNA template base at the -1 position. ppGpp, the starvation response regulator, strongly inhibits the initiation with cofactors, hypothetically due to competition for overlapping binding sites. Efficient RNA primer processing is a prerequisite for Okazaki fragments maturation, and we find that the efficiency of primer processing by DNA polymerase I in vitro is specifically affected by the cofactors on its 5'-end. Together these results indicate that utilization of cofactors as substrates by primase may influence regulation of replication initiation and Okazaki fragments processing.
    DOI:  https://doi.org/10.1093/nar/gkaa447
  37. Blood. 2020 May 26. pii: blood.2019001808. [Epub ahead of print]
    Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.
    Rushdia Yusuf, Borja Saez, Azeem Sharda, Nick van Gastel, Vionnie Yu, Ninib Baryawno, Elizabeth W Scadden, Sanket S Acharya, Shrikanta Chattophadhyay, Cherrie Huang, Vasanthi Viswanathan, Dana S'aulis, Julien Cobert, David Brian Sykes, Mark A Keibler, Sudeshna Das, John N Hutchinson, Michael Churchill, Siddharta Mukherjee, Dongjun Lee, Francois Emile Mercier, John Doench, Lars Bullinger, David J Logan, Stuart Schreiber, Gregory Stephanopoulos, William B Rizzo, David T Scadden.
      Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and generated from lipid peroxides underlying the non-caspase dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition, a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.
    DOI:  https://doi.org/10.1182/blood.2019001808
  38. Leukemia. 2020 May 23.
    Inhibition of ubiquitin-specific protease 7 sensitizes acute myeloid leukemia to chemotherapy.
    Maëlle Cartel, Pierre-Luc Mouchel, Mathilde Gotanègre, Laure David, Sarah Bertoli, Véronique Mansat-De Mas, Arnaud Besson, Jean-Emmanuel Sarry, Stéphane Manenti, Christine Didier.
      Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, single-cell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.
    DOI:  https://doi.org/10.1038/s41375-020-0878-x
  39. Mol Biol Cell. 2020 Jun 01. 31(12): 1201-1205
    Freedom of assembly: metabolic enzymes come together.
    Jacqueline C Simonet, Anika L Burrell, Justin M Kollman, Jeffrey R Peterson.
      Many different enzymes in intermediate metabolism dynamically assemble filamentous polymers in cells, often in response to changes in physiological conditions. Most of the enzyme filaments known to date have only been observed in cells, but in a handful of cases structural and biochemical studies have revealed the mechanisms and consequences of assembly. In general, enzyme polymerization functions as a mechanism to allosterically tune enzyme kinetics, and it may play a physiological role in integrating metabolic signaling. Here, we highlight some principles of metabolic filaments by focusing on two well-studied examples in nucleotide biosynthesis pathways-inosine-5'-monophosphate (IMP) dehydrogenase and cytosine triphosphate (CTP) synthase.
    DOI:  https://doi.org/10.1091/mbc.E18-10-0675
  40. DNA Repair (Amst). 2020 May 16. pii: S1568-7864(20)30115-4. [Epub ahead of print]91-92 102867
    Molecular and structural characterization of disease-associated APE1 polymorphisms.
    Amy M Whitaker, Wesley J Stark, Tony S Flynn, Bret D Freudenthal.
      Under conditions of oxidative stress, reactive oxygen species (ROS) continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. When the nucleobase structure is altered, its base-pairing properties may also be altered, promoting mutations. Consequently, oxidative DNA damage is a major source of the mutation load that gives rise to numerous human maladies, including cancer. Base excision repair (BER) is the primary pathway tasked with removing and replacing mutagenic DNA base damage. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central enzyme with AP-endonuclease and 3' to 5' exonuclease functions during BER, and therefore is key to maintenance of genome stability. Polymorphisms, or SNPs, in the gene encoding APE1 (APEX1) have been identified among specific human populations and result in variants of APE1 with modified function. These defects in APE1 potentially result in impaired DNA repair capabilities and consequently an increased risk of disease for individuals within these populations. In the present study, we determined the X-ray crystal structures of three prevalent disease-associated APE1 SNPs (D148E, L104R, and R237C). Each APE1 SNP results in unique localized changes in protein structure, including protein dynamics and DNA binding contacts. Combined with comprehensive biochemical characterization, including pre-steady-state kinetic and DNA binding analyses, variant APE1:DNA complex structures with both AP-endonuclease and exonuclease substrates were analyzed to elucidate how these SNPs might perturb the two major repair functions employed by APE1 during BER.
    Keywords:  APE1; Base excision repair; Enzyme kinetics; Oxidative stress; Polymorphisms; X-ray crystallography
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102867
  41. Nucleic Acids Res. 2020 May 26. pii: gkaa429. [Epub ahead of print]
    CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes.
    Neil J Rzechorzek, Steven W Hardwick, Vincentius A Jatikusumo, Dimitri Y Chirgadze, Luca Pellegrini.
      DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one β-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.
    DOI:  https://doi.org/10.1093/nar/gkaa429
  42. Nat Commun. 2020 May 27. 11(1): 2539
    Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes.
    Genome Aggregation Database Production Team
      Multi-nucleotide variants (MNVs), defined as two or more nearby variants existing on the same haplotype in an individual, are a clinically and biologically important class of genetic variation. However, existing tools typically do not accurately classify MNVs, and understanding of their mutational origins remains limited. Here, we systematically survey MNVs in 125,748 whole exomes and 15,708 whole genomes from the Genome Aggregation Database (gnomAD). We identify 1,792,248 MNVs across the genome with constituent variants falling within 2 bp distance of one another, including 18,756 variants with a novel combined effect on protein sequence. Finally, we estimate the relative impact of known mutational mechanisms - CpG deamination, replication error by polymerase zeta, and polymerase slippage at repeat junctions - on the generation of MNVs. Our results demonstrate the value of haplotype-aware variant annotation, and refine our understanding of genome-wide mutational mechanisms of MNVs.
    DOI:  https://doi.org/10.1038/s41467-019-12438-5
  43. J Vis Exp. 2020 May 08.
    Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates.
    Monica C Pillon, Robin E Stanley.
      Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.
    DOI:  https://doi.org/10.3791/61258
  44. Antiviral Res. 2020 May 24. pii: S0166-3542(20)30239-4. [Epub ahead of print] 104825
    Pemetrexed Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication through Blocking dTMP Synthesis.
    Jungang Chen, Haiwei Zhang, Xulin Chen.
      Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. In immunocompromised patients, KSHV infection is capable of causing severe and fatal diseases. Current antiviral treatments for KSHV infections consist mostly of nucleoside analogs, all of which target viral polymerases and are associated with adverse effects and drug resistance. By screening an FDA-approved drug library, we identified pemetrexed as a potent anti-KSHV agent, with an IC50 of 90 nM. Characterization of the antiviral properties of pemetrexed revealed that it interferes with the lytic replication of viral DNA, resulting in the reduction of infectious virions. The antiviral effect of pemetrexed depends on the dTMP synthesis pathway that requires the folate-dependent enzymes. Besides, pemetrexed shows a broad spectrum of anti-herpes virus activity. Thus, our findings suggest that pemetrexed inhibits the lytic replication of KSHV DNA by blocking dTMP synthesis. Pemetrexed has the potential to be utilized as an anti-KSHV agent.
    Keywords:  Anti-KSHV; Anti-herpesvirus; KSHV; Pemetrexed; dTMP synthesis; lytic replication
    DOI:  https://doi.org/10.1016/j.antiviral.2020.104825
  45. Genet Med. 2020 May 28.
    Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.
    Francisco Del Caño-Ochoa, Bobby G Ng, Malak Abedalthagafi, Mohammed Almannai, Ronald D Cohn, Gregory Costain, Orly Elpeleg, Henry Houlden, Ehsan Ghayoor Karimiani, Pengfei Liu, M Chiara Manzini, Reza Maroofian, Michael Muriello, Ali Al-Otaibi, Hema Patel, Edvardson Shimon, V Reid Sutton, Mehran Beiraghi Toosi, Lynne A Wolfe, Jill A Rosenfeld, Hudson H Freeze, Santiago Ramón-Maiques.
       PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy.
    METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype.
    RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype.
    CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.
    Keywords:  aspartate transcarbamoylase; carbamoyl phosphate synthetase; congenital disorder of glycosylation; de novo pyrimidine biosynthesis; dihydroorotase
    DOI:  https://doi.org/10.1038/s41436-020-0833-2
  46. Ann Hepatobiliary Pancreat Surg. 2020 May 31. 24(2): 127-136
    Human equilibrative nucleoside transporter-1 (hENT1) and ribonucleotide reductase regulatory subunit M1 (RRM1) expression; do they have survival impact to pancreatic cancer?
    Dae Wook Hwang, Eun Shin, Jai Young Cho, Ho-Seong Han, Yoo-Seok Yoon.
       Backgrounds/Aims: Gemcitabine is still one of adjuvant options in chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC). Integral membrane transporter protein and intracellular enzymes including human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), ribonucleotide reductase (RR) M1, and M2 are known as important factors for chemosensitivity of gemcitabine. We aimed to investigate the correlation between these key molecules and 5-year actual survival in PDAC patients.
    Methods: The expression of intratumoral hENT1, dCK, RRM1, and RRM2 was assessed immunohistochemically in 160 PDAC patients underwent surgical resection. Association between clininopathologic factors, immunohistochemical results, and overall survival were analyzed.
    Results: Adjuvant chemotherapy including concurrent chemoradiotherapy was not associated with overall survival (HR, 0.92; 95% CI, 0.65-1.31; p=0.658). High hENT1 expression group did not show statistical survival difference, compared with all others (HR, 1.16; 95% CI, 0.82-1.65, p=0.396). Gemcitabine therapy and high hENT1 group was compared with all other patients, and no difference in overall survival was identified (HR, 0.99; 95% CI, 0.68-1.42; p=0.940). And, gemcitabine therapy and high hENT1 group did not differ statistically from gemcitabine therapy and low hENT1 expression (HR, 0.92; 95% CI, 0.55-1.56; p=0.764). The intensity of dCK, RRM1, and RRM2 expression was not associated with overall survival (p=0.413, p=0.138 and p=0.061) in univariate analysis.
    Conclusions: The expression of hENT1, dCK, RRM1 and RRM2 may not be associated with overall survival for patients with pancreatic cancer on gemcitabine adjuvant therapy. These proteins and other factors that may interact with or confound these results should be investigated in the near future.
    Keywords:  Adjuvant chemotherapy; Gemcitabine; Overall survival; Pancreatic ductal adenocarcinoma; Surgery; hENT1
    DOI:  https://doi.org/10.14701/ahbps.2020.24.2.127
  47. J Mol Biol. 2020 May 21. pii: S0022-2836(20)30353-3. [Epub ahead of print]
    Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp.
    Brent W Anderson, Aili Hao, Kenneth A Satyshur, James L Keck, Jue D Wang.
      The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly-identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.
    Keywords:  PRPP; cooperativity; oligomerization; phosphoribosyltransferase; specificity
    DOI:  https://doi.org/10.1016/j.jmb.2020.05.013
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