DOCSLIB.ORG

Positional Cloning of the Werner's Syndrome Gene

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Positional Cloning of the Werner's Syndrome Gene Probes. These molecules have low photobleaching emission dipole orientation. However, the 1.25-NA (1977); J. Opt. Soc. Am. 67, 1607 (1977). rates(10-6 to 10-8 per excitation),near-unityquan- oil-immersion objective used here collected a calcu- 22. For a dipole on the air side of the interface,LlllLoois tum yield in a semirigid environment, and an absorp- lated 65% of the total light emitted by a molecule at 0.92, L~/Loo is 1.6,andthe radiativelifetimeIsde- tion cross section of 1.35 x 10-16 cm2 at 532 nm, the PMMA-alr interface [E. H. Helen and D. Axelrod, creased for a perpendicular emission dipole. estimated by us on the basis of the measured ab- J. Opt. Soc. Am. B 4, 337 (1987)], nearly indepen- 23. Aliphatic hydrocarbon immersion oil, type FF; sorption in methanol. dent of the emission-dipole orientation. The total col- Cargille Laboratories, Cedar Grove, NJ. 15. A quartz cover slip was first spin-coated with one lection or detection efficiency was 15%. 24. We deposited several micrometers of PMMA by spin- drop (0.2 ml) of 0.1 % by weight PMMA in chloroben- 18. A focused laser beam has a very small longitudinal coating several drops of a solution of 5% by weight zene and then spin-coated with one drop of a 1-nM field component along the z axis, on the order of PMMA in toluene and heating the sample to dry. solution of Dil'2C(12) in toluene. The nanomolar Elkw, whereEis the tangential laser field, k is 2'IT11.., 25. E. Akesson, V. Sundstrom, 1'. Gillbro, Chem. Phys. dye solution was freshly prepared from a micromo- and w is the focused spot size [M. Lax, W. H. Lett.121,513(1985). lar dye solution with each sample. Loulsell, W. B. McKnight, Phys. Rev. A 11, 1365 26. M. O. Scully, Appl. Phys. B 51,238 (1990). 16. Although the calculated saturation intensity is 1 MW (1975)]. 27. We obtained an ensemble measurement by raster- cm-2, in a separate study we found that the satura- 19. The emission dipole, which lies along the long axis of scanning a 36-fLm2 sample area prepared with 20 tion of the fluorescent transition was determined by the molecule that connects the two indole rings, is times higher coverage of molecules at a laser inten- transitions from the excited singlet state to a meta- oriented 28° from the absorption dipole [D. Axelrod, sity of 500 W cm-2, while continuously collecting stable state, probably the triplet state, with an inter- Biophys. J. 26, 557 (1979)]. fluorescence. It thus averaged over several hundred system crossing rate of about 0.15% and a triplet 20. The radiative lifetime for a broad molecular spectrum molecules. lifetime of 0.4 ms. is proportional to the spectrally averaged inverse 28. P. R. Bevington, Data Reduction and Error Analysis cube of the emission frequency [SoJ. Strickler and R. 17. The maximum fluorescence rate is Ro = TJ<PF(crf/hv), for the Physical Sciences (McGraw-Hili, New York, A. Berg,J. Chem. Phys. 37,814 (1962)] or, to a small where cr is the absorption cross section at the laser 1969), chap. 11. frequency v, <PFIs the fluorescence quantum yield, approximation, proportional to the cube of the peak I is the laser intensity, and TJis the collection efficien- fluorescence wavelength. cy. In general, both TJand <PFcan depend on the 21. W. Lukosz and R. E. Kunz, Opt. Commun. 20, 195 26 October 1995; accepted 19 January 1996 dem repeat polymorphism (STRP) markers Positional Cloning of the Werner's at the glutathione reductase (GSR) gene Syndrome Gene and D8S339 were shown to be in linkage disequilibrium with WS in Japanese WS Chang-En Yu,* Junko Qshima,* Ying-Hui Fu,* Ellen M. Wijsman, patients (9, 10), indicating that these mark- Fuki Hisama, Reid Alisch, Shellie Matthews, Jun Nakura, ers are most likely close to WRN. Tetsuro Miki, Samir Quais, George M. Martin, To clone the WS gene, we generated a map from yeast artificial chromosomes John Mulligan, Gerard D. Schellenbergt (YACs), PI clones, and cosmid contigs (Fig. 1), starting at GSR and extended by Werner's syndrome 0NS) is an inherited disease with clinical symptoms resembling walking methods to cover approximately 3 premature aging. Early susceptibility to a number of major age-related diseases is a key Mb (11). Eighteen STRP markers (Fig. 1B) feature of this disorder. The gene responsible for WS (known as WRN) was identified by were identified in the contig; probable re- positional cloning. The predicted protein is 1432 amino acids in length and shows sig- combinants were detected at D8S2194 nificant similarity to DNA helicases. Four mutations in WS patients were identified. Two (which excluded the region telomeric to of the mutations are splice-junction mutations, with the predicted result being the ex- this marker) and at D8S2186 [which ex- clusion of exons from the final messenger RNA. One of these mutations, which results in cluded the region centromeric to this mark- a frameshift and a predicted truncated protein, was found in the homozygous state in 60 er (12)], making the 1.2 to 1.4 Mb interval p~ent of Japanese WS patients examlnea.lfle other two mutations are nonsense mutations. I ne Identification of a mutated putative helicase as the gene product of the C.-E. Yu and S. Matthews, Geriatric Research Education WS gene suggests that defective DNA metabolism is involved in the complex process of and Clinical Center (182B), Veterans Affairs Puget Sound Health Care System, Seattle Division, 1660 South Co- aging in WS patients. lumbian Way, Seattle, WA 98108, USA, and Department of Neurology, University of Washington, Seattle, WA 98195, USA. J. Oshima, Geriatric Research Education and Clinical Center (182B), Veterans Affairs Puget Sound Health Care Werner's syndrome is a rare autosomal prevalent geriatric disorders such as Alz- System, Seattle Division, 1660 South Columbian Way, recessive disorder that is considered a par- heimer's disease and hypertension are not Seattle, WA 98108, USA, and Department of Pathology, tial model of human aging (1-3). WS observed in WS. Moreover, there are sub- University of Washington, Seattle, WA 98195, USA. Y.-H. Fu, R. Alisch, J. Mulligan, Darwin Molecular Corpo- patients prematurely develop a variety of tle discordances between WS and normal ration, 1631 220th Street, S.E., Bothell, WA 98021, USA. the major age-related diseases, including aging, such as a disproportionately severe E. M. Wijsman, Division of Medical Genetics, Department several forms of arteriosclerosis, malignant osteoporosis of the limbs relative to the of Medicine, and Department of Biostatistics, University of Washington, Seattle, WA 98195, USA. neoplasms, type II diabetes mellitus, osteo- trunk and the high prevalence of nonepi- F. Hisama, Department of Neurology, Yale University porosis', and ocular cataracts; these indi- thelial neoplasms in WS. Finally, there are School of Medicine, New Haven, CT 06510, USA. viduals also manifest early graying and loss unusual clinical features unrelated to ag- J. Nakura and 1'. Miki, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka Suita, of hair, skin atrophy, and a generally aged ing, including ulcerations around the an- Osaka 565, Japan. appearance. Growth retardation occurs, kles and soft tissue calcification (1, 2). S. Ouais, Section of Endocrinology, Damascus City Hos- typically around the time of puberty, but The WS locus (WRN) was initially lo- pital, Damascus, Syria. G. M. Martin, Department of Pathology, University of medical problems are rare during child- calized to 8p12 (5) by linkage analysis and Washington, Seattle, WA 98195, USA. hood. Cell culture studies also suggest a the genetic position refined by both meiotic G. D. Schellenberg, Geriatric Research Education and parallel between WS and aging; the repli- and homozygosity mapping (5-7). Initial Clinical Center (182B), Veterans Affairs Puget Sound mapping (6-8) placed WRN in an 8.3- Health Care System, Seattle Division, 1660 South Co- cative life-span of fibroblasts from WS lumbian Way, Seattle, WA 98108, USA, and the Depart- patients is reduced compared with age- centimorgan (cM) interval flanked by ments of Medicine, Neurology, and Pharmacology, Uni- matChed controls and is similar to the markers D8S137 and D8S87 (Fig. 1); versity of Washington, Seattle, WA 98195, USA. life-span of fibroblasts taken from more D8S339, located within this interval, was 'These authors contributed equally to this work. the closest marker. Subsequently, short tan- tTo whom correspondence should be addressed. , SCIENCE. VOL. 272 12 APRIL 1996 ~, ~ ;:'dYindivid"l; (4). How,v", wm, ~ Fig. 1. Genetic and physical maps of 088283 the WRN region. (A) The genetic map 088259 GSR 088278 088268 units are in centimorgans, assuming LPL 088136 088137 088131 088339 08887 FGFR1 088255 ANK1 sex-equal recombination rates (8). (B) The polymorphic loci are 8TRP mark- A ~~~~~ \ / , ers except for PPP2CB and 0882180 Telomere 8p I I I I I I I I I Centromere which are bi-allelic insertion-deletion 3.8 7.4 0.9 6.7 1.6 2.5 2.8 2.1 polymorph isms (11). The 8TRP loci at -IL... GSR and PPP2CB are 0882202 and 0882208, respectively. (C) The physi- r -, cal map has approximate distances in kilobases determined from sizes of 8 overlapping nonchimeric YACs, and from genomic ONA sequence from overlapping P1 clones 2233, 2253, 3833, 2236, and 3101. Marker and clone order was determined from the C 130 200 130 350 >200 sequence-tagged site (8T8) content of YACs, P1 clones, and cosmid clones and from genomic ONA sequence. (D) D 814E11:1080kb The YACs represent the minimal num- 896F4: 1200 kb ber of YACs to cover the WRN region 763A7: 800 kb and are the YACs used for cONA selec- 780E6: 500 kb tion experiments.
Load more

Recommended publications
  • Structure and Function of the Human Recq DNA Helicases
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2005 Structure and function of the human RecQ DNA helicases Garcia, P L Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-34420 Dissertation Published Version Originally published at: Garcia, P L. Structure and function of the human RecQ DNA helicases. 2005, University of Zurich, Faculty of Science. Structure and Function of the Human RecQ DNA Helicases Dissertation zur Erlangung der naturwissenschaftlichen Doktorw¨urde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultat¨ der Universitat¨ Z ¨urich von Patrick L. Garcia aus Unterseen BE Promotionskomitee Prof. Dr. Josef Jiricny (Vorsitz) Prof. Dr. Ulrich H ¨ubscher Dr. Pavel Janscak (Leitung der Dissertation) Z ¨urich, 2005 For my parents ii Summary The RecQ DNA helicases are highly conserved from bacteria to man and are required for the maintenance of genomic stability. All unicellular organisms contain a single RecQ helicase, whereas the number of RecQ homologues in higher organisms can vary. Mu- tations in the genes encoding three of the five human members of the RecQ family give rise to autosomal recessive disorders called Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome. These diseases manifest commonly with genomic in- stability and a high predisposition to cancer. However, the genetic alterations vary as well as the types of tumours in these syndromes. Furthermore, distinct clinical features are observed, like short stature and immunodeficiency in Bloom syndrome patients or premature ageing in Werner Syndrome patients. Also, the biochemical features of the human RecQ-like DNA helicases are diverse, pointing to different roles in the mainte- nance of genomic stability.
    [Show full text]
  • Plugged Into the Ku-DNA Hub: the NHEJ Network Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou
    Plugged into the Ku-DNA hub: The NHEJ network Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou To cite this version: Philippe Frit, Virginie Ropars, Mauro Modesti, Jean-Baptiste Charbonnier, Patrick Calsou. Plugged into the Ku-DNA hub: The NHEJ network. Progress in Biophysics and Molecular Biology, Elsevier, 2019, 147, pp.62-76. 10.1016/j.pbiomolbio.2019.03.001. hal-02144114 HAL Id: hal-02144114 https://hal.archives-ouvertes.fr/hal-02144114 Submitted on 29 May 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Progress in Biophysics and Molecular Biology xxx (xxxx) xxx Contents lists available at ScienceDirect Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio Plugged into the Ku-DNA hub: The NHEJ network * Philippe Frit a, b, Virginie Ropars c, Mauro Modesti d, e, Jean Baptiste Charbonnier c, , ** Patrick Calsou a, b, a Institut de Pharmacologie et Biologie Structurale, IPBS, Universite de Toulouse, CNRS, UPS, Toulouse, France b Equipe Labellisee Ligue Contre
    [Show full text]
  • Evolutionary History of Ku Proteins: Evidence of Horizontal Gene Transfer from Archaea to Eukarya
    Evolutionary history of Ku proteins: evidence of horizontal gene transfer from archaea to eukarya Ashmita Mainali Kathmandu University Sadikshya Rijal Kathmandu University Hitesh Kumar Bhattarai ( [email protected] ) Kathmandu University https://orcid.org/0000-0002-7147-1411 Research article Keywords: Double Stranded Breaks, Non-homologous End Joining, Maximum Likelihood Phylogenetic tree, domains, Ku protein, origin, evolution Posted Date: October 29th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-58075/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/24 Abstract Background The DNA end joining protein, Ku, is essential in Non-Homologous End Joining in prokaryotes and eukaryotes. It was rst discovered in eukaryotes and later by PSI blast, was discovered in prokaryotes. While Ku in eukaryotes is often a multi domain protein functioning in DNA repair of physiological and pathological DNA double stranded breaks, Ku in prokaryotes is a single domain protein functioning in pathological DNA repair in spores or late stationary phase. In this paper we have attempted to systematically search for Ku protein in different phyla of bacteria and archaea as well as in different kingdoms of eukarya. Result From our search of 116 sequenced bacterial genomes, only 25 genomes yielded at least one Ku sequence. From a comprehensive search of all NCBI archaeal genomes, we received a positive hit in 7 specic archaea that possessed Ku. In eukarya, we found Ku protein in 27 out of 59 species. Since the entire genome of all eukaryotic species is not fully sequenced this number could go up.
    [Show full text]
  • Biochemical Characterization of the Werner – Like Exonuclease From
    Biochemical characterization of the Werner – like exonuclease from Arabidopsis thaliana Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften an der Fakultät für Chemie und Biowissenschaften der Universität Karlsruhe (TH) genehmigte DISSERTATION von Diplom-Biochemikerin Helena Plchova aus Prag, Tschechische Republik Dekan: Prof. Dr. Manfred Metzler 1. Gutachter: Prof. Dr. Holger Puchta 2. Gutachter: Prof. Dr. Andrea Hartwig Tag der mündlichen Prüfung: 04.12.03 Acknowledgements This work was carried out during the period from September 1999 to June 2003 at the Institut for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany. I would like to express my sincere gratitude to my supervisor, Prof. Dr. H. Puchta, for giving me the opportunity to work in his research group “DNA-Recombination”, for his expertise, understanding, stimulating discussions and valuable suggestions during the practical work and writing of the manuscript. I appreciate his vast knowledge and skill in many areas and his assistance during the course of the work. I would like to thank also Dr. Manfred Focke for reading the manuscript and helpful discussions. My sincere gratitude is to all the collaborators of the Institute for Plant Genetics and Crop Plant Research (IPK) in Gatersleben, especially from the “DNA-Recombination” group for the stimulating environment and very friendly atmosphere during this work. Finally, I also thank all my friends that supported me in my Ph.D. study and were always around at any time. Table of contents Page 1. Introduction 1 1.1. Werner syndrome (WS) 1 1.2. Product of Werner syndrome gene 2 1.2.1. RecQ DNA helicases 3 1.2.2 Mutations in Werner syndrome gene 6 1.3.
    [Show full text]
  • An Overview of DNA Repair Mechanisms in Plants Compared to Mammals
    Cell. Mol. Life Sci. (2017) 74:1693–1709 DOI 10.1007/s00018-016-2436-2 Cellular and Molecular Life Sciences REVIEW Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals Claudia P. Spampinato1 Received: 8 August 2016 / Revised: 1 December 2016 / Accepted: 5 December 2016 / Published online: 20 December 2016 Ó Springer International Publishing 2016 Abstract The genome integrity of all organisms is con- methods and reporter systems for gene expression and stantly threatened by replication errors and DNA damage function studies. Here, I provide a current understanding of arising from endogenous and exogenous sources. Such base DNA repair genes in plants, with a special focus on A. pair anomalies must be accurately repaired to prevent thaliana. It is expected that this review will be a valuable mutagenesis and/or lethality. Thus, it is not surprising that resource for future functional studies in the DNA repair cells have evolved multiple and partially overlapping DNA field, both in plants and animals. repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mecha- Keywords DNA repair Á Photolyases Á BER Á NER Á nisms at the molecular level has been made by several MMR Á HR Á NHEJ talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from Introduction studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features All organisms are constantly exposed to environmental should be mentioned.
    [Show full text]
  • Rad18 and Rnf8 Facilitate Homologous Recombination by Two Distinct Mechanisms, Promoting Rad51 Focus Formation and Suppressing T
    Oncogene (2015) 34, 4403–4411 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining S Kobayashi1, Y Kasaishi1, S Nakada2,5, T Takagi3, S Era1, A Motegi1, RK Chiu4, S Takeda1 and K Hirota1,3 The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/− clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18−/−, RNF8−/− and RAD18−/−/RNF8−/− clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13−/−cells, they are more sensitive to camptothecin and olaparib than UBC13−/−cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18−/− and RNF8−/− cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation.
    [Show full text]
  • Crystal Structure of the Werner's Syndrome Helicase
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.075176; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Crystal Structure of the Werner’s Syndrome Helicase Joseph A. Newman1, Angeline E. Gavard1, Simone Lieb2, Madhwesh C. Ravichandran2, Katja Hauer2, Patrick Werni2, Leonhard Geist2, Jark Böttcher2, John. R. Engen3, Klaus Rumpel2, Matthias Samwer2, Mark Petronczki2 and Opher Gileadi1,* 1- Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford, United Kingdom. 2- Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria. 3- Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA. Abstract Werner syndrome helicase (WRN) plays important roles in DNA repair and the maintenance of genome integrity. Germline mutations in WRN give rise to Werner syndrome, a rare autosomal recessive progeroid syndrome that also features cancer predisposition. Interest in WRN as a therapeutic target has increased massively following the identification of WRN as the top synthetic lethal target for microsatellite instable (MSI) cancers. High throughput screens have identified candidate WRN helicase inhibitors, but the development of potent, selective inhibitors would be significantly enhanced by high-resolution structural information.. In this study we have further characterized the functions of WRN that are required for survival of MSI cancer cells, showing that ATP binding and hydrolysis are required for complementation of siRNA-mediated WRN silencing. A crystal structure of the WRN helicase core at 2.2 Å resolutionfeatures an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains.
    [Show full text]
  • Interactive Competition Between Homologous Recombination and Non-Homologous End Joining
    Vol. 1, 913–920, October 2003 Molecular Cancer Research 913 Interactive Competition Between Homologous Recombination and Non-Homologous End Joining Chris Allen,1 James Halbrook,2 and Jac A. Nickoloff1 1Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM, and 2ICOS Corporation, Bothell, WA Abstract distinct, non-conservative HR process that can result in DNA-dependent protein kinase (DNA-PK), composed of deletions when interacting regions are arranged as direct Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is repeats (1). Key HR proteins include RAD51, five RAD51 involved in double-strand break (DSB) repair by non- paralogues, RAD52, and RAD54 (2–8). Several other proteins homologous end joining (NHEJ). DNA-PKcs defects have poorly defined roles in HR, including replication protein confer ionizing radiation sensitivity and increase A (RPA), p53, ATM, BRCA1, and BRCA2 (9–14). In yeast, homologous recombination (HR). Increased HR is con- Rad50 and Mre11 have been implicated in HR, particularly in sistent with passive shunting of DSBs from NHEJ to HR. an early step involving end processing to 3V single-stranded tails We therefore predicted that inhibiting the DNA-PKcs (15). HR typically results in accurate repair, while defects in kinase would increase HR. A novel DNA-PKcs inhibitor HR proteins cause genome instability (6, 16–18). (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; desig- NHEJ often results in imprecise repair, yielding deletions or nated IC86621) increased ionizing radiation sensitivity insertions, yet NHEJ also plays a role in maintaining genome but surprisingly decreased spontaneous and DSB- stability (19, 20).
    [Show full text]
  • Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer
    Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer Ben Yang China-Japan Union Hospital of Jilin University Wang Ke China-Japan Union Hospital of Jilin University Yingchun Wan China-Japan Union Hospital of Jilin University Tao Li ( [email protected] ) China-Japan Union Hospital of Jilin University https://orcid.org/0000-0002-6981-2977 Primary research Keywords: Endometrial cancer, RNF8, cisplatin, doxorubicin, resistance Posted Date: September 2nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-57263/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Biochemical and Biophysical Research Communications on March 1st, 2021. See the published version at https://doi.org/10.1016/j.bbrc.2021.01.046. Page 1/19 Abstract Background Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major diculties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. Methods Cisplatin and doxorubicin resistant cell lines were acquired by continuous exposing parental EC cells to cisplatin or doxorubicin for 3 months. Cell viability was determined by using MTT assay. Protein Expression levels of protein were examined by western blotting assay. mRNA levels were measured by quantitative polymerase chain reaction (qPCR) assay. Ring nger protein 8 (RNF8) knockout cell lines were generated by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay.
    [Show full text]
  • Akt Promotes Post-Irradiation Survival of Human Tumor Cells Through Initiation, Progression, and Termination of DNA-Pkcs–Dependent DNA Double-Strand Break Repair
    Published OnlineFirst May 17, 2012; DOI: 10.1158/1541-7786.MCR-11-0592 Molecular Cancer DNA Damage and Cellular Stress Responses Research Akt Promotes Post-Irradiation Survival of Human Tumor Cells through Initiation, Progression, and Termination of DNA-PKcs–Dependent DNA Double-Strand Break Repair Mahmoud Toulany1, Kyung-Jong Lee3, Kazi R. Fattah3, Yu-Fen Lin3, Brigit Fehrenbacher2, Martin Schaller2, Benjamin P. Chen3, David J. Chen3, and H. Peter Rodemann1 Abstract Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site.
    [Show full text]
  • DNA Repair: How Ku Makes Ends Meet Aidan J
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector R920 Dispatch DNA repair: How Ku makes ends meet Aidan J. Doherty* and Stephen P. Jackson† The recently determined crystal structure of the Ku of NHEJ, structural and functional homologues of Ku70, heterodimer, in both DNA-bound and unbound forms, Ku80, ligase IV and XRCC4 exist in lower eukaryotes, has shed new light on the mechanism by which this including yeast. DNA-PKcs, however, seems to be restricted protein fulfills its key role in the repair of DNA to vertebrates. double-strand breaks. Biochemical studies have yielded some major insights into Address: *Cambridge Institute for Medical Research, Department of Haematology, University of Cambridge, Hills Road, the mechanisms of NHEJ. Most notably perhaps, the Cambridge CB2 2XY, UK. †Wellcome Trust and Cancer Research observation that Ku binds tightly and specifically to a Campaign Institute of Cancer and Developmental Biology and variety of DNA end-structures, including blunt ends, 5′ or Department of Zoology, University of Cambridge, Tennis Court Road, 3′ overhangs and DNA hairpins, suggests that it acts as the Cambridge CB2 1QR, UK. primary sensor of broken chromosomal DNA [3,5]. Once E-mail: *[email protected]; †[email protected] bound to a DNA double-strand break, Ku presumably Current Biology 2001, 11:R920–R924 then recruits DNA-PKcs, whose protein kinase function — which in vitro preferentially targets proteins bound in 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. cis on the same DNA molecule — could trigger changes in chromatin structure at the site of the DNA double-strand DNA double-strand breaks are generated by ionising breaks and/or regulate the activities of other repair factors radiation and radio-mimetic chemicals.
    [Show full text]
  • Ku Heterodimer Binds to Both Ends of the Werner Protein and Functional Interaction Occurs at the Werner N-Terminus
    Nucleic Acids Research, 2002, Vol. 30 No. 16 3583±3591 Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus Parimal Karmakar, Carey M. Snowden1, Dale A. Ramsden1 and Vilhelm A. Bohr* Laboratory of Molecular Gerontology, Box 1, National Institute on Aging, IRP, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA and 1Lineberger Comprehensive Cancer Center, Campus Box 7295, Mason Farm Road, Department of Biochemistry and Biophysics and Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599-7295, USA Received April 16, 2002; Revised June 21, 2002; Accepted July 2, 2002 ABSTRACT RecQ, yeast sgs1 and human Bloom (BLM) and Rothmund Thomson (RecQL) proteins. The striking difference between The human Werner syndrome protein, WRN, is a WRN and other members of the RecQ helicase family is the member of the RecQ helicase family and contains N-terminal exonuclease domain of WRN, which shares 3¢®5¢ helicase and 3¢®5¢ exonuclease activities. homology with RNA polymerase I and RNase D of Recently, we showed that the exonuclease activity Escherichia coli (4). of WRN is greatly stimulated by the human Ku Biochemical and genetic evidence suggests that WRN plays heterodimer protein. We have now mapped this an important role in DNA metabolism, possibly by partici- interaction physically and functionally. The Ku70 pating in DNA replication, transcription, repair and/or subunit speci®cally interacts with the N-terminus recombination. Puri®ed recombinant WRN displays both (amino acids 1±368) of WRN, while the Ku80 subunit 3¢®5¢ helicase and 3¢®5¢ exonuclease activity on a variety interacts with its C-terminus (amino acids 940± of DNA substrates (5,6).
    [Show full text]