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Reinvestigation of the Classification of Five Cell Strains of Xeroderma

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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Reinvestigation of the Classi®cation of Five Cell Strains of Xeroderma Pigmentosum Group E with Reclassi®cation of Three of Them Toshiki Itoh,*² Stuart Linn,² Tomomichi Ono,§ and Masaru Yamaizumi* *Department of Cell Genetics, Institute of Molecular Embryology and Genetics; and §Dermatology, Kumamoto University School of Medicine, Kumamoto, Japan; ²Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, U.S.A. Xeroderma pigmentosum is a photosensitive syn- however, remained assigned to xeroderma pigmen- drome caused by a defect in nucleotide excision tosum group E. With the exception of the Ddb+ repair or postreplication repair. Individuals of xero- strain XP89TO, which demonstrated defective derma pigmentosum group E (xeroderma pigmento- nucleotide excision repair, both Ddb± and Ddb+ xer- sum E) have a mild clinical form of the disease and oderma pigmentosum E cells exhibited the same their cells exhibit a high level of nucleotide excision levels of variation in unscheduled DNA synthesis that repair as measured by unscheduled DNA synthesis, were seen in normal control cells. By genome DNA as well as biochemical heterogeneity. Cell strains sequencing, the two Ddb± xeroderma pigmentosum from one group of xeroderma pigmentosum E E strains were shown to have mutations in the DDB2 patients have normal damage-speci®c DNA binding gene, con®rming previous reports for XP82TO and activity (Ddb+), whereas others do not (Ddb±). Using GM02415B, and validating the classi®cation of both a re®nement of a previously reported cell fusion cells. As only the Ddb± strains investigated remain complementation assay, the previously assigned classi®ed in the xeroderma pigmentosum E comple- Ddb+ xeroderma pigmentosum E strains, XP89TO, mentation group, it is feasible that only Ddb± cells XP43TO, and XP24KO, with various phenotypes in are xeroderma pigmentosum E and that mutations DNA repair markers, were reassigned to xeroderma in the DDB2 gene are solely responsible for the xero- pigmentosum group F, xeroderma pigmentosum derma pigmentosum E group. Key words: Cockayne variant, and ultraviolet-sensitive syndrome, respec- syndrome/damage-speci®c DNA binding protein/nucleotide tively. The Ddb± xeroderma pigmentosum E strains, excision repair/postreplication repair/ultraviolet±sensitive XP82TO, and GM02415B, which showed almost syndrome.J Invest Dermatol 114:1022±1029, 2000 normal cellular phenotypes in DNA repair markers, eroderma pigmentosum (XP) is a rare autosomal (RDS) after UV irradiation (Lehmann et al, 1975, 1977; Rude recessive disease characterized bya clinical and and Friedberg, 1977; Cleaver and Kraemer, 1995). Recently, the cellular hypersensitivity to ultraviolet (UV) light XPV gene was cloned, and identi®ed as DNA polymerase X (Cleaver and Kraemer, 1995). Patients exhibit (Masutani et al, 1999; Johnson et al, 1999). In the seven NER dermatologic abnormalities, including thickening defective groups, the genes which are defective in XP with severe and hyperpigmentation of sun-exposed skin, and develop sunlight- clinical manifestations (XPA, B, and G) and with the classical type induced malignancies at an earlier age. Bycell fusion analyses,XP of XP (XPC, D, and F) have been cloned (Friedberg et al, 1995; has been classi®ed into seven genetic complementation groups (A± Sijbers et al, 1996). The XPE gene, however, has yet to be cloned G), which are defective in nucleotide excision repair (NER). A because of several dif®culties: (i) the clinical manifestations in XPE separate group, XP variant (XPV) has pro®cient NER but patients are verymild; (ii) as cells derived from XPE patients have exaggerated delayin recoveryof replicative DNA synthesis high levels of unscheduled DNA synthesis (UDS), it is very dif®cult to distinguish between XPE, XPV, and normal cells; and (iii) the Manuscript received July1, 1999; revised January13, 2000; accepted for high level of UDS makes analysis of a complementation assay using publication January24, 2000. cell fusion to determine the classi®cation of XPE strains Reprint requests to: Dr. Toshiki Itoh, Department of Molecular and problematic. Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Biochemical heterogeneityin binding to damaged DNA has Hall, Universityof California, Berkeley,CA 94720-3202. Email: toshiki been reported for XPE cell free extracts (Kataoka and Fujiwara, @uclink4.berkeley.edu 1991; Keeney et al, 1992; Friedberg et al, 1995). Cell strains from Abbreviations: CS, Cockayne syndrome; DDB, damage-speci®c DNA binding; NER nucleotide-excision-repair; PRR, postreplication repair; two of 13 unrelated XPE individuals lack a damage-speci®c DNA- RDS, recoveryof replicative DNA synthesis;RRS, recoveryof RNA binding (DDB) activityin nuclear extracts (Chu and Chang, 1988; synthesis; TK, thymidine kinase; UVsS, ultraviolet-sensitive syndrome; Keeney et al, 1992) and are termed Ddb± XPE. Recently, three UDS, unscheduled DNA synthesis; XP, xeroderma pigmentosum. additional Ddb± XPE patients were reported (Otrin et al, 1998). 0022-202X/00/$15.00 ´ Copyright # 2000 byThe Societyfor Investigative Dermatology,Inc. 1022 VOL. 114, NO. 5 MAY 2000 RECLASSIFICATION OF XPE CELLS 1023 Mutations have been identi®ed in the DDB2 gene in the three and Ddb+ XPE strains actuallycomprise two separate XP XPE Ddb± strains examined, but not in XPE Ddb+ strains (Nichols complementation groups, with the DDB2 gene being responsible et al, 1996). The question has been proposed as to whether Ddb± for the NER defect in Ddb± XPE patients. To clarifythis question, Table I. Cell strains used in this study Cell strain Group Reference(s) Source Mori Normal Itoh et al, 1994, 1995a, 1995b, 1996a, 1996b Established in our laboratory. Turu Normal Itoh et al, 1995a, 1996b Established in our laboratory. Sono Normal Itoh et al, 1996b Established in our laboratory. Goryo Normal Established in our laboratory. Umi Normal Established in our laboratory. Mura Normal Established in our laboratory. XP24KO XPEa Ddb+ Fujiwara et al, 1985 Gift from Dr Fujiwara. XP43TO XPEa Ddb+ Kondo et al, 1989 Gift from Dr Kondo. XP82TO XPEa Ddb± Kondo et al, 1988 Gift from Dr Kondo. XP89TO XPEa Ddb+ Kondo et al, 1989 Gift from Dr Kondo. GM02415B XPEa Ddb± Bootsma et al, 1970; Kleijer et al, 1973; de Weerd-Kastelein NIGMS Human Genetic Mutant Cell Repository. et al, 1974; Kraemer et al, 1975a, 1975b Kps3 UVsSb Itoh et al, 1994, 1995a, 1996c Established in our laboratory. Kps5 XPDc Itoh et al, 1994, 1995b Established in our laboratory. Kps6 XPFd Itoh et al, 1995b Established in our laboratory. Nps8 XPFd Itoh et al, 1995b Established in our laboratory. Mps1 CSAe Itoh et al, 1994, 1995a, 1996a Established in our laboratory. CS1MO CSBf Itoh et al, 1994, 1995a, 1996a Human Science Research Resources Bank. GM10905 CSBf Itoh et al, 1996a. NIGMS Human Genetic Mutant Cell Repository. XP2SA XPVg Itoh et al, 1994, 1995a, 1996b Human Science Research Resources Bank. Ops2 XPAh Established in our laboratory. Ops3 XPAh Established in our laboratory. Ops12 XPAh Established in our laboratory. Ops24 XPAh Established in our laboratory. Ops34 XPAh Established in our laboratory. Ops35 XPAh Established in our laboratory. aXeroderma pigmentosum group E. bUVs syndrome. cXeroderma pigmentosum group D. dXeroderma pigmentosum group F. eCockayne syndrome group A. fCockayne syndrome group B. gXeroderma pigmentosum variant. hXeroderma pigmentousm group A. Table II. Unscheduled DNA synthesis Experiment Cell strain UDSa Range of relative rateb (%) I XP43TO/Mori 16.0 6 0.3/18.1 6 0.3c 85±92 XP82TO/Mori 15.4 6 0.4/20.4 6 0.4c 75±80 XP89TO/Mori 6.8 6 0.2/24.0 6 0.5c 27±30 XP24KO/Mori 14.0 6 0.3/11.7 6 0.3c 114±125 GM02415B/Mori 23.9 6 0.4/26.6 6 0.4c 87±93 II XP82TO/Turu 26.6 6 0.3/22.0 6 0.3c 118±124 GM02415B/Turu 30.4 6 0.4/27.7 6 0.4c 120±125 III Turu/Mori 23.2 6 0.3/25.9 6 0.3c 87±92 Sono/Mori 18.8 6 0.3/15.6 6 0.2c 110±113 IV Goryo/Mori 11.6 6 0.2/11.8 6 0.3d 94±103 Umi/Mori 8.3 6 0.2/11.2 6 0.3c 70±78 Mura/Mori 12.5 6 0.3/17.6 6 0.2c 69±73 V XP24KO/Morie 15.3 6 0.3/13.6 6 0.2c 109±116 VIf Ops2 (XPA)/Mori 2.9 6 0.8/35.7 6 2.6c 5±11 Ops3 (XPA)/Mori 2.2 6 0.4/26.8 6 1.7c 6±10 Ops12 (XPA)/Mori 2.9 6 0.4/30.1 6 1.0c 8±11 Ops24 (XPA)/Mori 4.5 6 0.5/24.5 6 1.2c 16±21 Ops34 (XPA)/Mori 3.8 6 0.5/40.6 6 1.7c 8±11 Ops35 (XPA)/Mori 1.4 6 0.3/28.8 6 1.1c 4±6 aTo measure UDS (unscheduled DNA synthesis), cells were irradiated with UV at a dose of 30 J per m2, and then immediatelylabeled with [ 3H]thymidine (50 mCi per ml) for 2.5 h. Exposure time was 24 h. UDS is given in grains per nucleus. Data are mean 6 SEM of 300 determinations except for experiment VI (50 determinations). bRange of relative rate is the range of UDS values (grain-numbers) of cell strains tested compared with those of the normal control (mori or Turu) cells. cp < 0.001. dp > 0.1. eThis experiment was performed at a dose of 10 J per m2. fThese experiments were control experiments using XPA cells. All cell strains were assigned to XPA bythe Kumamoto laboratory.Ops2, Ops3, and Ops24 ha ve homozygous G to C substitution at the 3¢ splice acceptor site in intron 3 of XPA gene (Itoh and Yamaizumi, unpublished data).
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  • Barbour-Thesis.Pdf (2.004Mb)

    Barbour-Thesis.Pdf (2.004Mb)

    Synthetically Lethal Interactions Classify Novel Genes in Postreplication Repair in Saccharomyces cerevisiae A thesis submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Microbiology and Immunology University of Saskatchewan Leslie Barbour, B.Sc. © Copyright Leslie Barbour, February 2005. All rights reserved. Permission to Use In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or make other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Microbiology and Immunology Health Sciences Building, 107 Wiggins Road University of Saskatchewan Saskatoon, SK Canada S7N 5E5 i Acknowledgments First I would like to thank my supervisor, Dr.