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Developmental, Tissue, and Sub-Tissue Specific Expression of the Mammalian Apurinic/Apyrimidinic Endonuclease

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Developmental, Tissue, and Sub-Tissue Specific Expression of the Mammalian Apurinic/Apyrimidinic Endonuclease Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1996 Developmental, Tissue, and Sub-Tissue Specific Expression of the Mammalian Apurinic/Apyrimidinic Endonuclease Teresa Mari Wilson Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Molecular Biology Commons Recommended Citation Wilson, Teresa Mari, "Developmental, Tissue, and Sub-Tissue Specific Expression of the Mammalian Apurinic/Apyrimidinic Endonuclease" (1996). Dissertations. 3398. https://ecommons.luc.edu/luc_diss/3398 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1996 Teresa Mari Wilson LOYOLA UNIVERSITY CHICAGO DEVELOPMENTAL, TISSUE, AND SUB-TISSUE SPECIFIC EXPRESSION OF THE MAMMALIAN APURINIC/APYRIMIDINIC ENDONUCLEASE A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN MOLECULAR BIOLOGY BY TERESA MARI WILSON CHICAGO, ILLINOIS JANUARY, 1996 LOYOLA UNfVEP~'TV w·~,,.. •' "''"'-'TCq LIBRARY Copyright by Teresa M. Wilson, 1995 All rights reserved ii ACKNOWLEDGEMENTS I would like to recognize the members of my committee for their helpful criticisms and assistance in developing a productive graduate school career: Dr. Nicholas Emanuele, Dr. Leonard Erickson, Dr. Hans-Martin Jack, Dr. Russ Pieper and my advisor Dr. Mark Kelley. I would also like to thank Dr. Kelley for his guidance, support and friendship. To my laboratory colleagues, I offer my appreciation for their help and friendship: Dr. James Carney, Dr. Margaret Halloran, Kent Hansen, Denise Scroggins, Dr. John Tentler, Dr. Shahab Uddin, Dr. David Wilson, III, Jonathan Yates, and Dr. Yi Xu. I would like to extend my love and thanks to my husband and family. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS . iii LIST OF ILLUSTRATIONS .. vii LIST OF TABLES . ix LIST OF ABBREVIATIONS x ABSTRACT . .xiii Chapter I. INTRODUCTION ....... 1 II. REVIEW OF RELATED LITERATURE 5 A. DNA Damage and Repair 5 B. AP Sites . 7 C. Base Bxcision Repair 8 1. DNA Glycosylases .. 8 2. DNA Glycosylases/AP Lyases 15 3. AP Endonucleases ... 17 D. Mammalian AP Endonucleases 22 1. Multifunctional Enzyme . 24 2. Gene Regulation/Expression . 28 E. Clinical Relevance of AP Sites 30 III. MATERIALS AND METHODS 34 A. Animals 34 B. Tissues 34 c. Antibody . 34 D. Oligonucleotides . 35 E. Screening-cDNA and Genomic Libraries . 35 F. Purification of lambda Phage DNA . • . 36 iv Chapter Page G. Subcloning rAPEN cDNA into pBlueScript 38 H. Sequencing . 39 I. Tissue Culture . 40 J. Total RNA Extraction . 42 K. Northern Analysis . 43 L. Reverse Transcription . 44 M. Polymerase Chain Reaction . 44 N. 5' End Mapping of the rAPEN Transcript 46 O. Genomic DNA Isolation 48 P. Biochemical Analysis of rAPEN Activity 49 Q. Purification of GST-rAPEN Protein from Inclusion Bodies . 50 R. Gel Purification of GST-rAPEN for Antibody Production . 51 s. Western Blot Analysis . 52 T. Purification of rAPEN Antibody . 53 U. Immunocytochemical Staining of A1235 Cells for hAPE . 53 v. Subcellular Fractionation 54 w. Immunohistochemistry . 55 X. Riboprobe Synthesis for In Situ Hybridization 56 Y. In Situ Hybridization 57 z. Construction of AP Endonuclease Vectors 58 AA. Protection of Mouse 3T3 Fibroblasts from Bleomycin induced DNA Damage by Transfected AP Endonuclease DNA Repair Vectors . • • . 61 IV. RESULTS 63 A. Cloning and Sequencing of the rAPEN cDNA . 63 v Chapter Page B. Genomic Organization of the rAPEN Gene . 64 c. 5' RACE of rAPEN Nb2 and Testicular mRNA . 67 D. Tissue Specific Expression of rAPEN mRNA . 67 E. In Situ Hybridization Analysis of rAPEN Gene Expression During Fetal Development 70 F. Distribution and Differential Expression of rAPEN mRNA in Adult Rat Brain . 74 G. Differential Expression of rAPEN mRNA in Adult Rat Testis . 77 H. Overexpression of Recombinant GST-rAPEN Fusion Protein • • • . • . 78 I. Biochemical Activity of rAPEN 80 J. Subcellular Expression of the rAPEN Protein 83 K. Specificity of the hAPE Antibody 83 L. Subcellular Localization of hAPE in Al235 Glioma Cells . 86 M. Subcellular Fractionation of the hAPE Protein 86 N. Expression of hAPE in Duodenum, Hepatocytes and Epidermis . 89 o. Distribution of hAPE Expression in Brain . 93 P. Affect of Apoptosis on hAPE Expression . 97 Q. Specificity of the hMSH2 Antibody 99 R. Expression of hMSH2 in Duodenum and Colon 99 S. Bleomycin Treatment of 3T3 Cells Transfected with the Apnl, hAPE and AhAPE Expression Vectors . 103 v. DISCUSSION . 106 LITERATURE CITED . 123 VITA ... 135 vi LIST OF ILLUSTRATIONS Figure Page 1. Schematic of excision repair 9 2. Base excision repair pathways . 16 3. Families of class II AP endonucleases . 23 4. Chemical structures of AP sites . 32 5. Schematic of AP endonuclease expression vectors . 62 6. · AP endonuclease sequence alignment· . 65 7. Genomic organization of the rAPEN gene . 66 8. Complete 5' sequence of the rAPEN mRNA ... 68 9. Tissue expression of rAPEN mRNA by RT-PCR . 69 10. Multiple tissue Northern blot analysis of rAPEN mRNA expression . 71 11. Multiple tissue Northern blot analysis of hAPE mRNA expression . 72 12. In situ hybridization analysis of rAPEN mRNA during fetal development . 73 13. In situ hybridization analysis of rAPEN mRNA in adult male saggital and coronal rat brain tissue sections . 75 14. Emulsion autoradiographs of rAPEN mRNA in situ hybridization products in adult rat brain ... 76 15. In situ hybridization analysis of rAPEN mRNA in adult rat testis . .. 79 16. SOS-PAGE analysis of affinity purified recombinant GST-rAPEN protein . 81 17. Western analysis of Nb2 and Hl9-7 subcellular fractions for rAPEN protein expression . 84 vii Figure Page 18. Demonstration of hAPE antibody specificity by Western analysis . 85 19. Subcellular localization of hAPE in A1235 glioma cells by irnrnunocytochemistry . 87 20. Western analysis of KCNR subcellular fractions for hAPE protein expression . 88 21. Irnrnunohistochemistry analysis of hAPE protein expression in duodenum . 90 22. Irnrnunohistochemistry analysis of hAPE protein expression in hepatocytes . 91 23. Irnrnunohistochemistry analysis of hAPE protein expression in epidermis . • . • . 92 24. Irnrnunohistochemistry analysis of hAPE protein expression in hippocampus . 94 25. Irnrnunohistochemistry analysis of hAPE protein expression in cerebellum . 95 26. Irnrnunohistochemistry analysis of hAPE protein expression in motor neurons of the hypoglossal nucleus of the brainstem . 96 27. hAPE protein expression during the apoptotic process . 98 28. Specificity of the hMSH2 antibody .100 29. Irnrnunohistochemistry analysis of hMSH2 protein expression in duodenum ...... 101 30. Irnrnunohistochemistry analysis of hMSH2 protein expression in colon . ..... 102 31. Enhanced survival of murine 3T3 fibroblasts transfected with AP endonuclease coding regions after treatment with bleomycin .... 105 viii LIST OF TABLES Figure Page 1. Rat AP endonuclease repair activity . 82 ix LIST OF ABBREVIATIONS A adenine aa amino acid AP apurinic/apyrimidinic APE human apurinic/apyrimidinic endonuclease APEX mouse apurinic/apyrimidinic endonuclease Apnl Saccharomyces cerevisiae AP endonuclease bp base pair BSA bovine serum albumin c carboxyl c cysteine cDNA complementary deoxyribonucleic acid Ci Curie CIAP calf intestinal alkaline phosphate DEPC diethyl pyrocarbonate DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DTT dithiothreitol EDTA ethylenediamine tetraacetic acid FBS fetal bovine serum g gram G guanine x GIT guanidinium isothiocyanate GST glutathione s-transferase H202 hydrogen peroxide h hour(s) hAPE , human apurinic/apyrimidinic endonuclease IPTG isopropyl-P-D-thio-galactosidase kb kilobases kDa kilodalton LB Luria broth M molar min minute(s) mg milligram ml milliliter mol mole(s) MOPS 3-(N-morpholino] propanesulfonic acid MPG methylpurine-DNA glycosylase mRNA messenger ribonucleic acid N amino PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol PMSF phenylmethylsulfonylf luoride rAPEN rat AP endonuclease RNA ribonucleic acid Rrpl recombination repair protein 1 xi s second(s) SDS sodium dodecyl sulfate T thymine TBE tris boric acid EDTA TBS tris buffered saline u units UDG uracil-DNA glycosylase µg microgram µl microliter µM micromolar UV ultraviolet x g times gravity xii ABSTRACT The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. Apurinic and apyrimidinic sites are among the most common DNA lesions and can lead to polymerase halting, and, therefore, aberrant transcripts and even cell death. Consequentially, AP endonucleases have a critical role in the maintenance of DNA integrity and cell survival. This type of lesion results from the hydrolysis of the glycosylic bond between the base and deoxyribose and may occur through a variety of mechanisms including the following: First, AP sites can be produced by the effects of ionizing radiation and by oxidative agents that are the by-products of biological processes such as oxidative respiration of aerobic organisms, superoxide generation by activated macrophages and other immune cells, and by radiomimetic agents such as
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