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Isolation and Characterization of DNA-Damage-Repair/Toleration Genes Fromarabidopsis Thaliana

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Isolation and Characterization of DNA-Damage-Repair/Toleration Genes Fromarabidopsis Thaliana AN ABSTRACT OF THE THESIS OF Qishen Pang for the degree of Doctor of Philosophy in Geneticspresented onDecember 17, 1992. Title: Isolation and Characterization of DNA-damage- repair/toleration Genes from Arabidopsis thaliana Redacted for privacy Abstract approved: Joh B. ays Arabidopsis thaliana was used as a model greenplant for studies of DNA damage and repair following UVirradiation. Research results indicated the following: (1) UV (254 nm) fluences that result in measurable cyclobutanepyrimidine dimer (CBPD) levels (about one CBPD per 2 X104 nucleotides) have significant physiological effects; (2)photoreactivation is the predominant pathway of UV-induceddamage repair in Arabidopsis; (3) Arabidopsis photolyase levels areincreased significantly by UV-B irradiation; (4) Arabidopsisphotolyase is markedly temperature-sensitive, both invitro and in vivo. A genetic technique was developed toisolate Arabidopsis cDNAs encoding putative plantDNA-damage-repair/toleration (DRT) activities by partial complemetation of E.coli DNA- repair-deficient mutations.The initial work yielded six unique cDNAs,probably encoding four distinct classesof activities: (1) DRT100 increased resistance ofmutant bacteria to UV irradiation, mitomycin C and methylmethanesulfonate (MMS) treatments, supported the growthof )red- gam- and P1 phages,and promoted conjugationalrecombination in RecA" bacteria; (2) the activities encoded by DRT101and DRT102 appear to beUV-specific, sincethey failtoprovide resistance to DNA damage by mitomycin Cand MMS;(3) Drt103 conferred UV resistance in a light-dependent manner,but its amino acid sequence shows only marginalsimilarity to those of microbial photolyases; and (4) DRT111 andDRT112 increased resistance of E.coli resolvase-deficientmutants to UV, mitomycin C,MMS and nitroquinolineoxide, and partially restored the ability of these mutants to carryout conjugal recombination. The transcript levels of theArabidopsis DRT genes were examined in response to various DNA-damagingagents. Levels of DRT100 mRNA increased in plantstreated with UV-B light, mitomycin C and MMS. The transcript levelsof DRT101, whose activity is specific for UV-induced DNAdamage in E.coli, became elevated in response not only to UV-Birradiation but also to mitomycin C treatment. Isolation and Characterization of DNA-damage-repair/toleration Genes fromArabidopsis thaliana by Qishen Pang A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed December 17, 1992 Commencement June 1993 APPROVED: Redacted for privacy Profe or of Agricultural Chemistryin charge of major Redacted for privacy Directo of netiCs Program Redacted for privacy Dean of Gradu Date thesis is presented December 17, 1992 Typed by researcher for Qishen Pang ACKNOWLEDGMENTS I am very grateful to my Professor,Dr. John Hays for his guidance and support. His creative ideaswhich initiated these investigations,his valuable criticisms, hisenthusiastic encouragement during this study arehighly appreciated. I would like to thank Dr.Walt Ream(Oregon State University) and Dr. David Mount (Universityof Arizona) for providing seeds of Arabidopsis thaliana,Dr. Stephen Lloyd (Vanderbilt University), Dr. Aziz Sancar(University of North Carolina)and Dr. A. P. M. Eker(Delft University) for providing phage T4 endonuclease V, E.coliphotolyase, and A. nidulans photolyase, respectively, Dr. Robert Lloyd (University of Nottingham)for E.coli resolvase-deficient strains, Dr. Louise Prakash (University ofRochester) and Dr. Errol Friedberg (University of TexasSouthwestern Medical Center) forprovidingyeast strains, Dr. Ronald Davis (Stanford University)for his generous gift ofthe RYES Arabidopsis cDNA library and ?KC phages,and Drs. R.J.Ferl (University of Florida) and R.L.Feinbaum (HarvardUniversity) for providing DNA clones ofArabidopsis actin gene and chalcone synthase gene, respectively. Many thanks tothe Professors in my degree program committee, Drs. AnitaAzarenko, Terri Lomax, DaleMosbaugh and Walt Ream, for theirvaluable advice and criticalreview of this thesis.I alsowish to thank Dr. George Pearson,the Director of GeneticsProgram, for his thoughtful concernand encouragement. Some of the results would notexist without the kind help I have had from Dr. Timothy Schaefer (John Hopkins University). Thanks are also due to allthe people in my laboratory for their help and cooperationwhich made my stay pleasant and unforgetable. Finally, I do not have words to expressgratitude to my wife, Liya, for her great patience insharing the troubles during these years in Corvallis for ourfuture. 1 TABLE OF CONTENTS Chapter Page LIST OF FIGURES iii LIST OF TABLES LIST OF ABBREVIATIONS vi 1 I. INTRODUCTION A. Mechanisms of DNA Repair 2 1. Direct Reversal of DNA Damage 2 2. Base Excision Repair 8 3. Nucleotide Excision Repair 12 4. Recombinational Repair -Postreplication Repair 18 5. Regulation of DNA Repair -Inducible Reppair 22 B. Repair and Toleration of DNA Damagein Plants 24 1. Photoreactivation in Plants 24 2. Excision Repair in Plants 26 3. Recombinational Repair in Plants 27 29 II. PROJECT REVIEW A. Research Objectives 29 B. Rationale and Significance 30 1. Solar UV-B and Plant Responses 30 2. Arabidopsis as a Model Plant forRepair Studies 32 3. Importance of the ResearchObjectives 33 III. EXPERIMENTAL MATERIALS AND APPROACHES 36 A. Bacterial, yeast, and PhageStrains 36 B. Buffers, Media and Chemicals 36 C. Growth, UV and Chemical Treatmentsof Plants 40 D. Assays of UV Damage and Repairin Arabidopsis 41 E. Assays for Arabidopsis Photolyase 42 F. Isolation of Arabidopsis DRT Genes 43 1. Selection by ComplementingRecA-UvrC-Phr" Mutants 43 2. Selection by Complementationof ruvC recG Mutations 47 G. Characterization of the IsolatedDRT Genes 48 1. Measurement of Resistance toDNA-damaging Agents 48 2. Determination of BacteriophagePlating Efficiencies 50 3. Measurement of ConjugalRecombination Frequencies 51 4. Determination of RecA-dependentSOS Induction 52 5. PCR Amplication of DRT cDNAs 53 6. Plasmid DNA Preparation,Restriction Mapping, Cloning and DeletionConstruction 54 7. Determination and Analysis of DNASequences 57 8. Southern HybridizationAnalysis 58 9. RNA Preparation and NorthernAnalysis 60 63 IV. RESULTS A. Photoreactivation and DarkRepair in Arabidopsis 63 B. Thermosensitivity and UV-BInducibility of Arabidopsis Photolyase 65 C. Selection and Isolation ofArabidopsis DRT cDNAs 71 D. Characterization of theArabidopsis DRT cDNAs 74 1. Complementation ofE.coli Repair-deficient Mutations 74 2. Complementation of RecA-Phenotypes by Drt100 Activity 83 3. Complementation of RecombinationDeficiency by Drt111 and Drt112 Activities 86 4. DNA and Protein SequenceAnalyses 87 5. Deletion Analyses of Drt100and Drt101 100 6. Southern HybridizationAnalysis 102 7. Northern HybridizationAnalysis 102 110 V. SUMMARY AND DISCUSSION ASummary of the Project Achievements 110 B. Temperature-sensitive andUV-B-inducible Photoreactivation in Arabidopsis 114 C. The Technical Feasibility ofIsolating Plant DRT Genes by BacterialComplementation 116 D. Some Implications forComplementation Efficiencies of the Isolated DRT Genes 120 E. DRT100 and DRT101 mRNA LevelsIncrease in Response to a Variety ofDNA-damaging Agents 123 128 VI. REFERENCES iii LIST OF FIGURES Figure Page 1. Model for enzyme-catalyzed photoreactivation 5 14 2. Model for nucleotide excision repairin E.coli 3. Model for repair of a post-replication gapby RecA-mediated sister strand exchange 20 4. Schematic representation of expressionvector AYES and cre-lox-mediatedautomatic subcloning 44 64 5. Effects of UV irradiation onArabidopsis 66 6. Repair of cyclobutane pyrimidine dimers invivo 67 7. Time course of photoreactivation invitro 8. Temperature sensitivity of Arabidopsis photolyase in vitro 69 9. Temperature sensitivity of in vivo photoreactivation in Arabidopsis 70 10. UV-B induction of Arabidopsis photolyase 72 11. Resistance to UV of E.coli mutantsexpressing Arabidopsis DRT cDNAs 75 76 12. Resistance to chemical DNA-damaging agents 13. Complementation ofE.coli DNA-damage- sensitivity mutations by DRT100 in high- copy-number expression vector 78 14. Resistance to DNA-damaging agents ofbacteria expressing Drtlll and Drt112 81 15. Complementation of yeast rad2 mutation by Drt101 83 16. Sequences of Arabidopsis DRT100 cDNAand of longest open reading frame 89 91 17. Protein motifs in Drt100 18. Sequences ofDRT101 cDNAand predicted Drt101 polypeptide 93 19. Sequences ofDRT102 cDNAand predicted Drt102 polypeptide 95 iv 20. Sequences of DRT103 cDNA and predicted Drt103 polypeptide 96 21. DNA and predicted protein sequences for DRT111 98 22. DNA and predicted protein sequences forDRT112 99 23. Hybridization of DRT100 cDNA probe to plant DNA 104 24. Northern blot analysis of DRT mRNA levelsin plants exposed to UV-B irradiation 107 25. DRT mRNA levels in plants treatedwith DNA-damaging chemicals 108 V LIST OF TABLES Table Page 37 1. E.coli, yeast strains and bacteriophages 80 2. Plating of UV-irradiated phages 3. Partial correction of Rec" phenotypes by DRT100 85 4. Increased conjugal complementation by Drtlll and Drt112 88 5. UV-resistance activity of partial DRT100 deletions 101 6. UV-resistance activity of partial DRT101 deletions 103 7. Quantitation of DRT mRNA levels by densitometric scanning 105 8. Putative activities of Arabidopsis DRT cDNAs 112 vi LIST OF ABBREVIATIONS Ap: ampicillin AP: apurinic/apyrimidinic ApR: ampicillin-resistant Aps: ampicillin-sensitive CBPD: cis syn-cyclobutane pyrimidinedieter
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