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University of Cincinnati UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ A thesis submitted to the Division of Graduate Studies and Research of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Department of Biological Sciences of the College of Arts and Sciences 2008 Targeted Mutation of Genes Implicated in DNA Replication and Repair in Sulfolobus acidocaldarius by Laura A. Runck B.S., Bowling Green State University, 2001 Committee Chair: Dr. Dennis W. Grogan Abstract The main goals of this research were to evaluate different experimental methods for gene cloning, disruption, and transfer to the Sulfolobus acidocaldarius genome, and to determine function of the genes in vivo. S. acidocaldarius and other hyperthermophilic archaea thrive at temperatures 80◦C and above which would typically render the DNA unstable. It is not well understood how the hyperthermophilic archaea such as Sulfolobus acidocaldarius maintain their DNA, but genomic analyses have found some homologs to known DNA repair and replication genes. These include genes implicated in nucleotide excision repair, photoreactivational repair, alternative excision repair, homologous recombinational repair, and DNA replication. Topoisomerase-mediated cloning of PCR products provided the most reliable cloning method, whereas disruption of cloned genes was more difficult. One cloned disruption (trpC::pyrE) made using restriction enzymes and ligase, and two linear PCR disruptions (phr::pyrE; uvde::pyrE) made by a direct PCR method, were tested for integration into the S. acidocaldarius genome. Analysis of successful transformants by PCR and sequencing indicate that all three disruptions have been integrated into the S. acidocaldarius via homologous recombination. In two cases the phenotypes have been confirmed. The trpC disruptant (DG251) is a tryptophan auxotroph and the phr disruptant (LR10) lacks photoreactivational repair. This work is significant because targeted gene disruption has not been reported for S. acidocaldarius and it provides more genetic tools for use in Sulfolobus spp. It also gives evolutionary insights into the diversity and similarities among the three domains of life, and even more specifically evolutionary insight into the molecular processes of the hyperthermophilic archaea. iii iv Acknowledgments I would first like to thank Dr. Dennis Grogan for being a wonderful advisor and teacher, and for all of the time and help throughout my graduate career. I would also like to thank my research advisory committee, Dr. Brian Kinkle and Dr. Charlotte Paquin for their help and suggestions in making this research a success. I would like to thank all past and present Grogan lab members – Reena Mackwan for helping me getting adjusted to the lab, Cynthia acidocaldarius Bellamy for starting our journal club, never ending support and help with everything and a great friendship, Dominic Mao for being the practical one in the lab, always helping me on the chalk board and being a great friend, and Janine Rockwood for keeping the lab in shape and always being entertaining. Good luck to all of you guys! I would also like to acknowledge my family and friends for their continued support even though you have no idea what I really do. Thanks! Finally, I would like to thank my lovely husband, Mike for being so supportive and patient throughout my graduate career. I’m finally done with school! v Table of Contents Committee Approval Page i Title Page ii Abstract iii Acknowledgments v Table of Contents vi List of Tables viii List of Figures ix List of Abbreviations xi Chapter I. DNA Replication and Repair in Archaea 1. Background of Archaea 1 2. DNA Replication in Archaea 3 3. DNA Repair in Archaea 6 4. Importance of Studying These Archaeal Processes 9 5. Biology of Sulfolobus acidocaldarius 11 6. Genes Implicated in DNA Repair in Sulfolobus acidocaldarius 12 Based on Sequence Similarity 7. Genes Implicated in DNA Replication in Sulfolobus acidocaldarius 24 Based on Sequence Similarity 8. Determining Functions of Sulfolobus Genes 25 9. Gene Disruption 30 10. References 31 Chapter II. Evaluating Techniques for Cloning Sulfolobus Genes vi 1. Methods Evaluated 45 2. Cloning by PCR, Restriction Digestion and Ligation 48 3. Cloning by PCR and –G (-A) Overhang Vectors 57 4. Summary 61 5. References 62 Chapter III. Evaluating Methods for Disruption of Sulfolobus Genes 1. Methods Evaluated 64 2. PCR, Restriction Digestion and Ligation 64 3. Overlap Extension PCR (OEP) 66 4. USER™ 68 5. Inverse PCR 70 6. Direct-Tailing by PCR 73 7. Summary 75 8. References 76 Chapter IV. Integration into the Sulfolobus acidocaldarius Genome 1. Introduction 79 2. Positive Control 79 3. Direct-Tailed Constructs 80 4. Phenotypic Determination of phr Mutant 82 5. Summary 84 6. Future Directions and Significance 85 7. References 86 vii List of Tables Number Title Page Table 2.1 Primers used to amplify genes cloned 46 Table 2.2 Summary of plasmids containing cloned genes with method 47 of cloning described Table 3.1 Primers used for direct-tailing by PCR 74 Table 4.1 Sulfolobus acidocaldarius strains used for phenotypic 80 determination experiments Table 4.2 Evidence for photoreactivational repair as a ratio between 84 photoreactivated:dark-maintained viability viii List of Figures Number Title Page Figure 2.1 Gel verification of amplification of Sulfolobus solfataricus pyrE 50 Figure 2.2 Gel verification of Sso pyrE ligation into pNEB193 to create pLK3a 50 Figure 2.3 Screening pLK3a with SspI and SpeI to verify cloning 51 Figure 2.4 Restriction enzyme analysis to verify insertion of cml gene into 53 pLK3a Figure 2.5 Map of pLK5a 53 Figure 2.6 Illustration of how the three homologs of Saccharomyces cerevisiae 55 RAD genes were cloned into pUC19 Figure 2.7 Restriction enzyme analysis verifying cloning of rad1 into pUC19, 56 creating pRAD1 Figure 2.8 Restriction enzyme analysis verifying cloning of rad2 into pUC19, 56 creating pLK6a Figure 2.9 Restriction enzyme analysis verifying cloning of rad25 into pUC19, 57 creating pRAD25 Figure 2.10 Restriction enzyme analysis verifying cloning of phr into 60 TOPO vector Figure 3.1 Map of pLK4A1f 63 Figure 3.2 General schematic of OEP 67 Figure 3.3 Schematic of USER method 70 Figure 3.4 Schematic of inverse PCR 72 Figure 3.5 Schematic of direct-tailing by PCR 75 ix Figure 4.1 Verification of phr-“tailed” Sso pyrE integration into PHR 81 locus via PCR screening of LR10 genomic DNA Figure 4.2 Verification of uvde-“tailed” Sso pyrE integration into UVDE 81 locus via PCR screening of LR12 genomic DNA Figure 4.3 Log survival of LR10, DG185, and DG251 after UV exposure 83 and white light illumination x List of Abbreviations and Symbols aa…..amino acid AER…..alternative excision repair amp…..ampicillin ampR…..ampicillin resistance ampS…..ampicilllin sensitive A-T…...Adenine - Thymine ATP…..adenosine 5’-triphosphate? bla…..betalactamase? bp…..base pairs cfu…..colony forming unit cml…..chloramphenicol acetyl transferase cmlR…..chloramphenicol acetyl transferase resistance cmlS…..chloramphenicol acetyl transferase sensitive C-terminal…..carboxy-terminal? CPDs…..cyclobutane pyrimidine dimers DGS….Aspartic acid, Glycine, Serine DNA…..deoxyribonucleic acid DNase…..deoxyribonuclease dNTPs…..deoxyribonucleotide triphosphates ds…..double-stranded DSB(s)…..double strand break(s) FAD…..flavin adenine dinucleotide xi Foa…..5-fluoroorotic acid Foar….. 5-fluoroorotic acid resistance G+C…..Guanine + Cytosine GY…..Glycine, Tyrosine HR…..Homologous recombination(al) hr…..hour IPTG…..Isopropyl-β-D-1-thiogalactopyranoside kan…..kanamycin kb…..kilobase Mbp…..mega base pairs MCM…..minichromosome maintenance proteins mg…..milligram Mg2+…..magnesium mins…..minutes ml…..milliliter MMR…..mismatch repair Mn2+…..manganese MTHF…..methenyltetrahydrofolate MW…..molecular weight standard N-terminal…..amino-terminal NEB…..New England Biolabs, Inc. (Ipswich, MA) NER……Nucleotide excision repair nm…..nanometers xii nt…..nucleotide OB fold…..oligonucleotide/oligosaccharide binding fold OEP…..Overlap extension PCR ORBs…..origin recognition boxes ORC…..origin recognition complex RE(s)…..restriction endonuclease(s) RNA…..ribonucleic acid RPA…..Replication Protein A PCNA…..proliferating cell nuclear antigen PCR…..Polymerase chain reaction PfuCx…..PfuTurbo® Cx Hotstart DNA Polymerase PRE…..photoreactivation Pyr<>Pyr…..pyrimidine dimer s…..second Sac…..Sulfolobus acidocaldarius Sdil…..Sulfolobus dilution buffer spp…...species ss…..single-stranded SSBs…..single stranded binding proteins Sso…..Sulfolobus solfataricus tRNA…..transfer ribonucleic acid U…..unit UDG…..Uracil DNA glycosylase xiii USER™…..Uracil-specific excision reagent UV…..ultraviolet UVDE…..UV DNA damage endonuclease µg…..microgram µl…..microliter WT…..wild type Xgal…..5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside XT…..xylose, tryptone media YT…..yeast extract tryptone media 2-D…..2 dimensional 3’…..three prime end of DNA with a terminal hydroxyl group 5’…..five prime end of DNA with a terminal phosphate group 6-4PPs…..pyrimidine-pyrimidone 6-4 photoproducts ◦C…..degrees Celsius Ψ-Ψ-C-G…..pseudouridine-pseudouridine-cytidine-guanine T- Ψ-C-G…..threonine-pseudouridine-cytidine-guanine
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