Proteomic analysis of the Rad18 interaction network in DT40 – a chicken B cell line Thesis submitted for the degree of Doctor of Natural Sciences at the Faculty of Biology, Ludwig-Maximilians-University Munich 15th January, 2009 Submitted by Sushmita Gowri Sreekumar Chennai, India Completed at the Helmholtz Zentrum München German Research Center for Environmental Health Institute of Clinical Molecular Biology and Tumor Genetics, Munich Examiners: PD Dr. Berit Jungnickel Prof. Heinrich Leonhardt Prof. Friederike Eckardt-Schupp Prof. Harry MacWilliams Date of Examination: 16th June 2009 To my Parents, Sister, Brother & Rajesh Table of Contents 1. SUMMARY ........................................................................................................................ 1 2. INTRODUCTION ............................................................................................................. 2 2.1. MECHANISMS OF DNA REPAIR ......................................................................................... 3 2.2. ADAPTIVE GENETIC ALTERATIONS – AN ADVANTAGE ....................................................... 5 2.3. THE PRIMARY IG DIVERSIFICATION DURING EARLY B CELL DEVELOPMENT ...................... 6 2.4. THE SECONDARY IG DIVERSIFICATION PROCESSES IN THE GERMINAL CENTER .................. 7 2.4.1. Processing of AID induced DNA lesions during adaptive immunity .................. 9 2.5. TARGETING OF SOMATIC HYPERMUTATION TO THE IG LOCI ............................................ 10 2.6. ROLE OF THE RAD6 PATHWAY IN IG DIVERSIFICATION PROCESSES ................................. 12 2.7. THE RAD6 PATHWAY ..................................................................................................... 12 2.7.1. Translesion Synthesis ............................................................................................. 13 2.7.2. Error free bypass .................................................................................................... 14 2.7.3. Interactions of components of the Rad6 pathway .................................................. 15 2.8. MOLECULAR INTERPLAY BETWEEN THE RAD6 PATHWAY AND CHECKPOINT SIGNALLING COMPONENTS ........................................................................................................................ 15 2.9. OTHER FUNCTIONS OF COMPONENTS OF THE RAD6 PATHWAY ........................................ 16 2.10. FUNCTIONAL CHARACTERISATION OF RAD18 PROTEIN DOMAINS ................................. 17 2.11. BIOCHEMICAL AND GENETIC APPROACHES TO STUDY CELLULAR NETWORKS ............... 18 2.11.1. The tandem affinity purification (TAP) technique ................................................ 19 2.11.2. The DT40 system .................................................................................................. 21 2.12. OBJECTIVES .................................................................................................................. 21 3. RESULTS ........................................................................................................................ 23 3.1. ESTABLISHMENT OF TAP IN THE DT40 CELL LINE USING AID-TAP FUSIONS ................ 23 3.1.1. Expression of the AID-TAP fusion proteins in the DT40 cell line ......................... 24 3.1.2. Tandem affinity purification in DT40 cells using the AID-TAP fusions ................ 28 3.1.2.1. Western blot analysis of the TAP fractions in DT40 cells .............................. 28 3.1.2.2. Silver Stain Analysis of the TAP fractions ..................................................... 30 3.1.3. Trypsin Digestion and nano-Liquid Chromatography of the final eluates ............ 30 3.1.4. MALDI TOF-TOF analysis of the peptides of the AID fusion eluates ................... 32 3.1.5. Generation of an NCBI Gallus database and MASCOT analysis for AID-TAP fusions ............................................................................................................................... 32 3.2. STUDY OF THE RAD18 INTERACTION NETWORK IN DT40 CELLS ..................................... 35 i Table of Contents 3.2.1. Exogenous expression of Rad18-HA-TAP fusions in rad18-/- DT40 cells .............. 35 3.2.2. Native PAGE of Rad18 fusion protein complexes .................................................. 36 3.2.3. Cellular localization of Rad18 fusion proteins ...................................................... 36 3.2.4. Functional Characterization of Rad18-HA-TAP fusions ....................................... 38 3.2.5. Isolation of Rad18-HA-TAP fusion complexes by the TAP method ....................... 39 3.2.6. Silver Stain analysis of the Rad18 TAP fractions .................................................. 40 3.2.7. LC-MALDI analysis of Rad18 TAP samples .......................................................... 41 3.2.8. MASCOT analysis for Rad18 fusions ..................................................................... 41 3.2.9. Immunoprecipitation attempts for confirmation of some potential Rad18 interaction partners .......................................................................................................... 44 3.2.10. Functional Link of Rad18 to the JNK pathway .................................................... 46 4. DISCUSSION .................................................................................................................. 50 5. MATERIALS ................................................................................................................... 64 5.1. CHEMICALS AND REAGENTS ........................................................................................... 64 5.2. EQUIPMENTS ................................................................................................................... 64 5.3. KITS................................................................................................................................ 65 5.4. BACTERIA ....................................................................................................................... 65 5.5. ENZYMES ........................................................................................................................ 65 5.6. ANTIBODIES .................................................................................................................... 66 5.7. OLIGONUCLEOTIDES ....................................................................................................... 67 5.8. PLASMIDS ....................................................................................................................... 68 5.9. CELL LINES ..................................................................................................................... 68 5.10. SOFTWARES .................................................................................................................. 69 6. METHODS ...................................................................................................................... 70 6.1. STANDARD METHODS ..................................................................................................... 70 6.2. CLONING OF THE RAD18-HA-TAP FUSIONS ................................................................... 70 6.3. CLONING OF RAD18-HA (R18-HA1) ............................................................................. 70 6.4. RNA ISOLATION AND CDNA SYNTHESIS ........................................................................ 71 6.5. SDS PAGE & WESTERN BLOT ANALYSIS ....................................................................... 71 6.6. NATIVE PAGE & BLOTTING ........................................................................................... 71 6.7. TAP EXTRACTION OF GOI-TAP FUSIONS ....................................................................... 72 6.8. TANDEM AFFINITY PURIFICATION .................................................................................. 72 6.9. TCA PROTEIN PRECIPITATION ......................................................................................... 73 6.10. SILVER STAIN ANALYSIS ............................................................................................... 73 ii Table of Contents 6.11. IN-GEL TRYPSIN DIGEST .............................................................................................. 74 6.12. REVERSE PHASE NANO-LIQUID CHROMATOGRAPHY .................................................... 74 6.13. MALDI TOF-TOF ....................................................................................................... 75 6.14. CO-IMMUNOPRECIPITATION IN THE RAD18-HA-TAP DT40 CELLS .............................. 77 6.15. ENDOGENOUS CO-IMMUNOPRECIPITATION IN RAJI CELLS ............................................. 77 6.16. CELL CULTURE ............................................................................................................. 77 6.17. TRANSFECTION OF DT40 CELLS ................................................................................... 78 6.18. COLONY SURVIVAL ASSAY FOR DT40 USING CHEMICAL AGENTS ................................. 79 6.19. NUCLEAR/CYTOPLASMIC FRACTIONATION ................................................................... 79 6.20. DNA DAMAGE INDUCED ACTIVATION OF THE JNK/AP1 PATHWAY .............................. 79 REFERENCES ....................................................................................................................... 80 APPENDIX .............................................................................................................................