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1. INTRODUCTION 19
1.1. Human RAD51 19
1.1.1. RAD51 in a repair mechanism 19
1.1.2. Interacting proteins with RAD51 22
1.1.3. Modification and expression of RAD51 in tumor cells 24
1.1.4. RAD51 in apoptosis 25
1.2. ASK1 as an apoptotic inducer 27
1.3. ABL-tyrosine kinase as a RAD51 modifier 30
1.4. Apoptosis as a "fail-safe" mechanism 33
1.5. The aim of this study 35
2. MATERIALS AND METHODS 38
2. 1. Construction of epitope-tagged RAD51 clones in mammalian expression vectors 38
2. 2. Construction of deletion and point mutants for RAD51 in mammalian expression vectors 38
2. 3. Construction of GST-tagged ASK1 expression clones 39
2. 4. In vitro binding assay for RAD51 and ASK1 40
2. 5. Cell culture and transfection 40
2. 6. Antibodies for the experimentation 41
2. 7. Immuno-fluorescence analysis using a fluorescence microscopy 42
2. 8. Subcellular fractionation and localization 43
2. 9. Co-immunoprecipitation 43
2. 10. Immunoblotting 44
2. 11. Caspase activity assay 45
2. 12. Morphological analysis of cells 46
2. 13. In vitro JNK and p38 kinase assays 46
2. 14. ASK1 kinase assay 47
2. 15. Constructions of Flag-tagged MAPK and dominant negative clones in mammalian expression vectors 48
2. 16. Inhibition of BCR/ABL tyrosine Kinase 49
3. RESULTS 50
3.1. Interaction of RAD51 with ASK1 50
3.1.1. RAD51 interacts to ASK1 in HEK 293 and 293T cells 50
3.1.2. RAD51 and ASK1 interaction occurs in the cytoplasm of HEK 293 cells 50
3.1.3. RAD51 interaction regions within ASK1 55
3.2. Inhibition of ASK1 kinase activity by RAD51 58
3.2.1. Direct inhibition of ASK1 kinase activity by RAD51 58
3.2.2. MEKK1 was not inhibited by RAD51 58
3.2.3. Down-regulation of ASK1 downstream MAP-Kinases activities by RAD51, in vitro 62
3.2.4. Inactivation of ASK1 downstream MAP kinases by RAD51 co-expression, in vivo 67
3.2.5. Dose-dependent ASK1 inhibition of RAD51 67
3.3. Inhibition of ASK1-mediated apoptosis 71
3.3.1. Inhibition of TNF-α induced apoptosis by RAD51 71
3.3.2. Effect of RAD51 on Caspase 3 activity stimulated by ASK1 expression 74
3.3.3. Inhibition of ASK1 dimerization by RAD51 78
3.3.4. RAD51 blocked JNK activation caused by TNF-α treatment 78
3.3.5. Reduced interaction of RAD51 with ASK1 after TNF-α treatment in 293 cell 82
3.4. Requirement of phosphorylation at Y315 of RAD51 forASK1 inhibition 84
3.4.1. Effects of RAD51 tyrosine mutants on C-Jun N-terminal kinase activation by ASK1 84
3.4.2. Effect of mutations on the conserved Walker type domains of RAD51 in ASK1 inhibition 87
3.4.3. Interactions of RAD51 mutants with GST-tagged ASK1 deletion series 89
3.4.4. RAD51 phosphorylation was decreased via ABL tyrosine kinase inactivation by STI571 (Gleevec) in HEK 293 cells 89
3.4.5. Effect of STI571 on ASK1 inactivation by RAD51 97
3.5. Cleaved RAD51 by Caspase-3 triggers apoptosis : a "Fail-Safe" mechanism 103
3.5.1. RAD51 was cleaved during apoptosis induced by various stress signals 103
3.5.2. Expression of RAD51 truncation mutants in HEK 293 cells 103
3.5.3. Localization of RAD51 truncation mutants 106
3.5.4. Activation of Jun N-terminal kinase by the RAD51 C-terminus expressed in HEK 293 cells 106
3.5.5. Caspase activation by the RAD51 C-terminus in HEK 293 cells 109
3.5.6. Apoptosis induced by the C-terminus of RAD51 in HEK 293 cells was independent with ASK1 and JNK activations 109
3.5.7. Mitochondria independency in apoptosis induced by the C-terminus of RAD51 in HEK 293 cells 113
3.5.8. Caspase-3 inhibitors blocked cell death induced by expression of the RA51-C terminus 118
4. DISCUSSION 121
5. REFERENCES 128
6. ABSTRACT IN KOREAN 138
7. ACKNOWLEDGEMENT 141
8. APPENDIX 142
Figure 1.1. Homologous recombination by RAD51 20
Figure 1.2. Various proteins interacting with RAD51 23
Figure 1.3. Phosphorylation of RAD51 by various kinases 26
Figure 1.4. Overview in ASK1 induced apoptosis 28
Figure 1.5. MAP Kinase cascade modules 29
Figure 1.6. Generation and Reaction of BCR/ABL 32
Figure 1.7. Structure and the reaction mechanism of STI 34
Figure 1.8. The relation of DNA repair and apoptosis 36
Figure 3.1. Interactions of RAD51 and ASK1 in HEK 293 and 293T cells 51
Figure 3.2. Localization of ASK1 and RAD51 proteins in HEK 293 cells 53
Figure 3.3. RAD51 interaction regions in ASK1 56
Figure 3.4. Inhibition of ASK1 kinase activity by RAD51 59
Figure 3.5. Inhibition of ASK1 but not MEKK1 by RAD51 60
Figure 3.6. In vitro kinase assays for the effect of RAD51 and ASK1 on JNK activity 63
Figure 3.7. In vitro kinase assays for the effect of RAD51 and ASK1 on p38 kinase activity 65
Figure 3.8. Inactivation of ASK1-downstream MAP kinases byRAD51 co-expression, in vivo 68
Figure 3.9. Dose-dependent ASK1 inhibition by RAD51 70
Figure 3.10. Inhibition of TNF-α induced apoptosis by RAD 72
Figure 3.11. Inhibitory effect of RAD51 on Caspase-3 activity activated by ASK1 expression 75
Figure 3.12. Inhibition of ASK1 dimerization by RAD51 79
Figure 3.13. Down-regulation of JNK activation after treatments of certain DNA damaging agents by RAD51 80
Figure 3.14. Reduced interaction of RAD51 with ASK1 after TNF-α treatment in 293 cells 83
Figure 3.15. Effects of the RAD51 tyrosine mutants on JNK activation by ASK1 over-expression 85
Figure 3.16. Effects of RAD51 tyrosine mutations on Caspase-3 activation by ASK1 88
Figure 3.17. Effects of the RAD51 mutations in the Walker type domains on JNK activation by ASK1 90
Figure 3.18. Dephosphorylation of RAD51 after STI571 treatment via ABL-inactivation in 293 cells 95
Figure 3.19. Effect of STI571 on ASK1 inactivation by RAD 99
Figure 3.20. Effect of STI571 in ASK1 inactivation by RAD315 tyrosine mutants (Y315F) 101
Figure 3.21. Hypothetical scheme of ASK1 inhibition by RAD ; in BCR/ABL positive cells 102
Figure 3.22. Endo-RAD51 cleavage by stress signals 104
Figure 3.23. Induction of apoptosis by the RAD51 C-terminus 107
Figure 3.24. Localization of RAD51 truncation mutants 108
Figure 3.25. Activations of JNK and p38 kinases by the RAD51 C- and N-terminal truncation mutants 110
Figure 3.26. Various caspase activities after over-expression of the RAD51-C terminus in HEK 293 cells 112
Figure 3.27. JNK and Caspase-3 activation by the RAD51-C terminus with co-expression of dominant negative forms of JNK upstream MAP kinases 114
Figure 3.28. Cell morphology after transient transfection of the RAD51 C-terminus with/without coexpression of dominant negative forms of MAPKs 116
Figure 3.29. Requirement of Caspase-3 in cell death induced by the RAD51 C-terminus 117
Figure 3.30. The caspase-3 inhibitors including ZVAD-fmk blocked cell death by overexpression of the RA51-C terminus 119
Figure 4.1. Scheme of anti-and pro-apoptotic roles of RAD 127
Table 1. The RAD51 paralogs 21
Table 2. Summary of the effects of RAD51 mutants in ASK1 inhibition 92
Table 3. Summary of in vitro binding assays for various RAD51 mutants and ASK1 93
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