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Contents
Abbreviations 14
I. Abstract 16
II. Introduction 18
Subject 1: The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals 22
Abstract 23
1. Introduction 25
2. Materials and methods 28
2.1. Yeast strains, cultures, cell cycle synchronization 28
2.2. Construction of BFA1 deletion mutants 28
2.3. Microscopic techniques and FACS analysis 29
2.4. Two-hybrid analyses 30
2.5. Protein techniques 30
2.6. Random mutagenesis of plasmid DNA 31
3. Results 32
3.1. Bfa1-D8391-574, the 184 amino acids in the C-terminus, is sufficient to arrest mitosis exit in response to spindle damage(이미지참조) 32
3.2. Bfa1-D8391-574 is sufficient to induce mitotic arrest by spindle misorientation(이미지참조) 34
3.3. Bfa1-D8391-574 is sufficient to arrest mitosis in response to DNA damage(이미지참조) 35
3.4. Only D8 can aggravate the defect of Ddc5p mutations that is suppressed when Bfa1p is deleted 36
3.5. D8391-574 and D161-376 are two independent domains that induce mitotic arrest when overexpressed 37
3.6. Only Bfa1-D8391-574 is necessary for Bfa1p localization to the SPB(이미지참조) 40
3.7. The mutant of Bfa1-D8, D8E438K, is deprived of the checkpoint function of D8(이미지참조) 41
4. Discussion 43
국문요약 73
Subject 2: The GAP activity of Bfa1p and its phosphorylation by Cdc5p are not essential to regulate the mitotic exit network in budding yeast 75
Abstract 76
1. Introduction 78
2. Materials and methods 81
2.1. Yeast strains, cultures, and cell cycle synchronization 81
2.2. Random mutagenesis of plasmid DNA 81
2.3. GTPase activity assays 82
2.4. Microscopy and FACS analysis 82
2.5. Yeast two-hybrid analyses 83
2.6. Protein analyses 83
3. Results 84
3.1. Bfa1p-D8M413I does not cause mitotic arrest when overexpressed(이미지참조) 84
3.2. Bfa1pM413I interacts with Tem1p and Bub2p on the SPB(이미지참조) 85
3.3. The Bfa1pM413I-Bub2p complex lacks GAP activity(이미지참조) 86
3.4. The GAP activity of Bfa1p-Bub2p complex is not essential to control the MEN 90
3.5. Cdc5p does not phosphorylate Bfa1pM413I(이미지참조) 91
3.6. Cdc5p-mediated phosphorylation of Bfa1p is essential not to promote mitotic exit but to direct Bfa1p to the SPB toward bud 93
3.7. BFA1M413I cells fail to induce mitotic arrest in response to spindle damage(이미지참조) 95
3.8. BFA1M413I cells induce mitotic arrest in response to spindle mis-orientation and DNA damage(이미지참조) 96
3.9. Bfa1pMB mutant contains GAP activity with Bub2p but is not be phosphorylated in vivo(이미지참조) 97
4. Discussion 100
4.1. Triggering mitotic exit in budding yeast 100
4.2. Cdc5p plays a role in asymmetrically localizing Bfa1p on SPBdaughter at anaphase onset(이미지참조) 101
4.3. The GAP activity of Bfa1p-Bub2p complex is required to arrest the cell cycle in response to spindle damage 104
5. Supplementary materials 135
국문요약 149
Subject 3: The GAP activity of Bfa1p-Bub2p is responsible for mitotic arrest in response to spindle damage and Bfa1p overexpression, but not to spindle misorientation and DNA damage 151
Abstract 152
1. Introduction 154
2. Materials and methods 157
2.1. Yeast strains and random mutagenesis of plasmid DNA 157
2.2. Random mutagenesis of plasmid DNA 157
2.3. GTPase activity assays 158
2.4. Protein analyses 158
2.5. Yeast two-hybrid analyses 158
2.6. Microscopy and FACS analysis 159
3. Results 160
3.1. Bfa1p-D8E438K does not cause mitotic arrest when overexpressed(이미지참조) 160
3.2. Bfa1pE438K interacts with Tem1p and Bub2p on the SPB(이미지참조) 160
3.3. Bfa1pE438K is phosphorylated by Cdc5p(이미지참조) 161
3.4. The GAP activity of Bfa1pE438K-Bub2p is slightly decreased(이미지참조) 162
3.5. Bfa1pE438K-Bub2p complex is capable of regulating mitotic exit in similar kinetics to Bfa1p-Bub2p(이미지참조) 163
3.6. BFA1E438K cells induce mitotic arrest in response to diverse checkpoint-activating signals(이미지참조) 165
3.7. BFA1E438K suppresses the growth of cdc5-1△bfa1 mutant, but not of cdc5-2△bfa1 166
4. Discussion 168
국문요약 189
III. Reference 191
IV. 국문요약 198
Table 1. Yeast strains used in this study 48
Table 2. Oligonucleotides used to construct the Bfa1 deletion mutants 49
Table 3. Construction of the BFA1 deletion mutants 50
Table 1. Yeast strains used in this study 106
Supplementary tabel 1. Yeast strains used in the supplementary study 148
Table 1. Yeast strains used in this study 171
Figure 1. Summary of the functional domains of Bfa1p 51
Figure 2. The C-terminal 184 amino acids of Bfa1p, Bfa1-D8, bear the ability of Bfa1p to… 53
Figure 3. Bfa1-D8 bears the ability of Bfa1p to generate M arrest in response to spindle misorientation 57
Figure 4. Bfa1p-D8 bears the ability of Bfa1p to induce M arrest in response to DNA damage 60
Figure 5. Bfa1-D8 restores the mitotic exit defects of Cdc5p mutations that are suppressed in cdc3 bfa1△ 62
Figure 6. Bfa1 can be bipartite into the N-terminal 376 amino acids (Bfa1-D16) and the… 64
Figure 7. Bfa1-D8 bears the SPB localization activity of Bfa1p 69
Figure 8. The mutant of Bfa1-D8391-574, D8E438K, is deprived of the checkpoint function of D8 for MEN regulation(이미지참조) 71
Figure 1. Bfa1p-D8M413I does not induce mitotic arrest when overexpressed(이미지참조) 108
Figure 2. Bfa1pM413I interacts with Tem1p and Bub2p on the SPB(이미지참조) 111
Figure 3. The Bfa1 pM413I-Bub2p complex lacks GAP activity(이미지참조) 114
Figure 4. The GAP activity of the Bfa1p-Bub2p complex is not essential to control the MEN 117
Figure 5. Cdc5p does not phosphorylate Bfa1pM413I(이미지참조) 120
Figure 6. Cdc5p-mediated phosphorylation of Bfa1p is essential not to promote mitotic exit but to direct Bfa1p to the SPB toward bud(이미지참조) 122
Figure 7. Bfa1pM413I cannot inhibit mitotic exit in response to spindle damage(이미지참조) 124
Figure 8. Bfa1pM413I induces mitotic arrest in response to spindle mis-orientation and DNA damage(이미지참조) 127
Figure 9. The characterization of Bfa1pMB mutant(이미지참조) 130
Supplementary figure 1. The phenotype of full-length bfa1pM413I or Bfa1pMB overexpressing cells(이미지참조) 135
Supplementary figure 2. Interactions between Bfa1p-D8M413I and Tem1p or Bub2p were…(이미지참조) 136
Supplementary figure 3. The localization of Tem1p-RFP in BFA1 or △bfa1 cells 137
Supplementary figure 4. The GTPase activity of Tem1p was measured in the presence… 138
Supplementary figure 5. The quantitative analysis of the interactions between Bfa1pMB…(이미지참조) 141
Supplementary figure 6. Bfa1pMB inhibits mitotic exit in response to spindle damage(이미지참조) 142
Supplementary figure 7. Bfa1pMB induces mitotic arrest in response to spindle mis-orientation(이미지참조) 143
Supplementary figure 8. Bfa1pMB induces mitotic arrest in response to DNA damage(이미지참조) 144
Supplementary figure 9. Bfa1p-11A-GFP is localized on only SPBdaughter in anaphase(이미지참조) 145
Supplementary figure 10. Bfa1pMB cannot be efficiently phosphorylated by Cdc5p(이미지참조) 146
Supplementary figure 11. Ability of BFA1M413I and BFA1MB to alleviate the mitotic exit defect of cdc5-1 and cdc5-2(이미지참조) 147
Figure 1. Bfa1p-D8E438K does not cause mitotic arrest when overexpressed(이미지참조) 172
Figure 2. Bfa1pE438K interacts with Tem1p and Bub2p on the SPB(이미지참조) 175
Figure 3. Cdc5p phosphorylates Bfa1pE438K(이미지참조) 177
Figure 4. The GAP activity of Bfa1pE438K-Bub2p complex is slightly decreased(이미지참조) 179
Figure 5. Bfa1pE438K-Bub2p complex is capable of regulating mitotic exit in similar…(이미지참조) 181
Figure 6. Bfa1pE438K can inhibit mitotic exit in response to diverse checkpoint-activating signals(이미지참조) 183
Figure 7. BFA1E438K suppresses the growth of cdc5-1△bfa1 mutant, but not of cdc5-2△bfa1(이미지참조) 187
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