在許多遺傳性乳癌及卵巢癌病患中常發現有BRCA1的基因異常。 在細胞中，BRCA1 N端RING domain會和BARD1 Ｎ端 RING domain相互形成heterodimer，並共同調控cell cycle checkpoints，DNA damage repair，以及centrosome regulation。結構上，BARD1的Ｃ端含有兩個串連的BRCT domains，這個domains 對於一些參與DNA修復的磷酸化蛋白結合具有重要的指標。在動物細胞中，中心體負責組織microtubule networks，並負責bipolar mitotic spindle的生成。在細胞週期中BRCA1/BARRD1會一同移動到中心體內部，因此，若中心體的調控失常，將會引起中心體的增生以及細胞分裂異常。 OLA1在細胞分裂時也會集中到中心體內部，研究指出， OLA1 的knockdown會引發centrosome的增生以及過度活化centrosomal aster的形成。此外，研究發現OLA1和BARD1 BRCT domains具有直接的interaction，因此我們利用核磁共振實驗辨認出BARD1 BRCT domains對於OLA1的possible binding sites，並利用分光光譜儀測試這些BRCT domains 上的critical residues對於OLA1的ATPase 活性有何影響。

The germline line mutations in BRCA1 (Breast cancer-associated susceptibility gene 1) can be found in patients who have heredity breast and ovarian cancer. As a tumor suppressor, BRCA1 interacts with BARD1 (BRCA1-associated RING domain protein 1) that together form a heterodimer complex and play an important role in cell cycle checkpoints, DNA damage repair, and centrosome regulation. The C-terminal BARD1 contains two tandem BRCT domains, which is essential for phosphopeptide-binding found in various proteins function in DNA damage repair. In animal cells, centrosomes organize microtubule networks and are critical for the establishment of bipolar mitotic spindle. Centrosomes duplicate once pre cell cycle. The misregulation of centrosome leads to centrosome amplification and cell division errors. The BRCA1/BARRD1 complex localizes to centrosomes throughout the cell cycle. OLA1 (Obg-like ATPase 1) also localizes to centrosomes in interphase and then moves to the spindle poles in the mitotic phase. Knockdown of OLA1 causes the centrosome amplification and overactive centrosomal aster formation. Moreover, the direct interaction between OLA1 and the BRCT domains of BARD1 had been identified.
In order to know the biochemical interplay between OLA1 and BARD1 BRCT domains, we mapped the 15N-BARD1 BRCT/OLA1 interaction by NMR and tested whether these possible OLA1 binding sites on BARD1 BRCT domains can regulate the ATPase activity of OLA1. In this study, we measured ATP hydrolysis by OLA1 using a spectrophotometer. Our data revealed that mutations on BARD1 BRCT reduce the OLA1 ATPase activity. Furthermore, a breast cancer-associated missense mutation on BARD1 BRCT weakens the ATPase activity of OLA1 much more obvious. Together, these data highlight the uniqueness of BARD1 BRCT domain, which uses a novel binding site to promote activity of a non-phosphorylated OLA1 to regulate centrosomes.

中文摘要 i
Abstract ii
誌謝 iv
Contents v
Abbreviation 1
Chapter 1. Introduction 3
1.1 BRCA1 and BARD1 3
1.2 Human Obg-like ATPase-1 (OLA1) 7
1.3 The BRCA1/BARD1/OLA1/γ-tubulin complex localizes to centrosomes. 8
1.4 Specific aim 9
Chapter 2. Materials and methods 10
2.1 Cloning 10
2.2 Protein expression and purification 11
2.3 BRCT-OLA1 and mutants 14
2.4 ATPase assay 16
2.5 Circular dichroism spectroscopy 17
2.6 Isothermal titration calorimetry 18
2.7 Enzyme kinetics assay 19
2.8 Size exclusion chromatography 19
Chapter 3. Results 20
3.1 The wild-type BARD1 BRCT domains enhanced the ATP hydrolysis rate of OLA1. 20
3.2 The wild-type BRCT-OLA1 fusion increased the ATP hydrolysis rate of OLA1. 21
3.3 The BARD1 BRCT domains increased the turn over number (kcat) of OLA1. 22
3.4 The binding affinity between OLA1 and ATP or ADP remained similar in the presence
of BARD1 BRCT domains. 23
3.5 Mutations on BARD1 BRCT domains weaken the ATP hydrolysis rate of OLA1. 24
3.6 Mutations on BARD1 BRCT domains diminished the stimulation of OLA1. 26
3.7 The conformation of BRCT-OLA1 mutants resembled that of the wild-type
BRCT-OLA1 (APO form) as shown by size exclusion chromatography. 27
3.8 The secondary structure content of BARD1 BRCT mutants were similar to that of
wild-type BARD1 BRCT, but the thermal stability was decreased. 27
3.9 The BRCA1/BARD1 RING domain enhanced the ATP hydrolysis rate of BRCT-OLA1. 29
Chapter 4. Conclusion and Discussion 31
4.1 Conclusion 31
4.2 Discussion 31
Figures 35
Reference 59

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