蛋白質複合體SWI5-SFR1(S5S1)是一個調整DNA同源重組修復過程的調控因子，而DNA同源重組修復可以修復DNA雙股斷裂，當DNA雙股斷裂發生時，重組酶RAD51會與單股DNA結合，形成突觸前長絲，之後ATP與RAD51-DNA 複合體結合，幫助其穩定並使單股DNA互換。由於RAD51的ATP水解活性，ATP會被水解成ADP導致複合體活性下降，而S5S1會加速水解後產物ADP從RAD51釋放出來的速度。最近，SWI5的兩個關鍵活性序列F83與L85被發現參與S5S1和RAD51的相互作用，為了研究這個相互作用的機制，我們利用酵母菌的S5S1結晶結構(PDB編號:3VIQ)為模板，模擬老鼠的S5S1分子結構，我們發現F83與L85皆位於SWI5 C端末端的疏水性核心之中，並且兩者都面向結構內部。因此，我們假設S5S1與RAD51作用時，S5最末端的α螺旋結構會打開，露出F83與L85與RAD51反應。
為了證明上述的假設，我們將S5 C端最末端的D89點突變成半胱胺酸，使其與S1的C53形成雙硫鍵，將S5最末端的部分固定起來，並且將S1的C59點突變成絲胺酸來避免其他雙硫鍵的發生。我們分別利用圓二色光譜(Circular dichroism)與小角度X光散射來觀察突變蛋白質S5D89CS1cC59S的二級與三級結構。在圓二色光譜中，顯示S5S1c與S5D89CS1cC59S的圖譜主要由α螺旋結構所構成，藉由圓二色光譜光譜分析網站DichroWEB的分析，發現兩者的α螺旋結構構成比例相似。利用小角度X光散射，我們分析了S5S1c與S5D89CS1cC59S的特性資料，並做出兩者的重頭起算模型(ab initio model)，結果顯示，S5S1c與S5D89CS1cC59S外觀相似。
最後，我們利用pull-down assay來分析S5S1c或S5D89CS1cC59S與RAD51的結合能力。比較S5S1c、雙硫鍵連接或未連接的S5D89CS1cC59S與RAD51的結合能力，結果顯示，具備雙硫鍵的S5D89CS1cC59S無法與RAD51作用，但若將雙硫鍵連接打斷(即F83與L85有可能暴露出來與RAD51作用)，S5D89CS1cC59S就會回復部分的結合能力。結論是，S5 C端的最後α螺旋結構能不能打開會影響到S5S1與RAD51的作用，此項S5S1與RAD51結合機制的探討, 可作為後續研究DNA同源重組修復作用的重要參考。

SWI5-SFR1 protein complex (S5S1) has been proven as an accessory factor of homologous recombination repair, which can restore the DNA double strands break (DSB). When DNA double strands break (DSB) happen, RAD51 recombinases (RAD51) as a key protein form a presynaptic filament via binding to a single-stranded DNA. Then, ATP would bind to RAD51 to stabilize the RAD51-DNA complex and induce single-stranded DNA exchange. Due to the hydrolytic activity of RAD51, ATP would be hydrolyzed to ADP and the activity of RAD51-DNA complex will decrease, and S5S1 can promote the ADP releasing rate by interaction between RAD51.
Recently, two active sites, F83 and L85, on the C-terminus of SWI5 (S5) have been reported to participate in the interaction between S5S1 and RAD51. In order to investigate their interaction, we use molecular modeling with the template “S5S1c from yeast (which has a known crystal structure, PDB code: 3VIQ)” to construct the model, we found that S5F83 and S5L85 are located in the hydrophobic core in the ending of S5 and faced inside the core. Hence, we speculate the two positions would expose and attach to RAD51 when the bundle structure on the S5 C-terminal alpha-helix was separated.
To validate our hypothesis, first, we fix the C-terminal loop by creating the disulfide bond S5D89C-S1C53 on the complex. In addition, to prevent the other disulfide bond take place in the molecular, we also replaced the S1C59 to Ser. Next, we compared the secondary and tertiary structure by circular dichroism (CD) and small-angle X-ray scattering (SAXS). CD analysis showed that both S5S1c and S5D89CS1cC59S presented mainly alpha-helical structure, and their secondary structure compositions are similar by analyzed on the DichroWEB website. For SAXS experiment, we determined the structure characteristic of S5S1c and mutant, and constructed the ab initio model of them. The results showed no huge difference between S5S1c and mutant.
Finally, the binding ability was determined by pull-down assay. We compared S5S1c, loop-fixed and unfixed S5D89CS1cC59S (without and with 2-ME treatment) The results showed that, compare to S5S1c, loop-fixed S5D89CS1cC59S cannot interact to RAD51, and it restored partially binding ability when freed the loop which indicate S5F83 and S5L85 could expose and bind to RAD51. In conclusion, we speculate the opening of S5 C-terminal loop is essential when S5S1c interact to RAD51. Our study provide information regarding to S5S1c binding to RAD51.

Abstract １
中文摘要 ３
Acknowledgements ４
Contents ５
Abbreviations ８
Chapter 1. Introduction １０
1.1 DNA damage and homologous recombination repair １０
1.2 SWI5-SFR1 protein complex １２
1.3 Interaction of SWI5-SFR1 protein complex and RAD51 １３
1.4 Small-angle X-ray scattering (SAXS) １４
1.4.1 SAXS background １４
1.4.2 Guinier Plot １５
1.4.3 Kratky plot １６
1.4.4 Pair distance distribution function １６
1.4.5 Dummy atom model １６
1.5 The aim of this thesis １７
Chapter 2. Materials and methods １８
2.1 Molecular modeling １８
2.2 Construction of the S5S1c mutant, S5D89CS1cC59S １８
2.3 Expression of S5S1c and S5D89CS1cC59S １９
2.4 Purification of S5S1c and S5D89CS1cC59S １９
2.5 Quantification of protein concentration ２０
2.6 SDS-PAGE analysis ２０
2.7 Circular dichroism spectroscopy ２１
2.8 His tag remove ２２
2.9 Small-Angle X-ray Scattering (SAXS) ２２
2.10 Disulfide bond reduced test ２３
2.11 Pull-down assay ２４
Chapter3. Results and Discussion ２５
3.1 Mouse S5S1c molecular model and structure characteristic ２５
3.2 Study of S5 C-terminal end ２５
3.3 Double mutagenesis, S5D89C and S1cC59S ２７
3.4 Expression and purification of S5S1c and S5D89CS1cC59S ２８
3.5 Protein characterization ２９
3.5.1 Circular dichroism (CD) spectroscopy ２９
3.5.2 SAXS data analysis ２９
3.5.3 SAXS structural Analysis ３０
3.6 Binding ability test ３１
Chapter 4. Conclusions ３３
Chapter 5. Figures and Table ３５
Figure 1.1 The DNA homologous recombination repair process ３５
Figure 1.2 Crystal structure of yeast SWI5SFR1c (DN177) protein complex ３６
Figure 1.3 Sequence and secondary structure of yeast S5S1c ３７
Figure 1.4 The schematic diagram of S5S1 interaction to RAD51 and ssDNA ３８
Figure 1.5 Kratky plot diagram. ３９
Figure 1.6 Illustration of pair distance distribution function ４０
Figure 1.7 Running process of DAMMIF ４１
Figure 1.8 Pull-down assay for binding ability of S5S1 and its variants ４２
Figure 2.1 The sequencing map of S5S1c ４３
Figure 2.2 Expression and purification process of S5D89CS1cC59S ４５
Figure 2.3 His tag remove test of S5D89CS1cC59S ４５
Table 2.1 primers for site-directed mutagenesis, S5D89C and S1cC59S ４６
Figure 3.1 Molecular model of mouse S5S1c ４７
Figure 3.2 Structure comparison of yeast and mouse S5S1c ４８
Figure 3.3.1 The leucine zipper motif in yeast S5S1c ４９
Figure 3.3.2 The leucine zipper motif in mouse S5S1c ５０
Figure 3.4.1 Multiple sequence alignment of SWI5 and SFR1c ５１
Figure 3.4.2 Sequence alignment of S5 between mouse and yeast and the last 40 residues alignment ５２
Figure 3.5 Three-dimensional structure of hydrophobic core in yeast and mouse S5S1c ５３
Figure 3.6 The critical residues mouse S5L85 and F83 and its conserved residues in yeast ５４
Figure 3.7 Schematic diagram of the residues S5D89, S1C53 and S1C59 ５５
Figure 3.8 DNA gel electrophoresis of S5D89CS1c and S5D89CS1cC59S ５６
Figure 3.9 DNA Sequence alignment of S5S1c and S5D89CS1cC59S. ５７
Figure 3.10 Purification of S5D89CS1cC59S by HPLC ５８
Figure 3.11 CD wavelength scans of S5S1c and S5D89CS1cC59S ５９
Figure 3.12 HPLC prerun spectra ６０
Figure 3.13 The SAXS of S5S1c (up) and S5D89CS1cC59S (down). ６１
Figure 3.14 The Logarithmic scale of S5S1c and S5D89CS1cC59S ６２
Figure 3.15 The Guinier plot of S5S1c and S5D89CS1cC59S ６３
Figure 3.16 Kratky plots scatter diagram of S5S1c and S5D89CS1cC59S. ６４
Figure 3.17 The Pair-distance distribution function of S5S1c and S5D89CS1cC59S ６５
Figure 3.18 Dummy atom model of S5S1c and S5D89CS1cC59S ６６
Figure 3.19 The ab initio envelop of S5S1c and S5D89CS1cC59S ６７
Figure 3.20 Disulfide bond breaking test by 2-ME ６８
Figure 3.21 Sample quality which was used in the binding ability test ６９
Figure 3.22 The pull-down assay result of S5S1c and S5D89CS1cC59S ７０
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