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研究生：	鄭政軒 Cheng, Cheng-Hsuan
論文名稱：	探討大腸桿菌之單股DNA結合蛋白與RecQ解旋酶在DNA修復中之分子交互作用 Study on the molecular interaction between Escherichia coli single-stranded DNA binding protein and RecQ helicase in DNA repair
指導教授：	王淑鶯 Wang, Shu-Ying
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	71
中文關鍵詞：	單股DNA結合蛋白、RecQ解旋酵素、DNA修復、抑制性突變
外文關鍵詞：	single-stranded DNA binding protein, RecQ helicase, DNA repair, suppressor mutation
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大腸桿菌 (E. coli) 之單股DNA結合蛋白 (SSB) 是一個重要的核酸交互作用蛋白，並且參與在DNA修復、重組和複製當中。SSB的關鍵功能之一是與各種交互作用蛋白 (SIP) 形成複合體，並協調SIP與單股DNA之間的交互作用。先前的研究表明，SSB與參與DNA修復的SIP之一RecQ有物理上的交互作用，並且先前已經證明SSB可以促進RecQ的活性。但是SSB-RecQ的複合物在兩個分子上的交互作用介面仍未被清楚解析，以及SSB是如何幫助RecQ 所介導的DNA解旋的分子機制仍是未知的。因此，在這項研究中，我們旨在鑑定參與SSB和RecQ之間交互作用的殘基。首先透過過共純化 (co-purification) 證實了SSB和帶有組胺酸標記的RecQ的確實會形成複合體。隨後，分別通過金屬離子親和層析法 (metal affinity chromatography) 和粒徑篩析層析法 (size-exclusion chromatography) 純化帶有組胺酸標記的重組的RecQ和SSB蛋白。我們透過以螢光共振能量轉移(FRET)為基礎的解旋試驗 (unwinding assay) 評估了純化的RecQ的解旋功能，並證明了SSB確實會增強RecQ的活性。接著，我們進行抑制性突變 (suppressor mutation) 實驗以大腸桿菌體內試驗 (in vivo) 的方式去鑑定參與SSB-RecQ複合物的交互作用的殘基。我們建構了條件性去活性 (conditional inactivated) SSB基因的大腸桿菌菌株 (R36)，並通過隨機突變生成了SSB突變庫 (mutant library)。透過對於紫外線的敏感性，去篩選帶有SSB突變庫的R36菌株。我們獲得了兩個對紫外線敏感的突變菌株，ssb (L113P)和ssb (W55R、Y71N)。這些具有紫外線敏感表現型的突變菌株將用於未來的抑制實驗中，以探測SSB-RecQ複合物的交互作用殘基。對SSB和RecQ分子交互作用的理解將為我們提供證據以闡明SSB如何與SIP交互作用以及此交互作用在基因組穩定性維持中的角色。

Escherichia coli (E. coli) single-stranded DNA binding protein (SSB) is an essential nucleic acid interacting protein involved in DNA repair, recombination, and replication. One of the crucial functions of SSB is to form complex with various interacting proteins (SIP) and coordinate the interaction between SIPs and single-stranded DNA (ssDNA). Previous studies showed that SSB physically interacts with RecQ, one of the SIPs participating in DNA repair, and SSB was previously proved to facilitate the activity of RecQ. However, the overall interacting surface of the SSB-RecQ complex and the molecular mechanism of how SSB stimulates RecQ-mediated DNA unwinding are still unknown. Therefore, in this study, we aim to identify the residues involved in the interaction between SSB and RecQ. The complex formation of SSB and His-tagged RecQ was confirmed by co-purification. Subsequently, recombinant His-tagged RecQ and recombinant His-tagged SSB were purified respectively by metal affinity chromatography and size-exclusion chromatography. We evaluated the helicase function of the purified RecQ by fluorescence resonance energy transfer (FRET)-based unwinding assay and proved the enhancement of the activity by SSB. Next, the suppressor mutation experiment was performed to identify interacting residues of the SSB-RecQ complex in vivo. We simultaneously constructed an SSB conditional inactivated E. coli strain (R36) and generated an SSB mutant library by random mutagenesis. The R36 harboring the SSB mutant library was screened by UV sensitivity. We obtained two UV-sensitive mutant strains, ssb (L113P), and ssb (W55R, Y71N). The mutant strains with UV-sensitive phenotype would be utilized in the future suppression experiment to probe the interacting residues of the SSB-RecQ complex. Understanding the molecular interaction of SSB and RecQ will provide us the evidence to clarify how SSB interacts with the SIPs and the role of the interactions in genomic stability maintenance.

中文摘要	I
Abstract	II
Acknowledgment	IV
Contents	V
List of figures	IX
List of tables	XI
Abbreviation	XII
Chapter 1 Introduction	1
1 Single-stranded DNA binding protein (SSB)	1
1.1 The physiological function of SSB	1
1.2 The function of E. coli SSB different domains	1
1.3 SSB interacting proteins and selectively interacting mechanism	3
2 RecQ helicase	3
2.1 The physiological function of RecQ	3
2.2 The interaction between SSB and RecQ	4
3 In vivo studies of SSB	4
3.1 SSB mutants and in vivo tools for study SSB	5
3.2 Phenotype-based probing of SSB-SIP interacting residues	5
4 Rationale and specific aims	6
Chapter 2 Materials and Methods	7
1 Materials	7
1.1 Bacterial strains	7
1.2 Plasmids	8
1.2 Plasmids (continued-1)	9
1.2 Plasmids (continued-2)	10
1.3 Chemicals and other materials	10
2 Methods	16
2.1 Construction	16
2.2 Protein overexpression and metal affinity purification of His-tagged SSB and His-tagged RecQ	17
2.3 Protein overexpression of SSB	18
2.4 Size-exclusion chromatography (SEC)	18
2.5 Complex formation by co-elution	18
2.6 Recombination-mediated genetic engineering	19
2.7 Unwinding assay	20
2.8 UV sensitivity test	21
2.9 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)	21
Chapter 3 Results	22
1 Overexpression and complex formation of SSB and His-tagged RecQ	22
1.1 Overexpression of SSB and His-tagged RecQ	22
1.2 Complex formation of SSB and His-tagged RecQ	22
2 The helicase function of RecQ and SSB-mediated enhancement of RecQ unwinding activity	22
2.1 Overexpression and purification of recombinant His-tagged SSB	22
2.2 Overexpression and purification of recombinant His-tagged RecQ	23
2.3 RecQ-mediated DNA unwinding	24
3 SSB-RecQ interacting residues probing and phenotype-based approach establishment	24
3.1 Construction of an SSB conditional-inactivated E. coli strain	25
3.2 SSB mutant library construction	25
3.3 UV sensitivity screening of SSB mutant library	25
3.4 SSB mutant identification	26
Chapter 4 Conclusion	28
Chapter 5 Discussion	29
1 The subsequent experiments of the suppressor mutation	29
2 The mutations on the OB domain might not be the protein interacting residues	30
3 The SSB mutants failed to interact with RecQ might not be the cause of UV-sensitive phenotype	31
4 The buffer condition of SSB-RecQ complex required further optimization to improve the stability	31
5 The purified SSB without His tag should be confirmed by protein identification	32
6 The DNA damage and repair pathway are UV-dependent	32
7 The uneven mutation rate of SSB different domains	33
References	34
Table	42
Figures	46
Appendix	61
                                    

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