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Biochemical Study of an Anti-CRISPR protein, AcrIE4-F7 : Anti-CRISPR 단백질, AcrIE4-F7 의 생화학적 연구
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 배의영 | - |
dc.contributor.author | 이규진 | - |
dc.date.accessioned | 2023-06-29T02:07:05Z | - |
dc.date.available | 2023-06-29T02:07:05Z | - |
dc.date.issued | 2023 | - |
dc.identifier.other | 000000174138 | - |
dc.identifier.uri | https://hdl.handle.net/10371/193568 | - |
dc.identifier.uri | https://dcollection.snu.ac.kr/common/orgView/000000174138 | ko_KR |
dc.description | 학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 2. 배의영. | - |
dc.description.abstract | To defend invasions of bacteriophages and plasmids, bacteria and archaea employ the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) defense system. Type I-E and I-F CRISPR-Cas systems exploit CRISPR-associated complex for antiviral defense (Cascade) to recognize the invading DNA. In turn, bacteriophages have evolved anti-CRISPR (Acr) proteins to neutralize CRISPR-mediated immunity. AcrIE4-F7 encoded by Pseudomonas citronellolis is a fused protein of AcrIE4 and AcrIF7 which inactivates both type I-E and I-F CRISPR-Cas system. In this study, I reported the target Cas proteins of AcrIE4-F7 by analyzing the interaction between AcrIE4-F7 and Cas proteins which comprise the type I-E and I-F Cascade complex. The N- and C-terminal domains of AcrIE4-F7 inhibit target DNA binding by interacting with the PAM recognition site of Cas8e and Cas8f, respectively. Pairwise sequence alignment and mutation analyses demonstrated that conserved negative-charged residues in each Acr domain are essential for interaction with their Cas8 target. These results suggest PAM recognition sites are the main targets of AcrIE4-F7, which is a common inhibition mechanism against divergent CRISPR-Cas types. | - |
dc.description.abstract | 박테리오파지와 플라스미드의 침입을 방어하기 위해서, 박테리아와 고세균은 CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats–CRISPR associated proteins) system 을 사용한다. Type I-E 와 I-F CRISPR-Cas system 은 침입한 DNA 를 인지하기 위해서 Cascade (CRISPR-associated complex for antiviral defense) 를 활용한다. 이에 대항하여, 박테리오파지는 CRISPR-관련 면역체계를 무력화하기 위해서 Acr (anti-CRISPR) 단백질을 진화시켰다. Pseudomonas aeruginosa 유래 AcrIE4-F7 은 AcrIE4 와 AcrIF7 의 fusion protein 으로 type I-E 와 I-F CRISPR-Cas system 을 모두 무력화시킨다. 본 연구에서는 type I-E 와 I-F Cascade complex 를 구성하는 Cas 단백질들과 AcrIE4-F7 사이의 상호작용을 분석하여 AcrIE4-F7 의 target Cas 단백질들을 보고했다. AcrIE4-F7 의 N-말단과 C-말단 도메인 각각이 Cas8e 와 Cas8f 의 PAM 인식 부위와 상호작용하여 target DNA 의 결합을 억제한다. Pairwise sequence alignment 와 mutation analyses 는 각각의 Acr 도메인에 존재하는 음전하 잔기들이 target Cas8 과의 상호 작용에 필수적이라는 것을 말해준다. 이러한 결과들은 PAM 인식 부위가 AcrIE4-F7 의 주요 target 이라는 것을 제안하고 이는 다양한 CRISPR-Cas type 들을 억제하는 일반적인 메커니즘이다. | - |
dc.description.tableofcontents | Introduction 1
Materials and Methods 12 Cloning 12 Protein expression and purification 15 Analytical size exclusion chromatography (SEC) 17 Circular dichroism (CD) 17 Pairwise sequence alignment 18 Results 19 Purification of AcrIE4-F7 19 Purification of type I-E Cas proteins 25 The N-terminal domain of AcrIE4-F7 binds to Cas8e 35 AcrIE4-F7 targets the PAM recognition site of Cas8e 40 Identification of AcrIE4-F7NTD binding interface for Cas8e binding 52 AcrIE4-F7CTD binds to Cas8f:Cas5f heterodimer 59 The binding interface between AcrIE4-F7CTD and Cas8f 62 Discussion 72 References 82 Abstract in Korean 85 | - |
dc.format.extent | vii, 86 | - |
dc.language.iso | eng | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | CRISPR-Cas | - |
dc.subject | Anti-CRISPR | - |
dc.subject | AcrIE4-F7 | - |
dc.subject | Cascade complex | - |
dc.subject | Pseudomonas aeruginosa | - |
dc.subject | PAM recognition site | - |
dc.subject.ddc | 630 | - |
dc.title | Biochemical Study of an Anti-CRISPR protein, AcrIE4-F7 | - |
dc.title.alternative | Anti-CRISPR 단백질, AcrIE4-F7 의 생화학적 연구 | - |
dc.type | Thesis | - |
dc.type | Dissertation | - |
dc.contributor.AlternativeAuthor | Lee Gyujin | - |
dc.contributor.department | 농업생명과학대학 농생명공학부 | - |
dc.description.degree | 석사 | - |
dc.date.awarded | 2023-02 | - |
dc.identifier.uci | I804:11032-000000174138 | - |
dc.identifier.holdings | 000000000049▲000000000056▲000000174138▲ | - |
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