화농성연쇄상구균 유래 Type II-A 크리스퍼 관련 단백질들의 구조 및 상호작용 연구

학위논문(박사)--서울대학교 대학원 :농업생명과학대학 농생명공학부,2019. 8. 배의영.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (cas) genes constitute a prokaryotic adaptive immune system against invading foreign genetic elements like phages and plasmids. CRISPR acts as genetic memory storing short unique DNA sequences that match up with nucleic acid sequences from the intruders that previously tried to attack the host cell. The CRISPR array is accompanied by a set of cas genes encoding the proteins for CRISPR-mediated immunity. Cas1 and Cas2, universally conserved Cas proteins, form a complex and enables memorizing intruders by integrating short fragments from the foreign nucleic acids into the host CRISPR array. In addition, Cas9 and Csn2 are also essential for the immunization process in the type II-A CRISPR-Cas system. In this thesis, I reported the X-ray crystal structures of type II-A Cas1 and Cas2 from Streptococcus pyogenes, and characterized the interaction network between four type II-A Cas proteins. Csn2, type II-A specific Cas protein forms a larger complex with the Cas1–Cas2 complex and also interacts directly with Cas9, while the Cas9 does not interact with the Cas1–Cas2 complex. The stoichiometry and the binding affinity of the interaction network among the four type II-A Cas proteins were determined. These results provide experimental evidence to infer the role of Csn2 and how Cas9 and Cas1–Cas2 integrase module are connected in the type II-A CRISPR-Cas systems.CRISPR (Clustered regularly interspaced shrot palindromic repeats, 주기적으로 반복되는 짧은 길이의 회문 구조를 가진 염기서열 집합체) 및 CRISPR 연관 유전자 (CRISPR-associated; cas gene)는 파아지 및 플라스미드와 같은 외래 유전 물질들의 침입에 대항하는 원핵생물의 적응 면역체계를 구성한다. CRISPR 배열은 이전에 숙주 세포를 공격하려고 시도하였던 침입자의 핵산 서열과 일치하는 짧은 고유 DNA 서열을 저장하는 유전적 기억 장치 역할을 한다. CRISPR 배열은 일련의 cas 유전자를 동반하며, 이 유전자들은 CRISPR-Cas 체계의 동작에 중요한 역할을 하는 단백질을 암호화 하고 있다. 여러 CIRPSR-Cas 체계 보편적으로 보존 된 2 개의 Cas 단백질인 Cas1 과 Cas2 단백질은 복합체를 형성하여 외래 핵산 단편을 숙주 CRISPR 배열로 통합시킨다. 유형 II-A CRISPR-Cas 시스템에서는 Cas1 및 Cas2 이외에도 다른 Cas 구성 요소인 Cas9 및 Csn2의 존재가 면역화 과정에 필수적이다. 이 논문에서는 Streptococcus pyogenes 의 type II-A Cas1 과 Cas2 단백질의 X 선 결정 구조를 보고하고 두 단백질 간의 상호 작용 특성을 규명하였다. 타입 II-A 특이 Cas 단백질로 알려진 Csn2 는 Cas1-Cas2 복합체와 함께 더 큰 복합체를 형성함을 확인되었다. Csn2 는 또한 Cas9와 직접 상호작용하는 반면, Cas9 은 Cas1-Cas2 복합체 또는 복합체의 개별 구성체와는 상호작용을 하지 않았다. 마지막으로 4 가지 II-A Cas 단백질 사이의 상호 작용들의 화학량론 및 결합 친화도를 측정 하였다. 이러한 결과는 지금까지 기술되지 않은 Csn2 의 역할과 Cas9 및 Cas1-Cas2 인테그라아제 모듈이 II-A CRISPR-Cas 시스템에서 어떻게 연결되는지를 보여주는 실험적 증거를 제공한다.Abstract ..................................................................................................................... i Table of contents .................................................................................................... ii List of Tables .......................................................................................................... iv List of Figures .......................................................................................................... v List of Abbreviations ........................................................................................... viii Introduction ............................................................................................................. 1 CRISPR-Cas system .......................................................................................... 1 Classification of the CRISPR-Cas systems ....................................................... 4 Mechanism of the CRISPR-Cas system ............................................................ 8 Type II-A CRISPR-Cas system ....................................................................... 12 Type II-A CRISPR adaptation ......................................................................... 13 Materials and Methods ......................................................................................... 17 Cloning of type II-A cas genes ........................................................................ 17 Purification of S. pyogenes II-A Cas proteins ................................................. 17 Crystallization and structure determination of the S. pyogenes II-A Cas1 and Cas2 ................................................................................................................. 22 Nuclease activity assay of S. pyogenes II-A Cas1 ........................................... 24 Analytical size exclusion chromatography (SEC) ........................................... 26 Isothermal titration calorimetry (ITC) experiment .......................................... 26 In vitro transcription and purification of the sgRNA ...................................... 28 Surface plasmon resonance (SPR) experiment ................................................ 29 Results .................................................................................................................... 31 Structural analysis of S. pyogenes II-A Cas1 .................................................. 31 Disruption of putative metal binding sites in S. pyogenes II-A Cas1 .............. 37 Structural analysis of S. pyogenes II-A Cas2 .................................................. 45 Characterization of S. pyogenes II-A Cas1–Cas2 complex formation ............ 51 Complex formation of S. pyogenes II-A Cas1 with a type II-A specific Cas protein, Csn2 ................................................................................................... 56 Identification of the binding interface of S. pyogenes II-A Cas1–Csn2 complex ......................................................................................................................... 63 Three-component complex formation in S. pyogenes Type II-A CRISPR-Cas system .............................................................................................................. 69 Interaction between S. pyogenes Cas9 and Csn2 ............................................. 79 Discussion ............................................................................................................... 94 Structural feature of S. pyogenes II-A Cas1 and Cas2..................................... 94 The formation of type II-A CRISPR-Cas adaptation module ....................... 101 The role of Csn2 in type II-A CRISPR adaptation mechanism ..................... 107 References ............................................................................................................ 114 Supplementary Materials ................................................................................... 119 Accession numbers .............................................................................................. 120 Abstract in Korean .............................................................................................. 121 Acknowledgement ............................................................................................... 123Docto