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Structural and Mechanistic Studies of the Type IIS Restriction Endonuclease PaqCI and De Novo Designed Circular Tandem Repeat Proteins- [electronic resource]
Structural and Mechanistic Studies of the Type IIS Restriction Endonuclease PaqCI and De Novo Designed Circular Tandem Repeat Proteins- [electronic resource]
상세정보
- 자료유형
- 학위논문파일 국외
- 최종처리일시
- 20240214101214
- ISBN
- 9798379913328
- DDC
- 574.191
- 서명/저자
- Structural and Mechanistic Studies of the Type IIS Restriction Endonuclease PaqCI and De Novo Designed Circular Tandem Repeat Proteins - [electronic resource]
- 발행사항
- [S.l.]: : University of Washington., 2023
- 발행사항
- Ann Arbor : : ProQuest Dissertations & Theses,, 2023
- 형태사항
- 1 online resource(143 p.)
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-01, Section: B.
- 주기사항
- Advisor: Stoddard, Barry L. .
- 학위논문주기
- Thesis (Ph.D.)--University of Washington, 2023.
- 사용제한주기
- This item must not be sold to any third party vendors.
- 초록/해제
- 요약This thesis spans the work completed on two parallel projects, to (i) study the structure and mechanism of the Type IIS restriction endonuclease PaqCI, and (ii) participate in the characterization and engineering of de novo designed circular tandem repeat proteins ('cTRPs') for the development of novel liganddependent protein dimerization systems. Through the latter project, I gained early intensive training in protein crystallography and engineering, contributing to my independent study of PaqCI. Restriction endonucleases are an essential component of innate, 'preprogrammed' phage restriction systems that protect bacteria from foreign DNA. Type II restriction endonucleases are invaluable tools in research because of their ability to identify and cleave specific DNA sequences with extremely high fidelity, as well as their unique mechanisms of cleavage. The most well-studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or related enzymes form a DNA-bound reaction complex have not been described at atomic resolution. Here I describe a series of crystallographic and CryoEM structures in the presence and absence of bound DNA targets that reveal aspects of DNA recognition and cleavage by the Type IIS PaqCI restriction endonuclease. The structures illustrate the enzyme's tetrameric domain organization in the absence of bound substrate and the subsequent formation of a tetrameric reaction complex poised to deliver the first of a series of double-strand breaks. Understanding the structure of the Type IIS restriction endonucleases PaqCI reveals (i) the requirement for multiple DNA targets to be pulled together for enzyme activation, (ii) that enzymatic domains are sterically constricted and can only correctly orient for cleavage at 4/8 bases from the target site, and (iii) that the orientation of the target recognition domain on the DNA determines the motion required of the endonuclease domains to engage the cleavage site. These results bolster the dominant hypothesis that Type II restriction enzymes require the engagement of multiple unmodified targets to bias cleavage towards unprotected foreign DNA. Through these two projects I gained expertise in crystallography and cryoEM, enzymatic analyses, and protein engineering.
- 일반주제명
- Biophysics.
- 일반주제명
- Biochemistry.
- 키워드
- CryoEM
- 키워드
- DNA binding
- 키워드
- DNA cleavage
- 기타저자
- University of Washington Biochemistry
- 기본자료저록
- Dissertations Abstracts International. 85-01B.
- 기본자료저록
- Dissertation Abstract International
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520240214101214
■006m o d
■007cr#unu||||||||
■020 ▼a9798379913328
■035 ▼a(MiAaPQ)AAI30525864
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574.191
■1001 ▼aKennedy, Madison Arza.
■24510▼aStructural and Mechanistic Studies of the Type IIS Restriction Endonuclease PaqCI and De Novo Designed Circular Tandem Repeat Proteins▼h[electronic resource]
■260 ▼a[S.l.]:▼bUniversity of Washington. ▼c2023
■260 1▼aAnn Arbor :▼bProQuest Dissertations & Theses, ▼c2023
■300 ▼a1 online resource(143 p.)
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-01, Section: B.
■500 ▼aAdvisor: Stoddard, Barry L. .
■5021 ▼aThesis (Ph.D.)--University of Washington, 2023.
■506 ▼aThis item must not be sold to any third party vendors.
■520 ▼aThis thesis spans the work completed on two parallel projects, to (i) study the structure and mechanism of the Type IIS restriction endonuclease PaqCI, and (ii) participate in the characterization and engineering of de novo designed circular tandem repeat proteins ('cTRPs') for the development of novel liganddependent protein dimerization systems. Through the latter project, I gained early intensive training in protein crystallography and engineering, contributing to my independent study of PaqCI. Restriction endonucleases are an essential component of innate, 'preprogrammed' phage restriction systems that protect bacteria from foreign DNA. Type II restriction endonucleases are invaluable tools in research because of their ability to identify and cleave specific DNA sequences with extremely high fidelity, as well as their unique mechanisms of cleavage. The most well-studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or related enzymes form a DNA-bound reaction complex have not been described at atomic resolution. Here I describe a series of crystallographic and CryoEM structures in the presence and absence of bound DNA targets that reveal aspects of DNA recognition and cleavage by the Type IIS PaqCI restriction endonuclease. The structures illustrate the enzyme's tetrameric domain organization in the absence of bound substrate and the subsequent formation of a tetrameric reaction complex poised to deliver the first of a series of double-strand breaks. Understanding the structure of the Type IIS restriction endonucleases PaqCI reveals (i) the requirement for multiple DNA targets to be pulled together for enzyme activation, (ii) that enzymatic domains are sterically constricted and can only correctly orient for cleavage at 4/8 bases from the target site, and (iii) that the orientation of the target recognition domain on the DNA determines the motion required of the endonuclease domains to engage the cleavage site. These results bolster the dominant hypothesis that Type II restriction enzymes require the engagement of multiple unmodified targets to bias cleavage towards unprotected foreign DNA. Through these two projects I gained expertise in crystallography and cryoEM, enzymatic analyses, and protein engineering.
■590 ▼aSchool code: 0250.
■650 4▼aBiophysics.
■650 4▼aBiochemistry.
■653 ▼aCryoEM
■653 ▼aDNA binding
■653 ▼aDNA cleavage
■653 ▼aProtein engineering
■653 ▼aRestriction endonuclease
■653 ▼aX-ray crystallography
■690 ▼a0786
■690 ▼a0487
■71020▼aUniversity of Washington▼bBiochemistry.
■7730 ▼tDissertations Abstracts International▼g85-01B.
■773 ▼tDissertation Abstract International
■790 ▼a0250
■791 ▼aPh.D.
■792 ▼a2023
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T16933186▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
■980 ▼a202402▼f2024
