서브메뉴
검색
Homology-Directed Repair of One-and Two-Ended DNA Double-Strand Breaks- [electronic resource]
Homology-Directed Repair of One-and Two-Ended DNA Double-Strand Breaks- [electronic resource]
상세정보
- 자료유형
- 학위논문파일 국외
- 최종처리일시
- 20240214101234
- ISBN
- 9798379780401
- DDC
- 575
- 서명/저자
- Homology-Directed Repair of One-and Two-Ended DNA Double-Strand Breaks - [electronic resource]
- 발행사항
- [S.l.]: : Columbia University., 2023
- 발행사항
- Ann Arbor : : ProQuest Dissertations & Theses,, 2023
- 형태사항
- 1 online resource(178 p.)
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-01, Section: B.
- 주기사항
- Advisor: Symington, Lorraine.
- 학위논문주기
- Thesis (Ph.D.)--Columbia University, 2023.
- 사용제한주기
- This item must not be sold to any third party vendors.
- 초록/해제
- 요약DNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context-dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair.To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events.To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.
- 일반주제명
- Genetics.
- 일반주제명
- Molecular biology.
- 일반주제명
- Cellular biology.
- 키워드
- DNA repair
- 키워드
- End resection
- 기타저자
- Columbia University Biological Sciences
- 기본자료저록
- Dissertations Abstracts International. 85-01B.
- 기본자료저록
- Dissertation Abstract International
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
008240612s2023 us |||||||||||||||c||eng d■001000016933334
■00520240214101234
■006m o d
■007cr#unu||||||||
■020 ▼a9798379780401
■035 ▼a(MiAaPQ)AAI30527709
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a575
■1001 ▼aKimble, Michael T.
■24510▼aHomology-Directed Repair of One-and Two-Ended DNA Double-Strand Breaks▼h[electronic resource]
■260 ▼a[S.l.]:▼bColumbia University. ▼c2023
■260 1▼aAnn Arbor :▼bProQuest Dissertations & Theses, ▼c2023
■300 ▼a1 online resource(178 p.)
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-01, Section: B.
■500 ▼aAdvisor: Symington, Lorraine.
■5021 ▼aThesis (Ph.D.)--Columbia University, 2023.
■506 ▼aThis item must not be sold to any third party vendors.
■520 ▼aDNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context-dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair.To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events.To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.
■590 ▼aSchool code: 0054.
■650 4▼aGenetics.
■650 4▼aMolecular biology.
■650 4▼aCellular biology.
■653 ▼aDNA repair
■653 ▼aDNA double-strand breaks
■653 ▼aEnd resection
■653 ▼aHomologous recombination
■653 ▼aReplication fork collapse
■690 ▼a0369
■690 ▼a0307
■690 ▼a0379
■71020▼aColumbia University▼bBiological Sciences.
■7730 ▼tDissertations Abstracts International▼g85-01B.
■773 ▼tDissertation Abstract International
■790 ▼a0054
■791 ▼aPh.D.
■792 ▼a2023
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T16933334▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
■980 ▼a202402▼f2024
