The Interplay of Replication and Transcription in Fragility of Structured DNA

复制和转录在结构 DNA 脆弱性中的相互作用

• 批准号: 1817499
• 负责人: Catherine Freudenreich
• 金额: $ 75万
• 依托单位: Tufts University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817499&HistoricalAwards=false
• 关键词: Interplay; Replication; Transcription; Fragility; Structured

The integrity of DNA is fundamental to life, but sometimes DNA can break, leading to loss of genetic material or even cell death. Interestingly, certain areas of chromosomes, known as fragile sites, tend to break more frequently. These fragile sites are a frequent source of DNA mutations, for example loss or gain of genetic material. Thus, an understanding of the basis of chromosome fragility will provide insight into causes of disease and of genome evolution. This topic is of great interest to college students studying molecular biology and genetics, and a Molecular Genetics Project Laboratory was developed to provide a research experience to 16 students a year, investigating the causes of chromosome fragility using a yeast model system. The goal of the course is to identify new pathways that protect against fragility, and give the students an authentic research experience. This project will support both the laboratory class as well as follow-up experiments by undergraduate and graduate students, to gain new knowledge about important pathways that prevent deleterious mutations. Many fragile sites in the human genome are associated with both DNA structures that interfere with replication and transcribed regions. It is suspected that the co-incidence of structure-forming DNA sequences, transcription, and replication may combine to cause these regions to be prone to fragility, however the interplay between them is poorly understood. Genetic systems have been developed in the yeast Saccharomyces cerevisiae to measure chromosome breaks that are induced at specific structure-forming repetitive DNA sequences that cause DNA fragility. In this system, transcription state and replication timing through the DNA structure can be controlled, to directly measure the consequences for DNA breakage. The role of proteins in causing or protecting against DNA breaks will be determined, including the action of identified nucleases Mlh1-Mlh3 and Mus81. These tools will be employed to elucidate the contributions and interplay of transcription and replication in causing fragility at two physiologically relevant fragile sequences: (1) expanded CAG repeats and (2) a long AT repeat from the human fragile site FRA16D. The knowledge gained will enhance understanding of mechanisms of fragility and the resulting genome instability that leads to chromosomal deletions and rearrangements.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

DNA 的完整性是生命的基础，但有时 DNA 会断裂，导致遗传物质丢失甚至细胞死亡。有趣的是，染色体的某些区域（称为脆弱位点）往往更容易断裂。这些脆弱位点是 DNA 突变的常见来源，例如遗传物质的丢失或获得。因此，了解染色体脆弱性的基础将有助于深入了解疾病和基因组进化的原因。这个课题引起了学习分子生物学和遗传学的大学生的极大兴趣，因此建立了分子遗传学项目实验室，每年为 16 名学生提供研究经验，利用酵母模型系统研究染色体脆性的原因。该课程的目标是找出防止脆弱性的新途径，并为学生提供真实的研究体验。该项目将支持本科生和研究生的实验室课程以及后续实验，以获得有关防止有害突变的重要途径的新知识。人类基因组中的许多脆弱位点与干扰复制和转录区域的 DNA 结构相关。人们怀疑结构形成 DNA 序列、转录和复制的同时发生可能导致这些区域容易脆弱，但人们对它们之间的相互作用知之甚少。酿酒酵母中已开发出遗传系统，用于测量在特定结构形成重复 DNA 序列处诱导的染色体断裂，从而导致 DNA 脆弱。在该系统中，可以通过DNA结构控制转录状态和复制时间，从而直接测量DNA断裂的后果。将确定蛋白质在引起或防止 DNA 断裂中的作用，包括已鉴定的核酸酶 Mlh1-Mlh3 和 Mus81 的作用。这些工具将用于阐明转录和复制在导致两个生理相关脆弱序列脆弱性方面的贡献和相互作用：(1) 扩展的 CAG 重复序列和 (2) 来自人类脆弱位点 FRA16D 的长 AT 重复序列。所获得的知识将增强对脆弱性机制以及由此产生的导致染色体缺失和重排的基因组不稳定的理解。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae

研究酿酒酵母重复脆弱性的遗传分析

• DOI: 10.1007/978-1-4939-9784-8_5
• 发表时间: 2019
• 期刊: Methods in molecular biology
• 作者: Polleys, Erica J.;Freudenreich, Catherine H
• 通讯作者: Freudenreich, Catherine H

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

• DOI: 10.1074/jbc.ra120.014161
• 发表时间: 2020-10-02
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Laverde, Eduardo E.;Lai, Yanhao;Liu, Yuan
• 通讯作者: Liu, Yuan

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D

• DOI: 10.1016/j.celrep.2019.03.103
• 发表时间: 2019-04-23
• 期刊: CELL REPORTS
• 影响因子: 8.8
• 作者: Kaushal, Simran;Wollmuth, Charles E.;Freudenreich, Catherine H.
• 通讯作者: Freudenreich, Catherine H.

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

THO 和 TRAMP 复合物可防止转录复制冲突、DNA 断裂和 CAG 重复收缩。

• DOI: 10.1101/2022.03.22.4853861
• 发表时间: 2022
• 期刊: bioRxiv
• 作者: Rebecca E. Brown;Xiaofeng A. Su;Stacey Fair;Katherine Wu;Lauren Verra;Robyn Jong;Kristin Andrykovich;Catherine H. Freudenreich
• 通讯作者: Catherine H. Freudenreich