Chemical and biochemical determinants of phosphorothioate stability and location in bacterial genomes

硫代磷酸盐稳定性和细菌基因组位置的化学和生化决定因素

• 批准号: 1709364
• 负责人: Peter Dedon
• 金额: $ 51万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1709364&HistoricalAwards=false
• 关键词: Chemical; biochemical; determinants; phosphorothioate; stability

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Prof. Peter Dedon from the Massachusetts Institute of Technology and Dr. James Galagan from Boston University. The project involves the study of a newly discovered chemical modification of DNA in bacteria. This modification involves replacement of an oxygen atom in the sugar-phosphate backbone of the DNA with a sulfur atom. The resulting chemical structure is known as a phosphorothioate (PT). The goal of the project is to understand the biological chemistry that is involved in these DNA modifications. The investigators are using chemical tools to examine the biochemistry of PT modification to bacterial DNA. The importance of this project is two-fold. First, the results advance the understanding of an important and fundamental new feature of microbial physiology known as epigenetics. The studies are significant for their practical impact as well. They contribute to understanding the mechanisms by which DNA-modifying enzymes find their DNA targets. The project findings can be exploited to develop new tools for biotechnology and synthetic biology research. The new tools can be used for enhancing the productivity of industrial microorganisms. The project provides cutting-edge interdisciplinary training at the interface of chemistry and biology. Training is provided for high school, undergraduate and graduate students and postdoctoral scientists. The project also develops novel epigenetics educational materials and curricula for high school science classes and teacher workshops. The goal of this project is to understand the biological chemistry of redox-sensitive sulfur-containing DNA modifications widespread in bacteria: phosphorothioates (PT). As a widespread epigenetic mark in all bacterial genera, PTs are incorporated into DNA by a 5-member dnd gene cluster (dndA-E). This gene cluster inserts S in place of a non-bridging oxygen in the DNA backbone as a sequence-specific PT. PTs function in restriction-modification (R-M) systems in many bacteria with Dnd proteins F-H. They are also present in bacteria lacking restriction genes, which suggests non-R-M epigenetic functions in gene expression. One highly unusual feature of PTs is that only 12-14% of short consensus sequences are modified in the genome. This fact raises questions about how the enzymes find their targets. The studies explore PT dynamics and function with the use of innovative sequencing, genomics, informatics and analytical technologies. The project uses a novel chemical cleavage/nick translation sequencing technology. Single-molecule real-time sequencing is used to quantify changes in PT location in response to oxidative stress and cell growth. The problem of genomic target selection is addressed by mining existing genomic maps of PTs relative to genomic landmarks. In addition, PT location is correlated with binding sites for Dnd proteins using ChIP-seq to map protein binding sites in the bacterial genomes. The mechanism of target selection by Dnd restriction enzymes is addressed by mapping restriction-induced cleavage sites in bacterial genomes. The results have a broad impact on understanding basic microbial physiology and epigenetics. The project is developing new enzymatic tools for inserting nuclease-resistant PTs into DNA in biotechnology applications.

凭借该奖项，化学部的生命过程化学项目将资助麻省理工学院的 Peter Dedon 教授和波士顿大学的 James Galagan 博士。该项目涉及对细菌中新发现的 DNA 化学修饰的研究。这种修饰涉及用硫原子取代 DNA 糖磷酸主链中的氧原子。所得化学结构称为硫代磷酸酯 (PT)。该项目的目标是了解这些 DNA 修饰所涉及的生物化学。研究人员正在使用化学工具来检查细菌 DNA PT 修饰的生物化学。该项目的重要性有两个。首先，这些结果促进了对微生物生理学的一个重要且基本的新特征（即表观遗传学）的理解。这些研究的实际影响也很重要。它们有助于理解 DNA 修饰酶找到 DNA 靶标的机制。该项目的研究成果可用于开发生物技术和合成生物学研究的新工具。新工具可用于提高工业微生物的生产力。该项目提供化学和生物学交叉领域的尖端跨学科培训。为高中生、本科生、研究生以及博士后科学家提供培训。该项目还为高中科学课程和教师讲习班开发新颖的表观遗传学教育材料和课程。该项目的目标是了解细菌中广泛存在的氧化还原敏感的含硫 DNA 修饰的生物化学：硫代磷酸酯 (PT)。作为所有细菌属中广泛存在的表观遗传标记，PT 通过 5 成员 dnd 基因簇 (dndA-E) 整合到 DNA 中。该基因簇插入 S 代替 DNA 主链中的非桥接氧作为序列特异性 PT。 PT 在许多具有 Dnd 蛋白 F-H 的细菌的限制性修饰 (R-M) 系统中发挥作用。它们也存在于缺乏限制性基因的细菌中，这表明基因表达中具有非 R-M 表观遗传功能。 PT 的一个非常不寻常的特征是，基因组中只有 12-14% 的短共有序列被修改。这一事实引发了关于酶如何找到目标的问题。这些研究利用创新的测序、基因组学、信息学和分析技术探索 PT 动力学和功能。该项目使用新型化学切割/切口翻译测序技术。单分子实时测序用于量化 PT 位置因氧化应激和细胞生长而发生的变化。基因组目标选择的问题是通过挖掘现有的 PT 相对于基因组标志的基因组图谱来解决的。此外，使用 ChIP-seq 绘制细菌基因组中的蛋白质结合位点图谱，将 PT 位置与 Dnd 蛋白质的结合位点相关联。通过绘制细菌基因组中限制性诱导的切割位点来解决 Dnd 限制性酶的靶标选择机制。研究结果对理解基本微生物生理学和表观遗传学具有广泛影响。该项目正在开发新的酶工具，用于在生物技术应用中将核酸酶抗性 PT 插入 DNA 中。

项目成果

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

• DOI: 10.3389/fmicb.2020.01960
• 发表时间: 2020-08-18
• 期刊: FRONTIERS IN MICROBIOLOGY
• 影响因子: 5.2
• 作者: Lutz, Thomas;Czapinska, Honorata;Xu, Shuang-yong
• 通讯作者: Xu, Shuang-yong

The road less traveled: A new phosphorothioate antiviral defense mechanism discovered in Archaea

• DOI: 10.1016/j.synbio.2019.06.002
• 发表时间: 2019-06
• 期刊: Synthetic and Systems Biotechnology
• 影响因子: 4.8
• 作者: M. DeMott;P. Dedon