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

通过这个奖项，化学学部的生命过程化学项目资助了麻省理工学院的Peter Dedon教授和波士顿大学的James Galagan博士。该项目涉及研究一种新发现的细菌DNA的化学修饰。这种修饰包括用硫原子取代DNA的糖-磷酸主链中的氧原子。由此产生的化学结构被称为硫代磷酸酯（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PT修饰细菌DNA的生物化学。这个项目的重要性是双重的。首先，这些结果促进了对微生物生理学的一个重要和基本的新特征的理解，即表观遗传学。这些研究的实际影响也很重要。它们有助于理解DNA修饰酶找到其DNA靶标的机制。项目成果可用于开发生物技术和合成生物学研究的新工具。这些新工具可用于提高工业微生物的生产率。该项目在化学和生物学的界面上提供尖端的跨学科培训。培训对象包括高中生、本科生、研究生和博士后科学家。该项目还为高中科学课程和教师研讨会开发新的表观遗传学教材和课程。该项目的目标是了解细菌中广泛存在的氧化还原敏感含硫DNA修饰的生物化学：硫代磷酸酯（PT）。作为一种广泛存在于所有细菌属的表观遗传标记，PTs通过一个由5个成员组成的DNA基因簇（dda - e）整合到DNA中。该基因簇在DNA主干中插入S代替非桥接氧作为序列特异性PT。PTs在许多含有Dnd蛋白F-H的细菌的限制性修饰（R-M）系统中起作用。它们也存在于缺乏限制性基因的细菌中，这表明基因表达具有非r - m表观遗传功能。PTs的一个非常不寻常的特征是，只有12-14%的短共识序列在基因组中被修改。这一事实提出了关于酶如何找到它们的目标的问题。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的动态和功能。该项目采用了一种新的化学裂解/缺口翻译测序技术。单分子实时测序用于量化PT位置的变化，以响应氧化应激和细胞生长。基因组目标选择的问题是通过挖掘与基因组标记相关的现有PTs基因组图来解决的。此外，PT位置与Dnd蛋白的结合位点相关，使用ChIP-seq绘制细菌基因组中的蛋白质结合位点。Dnd限制性内切酶的靶标选择机制是通过定位细菌基因组中限制性内切诱导的切割位点来解决的。这些结果对理解基础微生物生理学和表观遗传学具有广泛的影响。该项目正在开发新的酶工具，用于在生物技术应用中将耐核酸酶PTs插入DNA中。

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.