The Chemical Biology of Phosphorothioate Modifications of DNA in Bacteria

生命过程化学(CLP)项目的这一奖项支持麻省理工学院的Peter Dedon教授开展DNA硫代修饰化学生物学的基础研究，在该研究中，非桥磷氧被硫原子取代。硫代硫酸盐(PT)长期以来一直被认为是一种合成修饰，可以稳定寡聚脱氧核苷酸，防止核酸酶降解。然而，PI最近发现，PT修饰自然发生在细菌的DNA中。为了探索PT在细菌DNA中的生物学功能，我们将建立一种高灵敏的液相色谱质谱联用方法来定量PT的修饰并定义两个核苷酸的序列背景。还将开发一种亲和纯化方法，以分离含有PT的DNA片段，用于随后的序列分析，以确定更大的序列背景、基因组位置和细菌物种形成。这些方法将被用来(1)发现新的含有PT的细菌；(2)检验PT修饰是限制修饰系统的一部分的假设；(3)表征DND蛋白的生化功能；以及(4)检验PT修饰赋予细菌选择性优势的假设。该项目将为博士后科学家和本科生和高中生提供将化学工具和原理应用于生物系统的跨学科培训机会，该项目将作为高中化学课上关于看不见的微生物世界的化学生物学年度研讨会的主题。细菌和其他生物中DNA的化学修饰在控制基因表达、促进微生物生存和防止其他微生物的病理性入侵方面发挥着关键作用。这些研究将有助于理解新发现的和广泛分布的PT修饰的基本生物学功能，以及微生物在不同环境中生存并与真核宿主相互作用的机制，以及在活体中操纵硫的新的生化途径的发现。

This award in the Chemistry of Life Processes (CLP) program supports work by Professor Peter Dedon at the Massachusetts Institute of Technology to carry out fundamental studies on the chemical biology of phosphorothioate modifications of DNA, in which a non-bridging phosphate oxygen is replaced with a sulfur atom. Phosphorothioates (PT) have long been known as synthetic modifications that stabilize oligodeoxynucleotides against nuclease degradation. However, the PI recently discovered that PT modifications occur naturally in DNA from bacteria. To explore the biological function of PT in bacterial DNA, a highly sensitive liquid chromatography-coupled mass spectrometric method will be developed to quantify PT modifications and to define the two-nucleotide sequence context. An affinity purification method will also be developed to allow isolation of PT-containing DNA fragments for subsequent sequence analysis to define larger sequence contexts, genomic location and bacterial speciation. These methods will be used to (1) discover new PT-containing bacteria; (2) to test the hypothesis that PT modifications are part of a restriction-modification system; (3) to characterize the biochemical functions of the Dnd proteins; and (4) to test the hypothesis that PT modifications confer a selective advantage on bacteria. The project will provide opportunities for cross-disciplinary training of postdoctoral scientists and undergraduate and high school students in applying chemical tools and principles to biological systems, with the project used as a theme for an annual workshop on the chemical biology of the unseen world of microbes for high school chemistry classes.Chemical modifications of DNA in bacteria and other organisms play critical roles in controlling the expression of genes, promoting microbial survival, and preventing the pathological invasion of other microbes. The studies will lead to an understanding of the fundamental biological function of the newly discovered and widely distributed PT modifications, with implications for mechanisms by which microbes survive in different environments and interact with eukaryotic hosts, as well as the discovery of new biochemical pathways by which sulfur is manipulated in living organisms.