The Chemical Biology of Phosphorothioate Modifications of DNA in Bacteria

生命过程化学（CLP）计划中的这一奖项支持马萨诸塞州理工学院的Peter Dedon教授的工作，对DNA硫代磷酸酯修饰的化学生物学进行基础研究，其中非桥接磷酸氧被硫原子取代。 硫代磷酸酯（PT）长期以来被认为是稳定寡脱氧核苷酸以抵抗核酸酶降解的合成修饰。 然而，PI最近发现PT修饰天然存在于细菌DNA中。 为了探索PT在细菌DNA中的生物学功能，将开发一种高灵敏度的液相色谱-质谱联用方法来定量PT修饰并定义两个核苷酸序列背景。 还将开发亲和纯化方法，以分离含PT的DNA片段，用于后续序列分析，以确定更大的序列背景、基因组位置和细菌物种形成。 这些方法将用于（1）发现新的含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修饰赋予细菌选择优势的假设。 该项目将为博士后科学家以及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，该项目被用作高中化学课上关于微生物看不见的世界的化学生物学年度研讨会的主题。细菌和其他生物体中DNA的化学修饰在控制微生物的表达方面起着关键作用。基因，促进微生物的生存，并防止其他微生物的病理入侵。 这些研究将有助于了解新发现的和广泛分布的PT修饰的基本生物学功能，并对微生物在不同环境中生存并与真核宿主相互作用的机制产生影响，以及发现新的生物化学途径，通过这些途径在活生物体中操纵硫。

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.