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.
The funding provided by this grant has had a significant and global impact on scientific discovery and research training in our studies of the biological function of phosphorothioate (PT) modifications of DNA in bacteria. These sulfur modifications of the DNA backbone are present in hundreds of bacterial strains in virtually every environment, including dozens of human pathogenic bacteria and bacteria in the intestinal microbiome. The methods developed in this project provide researchers with the first tools to systematically and quantitatively study PT in any organism. The results, which were shared in several high-profile publications and in publically accessible databases, revealed that PTs function in in both a novel restriction-modification system to protect the host against other microbes and in the control of gene expression. Future applications of the affinity purification and mapping methods include metagenomic analysis of PT-containing bacterial strains in the human microbiome and their role in pathogenicity. The methods are now being applied to study PT in RNA, since all known DNA modifications also occur in RNA molecules such as rRNA and tRNA. The studies performed under this grant also had a significant impact on research training and technology transfer on a global scale. In addition to substantive career mentoring of postdoctoral scientists, the project provided opportunities for cross-disciplinary training of postdoctoral scientists, graduate students and undergraduate and high school students in applying chemical tools and principles to biological systems. The studies were performed in collaboration with students and scientists at Jiao Tong University in Shanghai, with research training in United States for two graduate students and a professor.
