The Chemical Biology of Phosphorothioate Modifications of DNA in Bacteria

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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.