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

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

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.