DNA glycosylases involved in interstrand crosslink repair and antibiotic self-resistance

DNA is chemically altered, or damaged, by its interaction with molecules found in the cell and in the environment. All organisms contain enzymes that repair damaged DNA to protect the integrity of the genetic information. This research project will determine how a newly discovered DNA repair enzyme found in bacteria works to repair damage created by specific toxins, which some bacteria produce as defense mechanisms. Because of their toxicity, these bacteria-derived toxins often have antimicrobial and antitumor properties, making them important in agriculture, industry, and medicine. The project will benefit society by providing 1) undergraduate and graduate students with hands-on experience in methods used to determine atomic structures of proteins that are important for discovery and innovation in biotechnology, 2) summer research opportunities to undergraduates of Fisk University, a nearby historically black college/university, 3) teaching and mentoring opportunities for all trainees, 4) exposure of high school students interested in STEM to biomedical research, and 5) community outreach. These activities are facilitated through a collaborative and inclusive training environment at the intersection of the College of Arts and Science and the School of Medicine at Vanderbilt University. Interstrand DNA crosslinks (ICLs) are among the most cytotoxic forms of DNA damage because they covalently tether the two DNA strands and therefore interfere with DNA replication and transcription. A new ICL repair pathway was recently discovered in both eukaryotes and prokaryotes, in which a DNA glycosylase liberates one of the crosslinked nucleobases from the DNA backbone. The bacterial ICL glycosylases belong to a family of uncharacterized proteins prevalent in antibiotic producers and pathogens. The PI’s laboratory discovered that E. coli YcaQ initiates an ICL repair pathway by unhooking chemically diverse ICLs, and characterized a related enzyme (AlkZ) that provides Streptomyces with self-resistance to the toxicity of one of its natural products, azinomycin B, by unhooking resulting azinomycin B-ICLs. The long-term goals of this project are to elucidate the ICL repair pathway in bacteria, to understand how microbes utilize DNA repair to protect against genotoxic natural products in nature and within the microbiome, and to discover new genotoxic agents with beneficial applications. The short-term goals are to understand the molecular basis for ICL unhooking by YcaQ and for the specificity of AlkZ-related enzymes for highly functionalized crosslinking and intercalating natural products, and to characterize the bioactivities of putative genotoxins. A multidisciplinary approach integrating structural biology, biochemistry, genetics, cell biology, and metabolomics will be employed to achieve these goals. This project is supported by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences/Directorate for Biological Sciences and the Chemistry of Life Processes program in the Division of Chemistry/Directorate for Mathematical and Physical Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.