Project Summary/Abstract Bacteria and the viruses that infect them, termed bacteriophages or simply phages, are locked in a never-ending evolutionary battle. To protect themselves against phages, bacteria have evolved numerous anti-phage defense systems. Some, like restriction-modification systems or CRISPR, have been well characterized and harnessed for biotechnological and therapeutic applications. Recently it has become apparent that there exist many other anti-phage defense systems and that most of them remain poorly understood. Some newly discovered anti-phage defense systems use Overcoming Lysogenization Defect (OLD) proteins, which possess a Toprim domain that can cleave DNA and an ABC-family ATPase domain that can hydrolyze nucleotide. Our long-term goal is to conduct basic research on OLD proteins to determine how they interact with DNA and elucidate the molecular mechanisms by which they contribute to anti-phage defense. OLD proteins can be classified by their surrounding genetic context, in which Class 1 OLD proteins exist in isolation in the genome while Class 2 OLD proteins are found immediately proximal to a predicted helicase gene and form a defense system called the Gabija system. The primary goal of the proposed research is to test hypotheses about the molecular mechanisms of both Class 1 and Class 2 OLD proteins, as well as the molecular interactions between Class 2 OLD proteins and their associated helicases. We will do so through a combination of biochemical assays characterizing DNA cleavage and nucleotide hydrolysis, and genetic plaque assays and mutational analysis for phage defense. These experiments will determine whether Class 1 OLD proteins provide anti-phage defense on their own, determine whether predicted Gabija helicases demonstrate helicase activity, and identify OLD protein amino acids that play functional roles in phage defense. In addition to the biotechnological opportunities studying anti-phage defense systems has brought, deepening our understanding of such systems could, in principle, also help set the stage for the development of potential therapeutic interventions such as phage therapy.