OLD family nuclease function across diverse anti-phage defense systems - 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.
