Everything OLD is new again: How structural, functional, and bioinformatic advances have redefined a neglected nuclease family

Overcoming lysogenization defect (OLD) proteins are a conserved family of ATP‐powered nucleases that function in anti‐phage defense. Recent bioinformatic, genetic, and crystallographic studies have yielded new insights into the structure, function, and evolution of these enzymes. Here we review these developments and propose a new classification scheme to categorize OLD homologs that relies on gene neighborhoods, biochemical properties, domain organization, and catalytic machinery. This taxonomy reveals important similarities and differences between family members and provides a blueprint to contextualize future in vivo and in vitro findings. We also detail how OLD nucleases are related to PARIS and Septu anti‐phage defense systems and discuss important mechanistic questions that remain unanswered. OLD family nucleases are conserved ATP‐powered enzymes that function broadly in anti‐phage defense. In this review, we summarize our current understanding of OLD structure, function, and evolution, highlighting important advances gleaned from bioinformatics, genetics, and X‐ray crystallography. We propose a new classification scheme to categorize OLD homologs and explore structural and mechanistic features that are shared with the PARIS and Septu anti‐phage defense systems.