Abstract Modification-dependent restriction systems (MDRs) recognize and cleave modified foreign DNA. These proteins are thought to play a role in establishing the epigenetic landscape of bacterial genomes and are especially important in protecting against predatory bacteriophage viruses, many of which incorporate modified bases into their DNA to evade detection by other defense systems. While MDRs can be found in most antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, and carbapenem-resistant enterobacteriaceae like Klebsiella pneumoniae, no eukaryotic homologs exist, making them promising targets for drug design. Inhibiting these systems has the potential to enhance the efficacy of phage-mediated bacterial killing, thus providing new therapeutic strategies to combat persistent, antibiotic resistant microbial infections. It is our long-term goal to study the basic biology and mechanisms of MDRs and use this knowledge to improve current phage therapy approaches. This proposal examines the structure and function of the McrBC restriction system, a two-component MDR that targets DNA containing methylated cytosines. E. coli McrB contains an N-terminal DNA binding domain and a C- terminal AAA+ motor domain that hydrolyzes GTP and mediates nucleotide-dependent oligomerization. McrB’s basal GTPase activity is stimulated via interaction with its partner endonuclease McrC. Biochemical studies suggest a model for DNA cleavage in which McrB and McrC assemble together at two distant methylated sites and translocate in a manner dependent on stimulated GTP hydrolysis. Collision of these McrBC assemblies triggers cleavage of both DNA strands. Despite this model, the molecular and mechanistic details underlying McrBC function remain poorly defined. In Aim 1, we will dissect the species- specific determinants of DNA binding in different McrB homologs using X-ray crystallography and biochemistry. We will also generate chimeras that exchange the DNA binding domains between different McrB homologs to test the hypothesis that the core hydrolysis and cleavage machineries in McrBC are conserved and have adapted to different evolutionary pressures via a modular design. In Aim 2, we will use mutagenesis and kinetic assays to identify the critical catalytic components responsible for McrC-stimulated GTPase activity. In Aim 3, we will determine the structure and architectural organization of the McrBC restriction complex at atomic resolution by X-ray crystallography and cryo-electron microscopy. These efforts will provide new insights into how McrBC complexes bind DNA, assemble, and hydrolyze GTP.