Functional implications of arginine‐121 in <scp>RuvA</scp> oligomerisation and <scp>RuvAB</scp> ‐mediated branch migration in the Gram‐positive <i>Listeria monocytogenes</i>

In prokaryotes, the RuvAB complex drives Holliday junction (HJ) branch migration, but the relative importance of RuvA tetramers versus octamers remains debatable and unexplored in Gram-positive bacteria. In this study, we aimed to determine whether RuvA from Listeria monocytogenes (LmRuvA) is active as a tetramer or octamer in branch migration. We identified arginine-121 as being critical for the formation of the tetramer-tetramer interface. Mutation of arginine-121 to aspartate results in a protein that exists in a dimer to tetramer equilibrium in solution (unlike other octamer-deficient mutants from earlier studies), binds to the HJ as a tetramer only, interacts poorly with RuvB, and cannot catalyse branch migration. Collectively, these findings suggest that the ability of LmRuvA to bind HJs as an octamer is critical to branch migration. Impact statement The study highlights the role of DNA repair protein RuvA in Gram-positive Listeria monocytogenes, demonstrating that its mutation at arginine-121 leads to branch migration deficiency. Hence, targeting RuvA offers a promising therapeutic strategy to slow down the development processes of antibiotic resistance in deadly foodborne pathogens of public concern.