Tyrosine partners coordinate DNA nicking by the Salmonella typhimurium plasmid pCU1 relaxase enzyme

Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demonstrate that the pCU1 relaxase preferentially uses Y26 or a combination of Y18 + 19 to nick DNA at wild type levels, and that an adjacent aspartic acid deprotonates these tyrosines to activate them for attack. Our findings illustrate the unique modifications that the pCU1 relaxase has introduced into the traditional relaxase-mediated DNA nicking mechanism.

► The pCU1 relaxase contains four potential DNA nicking tyrosines.
► Mutation of individual tyrosines reveals the relative DNA nicking activity of each.
► The pCU1 relaxase can efficiently nick DNA with either Tyr26 or Tyr18 + 19.
► Aspartic acid 84 activates these tyrosines for attack on the DNA scissile phosphate.