Abstract
Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a β-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.