An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme.

Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.