Type I restriction enzymes use two motors to translocate DNA before carrying out 
DNA cleavage. The motor function is accomplished by amino-acid motifs typical 
for superfamily 2 helicases, although DNA unwinding is not observed. Using a 
combination of extensive single-molecule magnetic tweezers and stopped-flow bulk 
measurements, we fully characterized the (re)initiation of DNA translocation by 
EcoR124I. We found that the methyltransferase core unit of the enzyme loads the 
motor subunits onto adjacent DNA by allowing them to bind and initiate 
translocation. Termination of translocation occurs owing to dissociation of the 
motors from the core unit. Reinitiation of translocation requires binding of new 
motors from solution. The identification and quantification of further 
initiation steps--ATP binding and extrusion of an initial DNA loop--allowed us 
to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation 
of motors during translocation allows dynamic control of the restriction process 
by the availability of motors. Direct evidence that this control mechanism is 
relevant in vivo is provided.