Measuring motion on DNA by the type I restriction endonuclease EcoR1241 using triplex displacement

The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triples, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease, Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. iis the distance between type I and triples sites was shortened, the lag time decreased whilst the displacement reaction remained exponential, This is indicative of processive DNA translocation followed by collision with the tripler and oligonucleotide displacement. A linear relationship between lag duration and intersite distance gives a translocation velocity of 400 +/- 32 bp/s at 20 degrees C, Furthermore, the data can only be explained by hi-directional translocation, An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive. but can 'reset' in either direction whenever the DNA is released.