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

The type I restriction enzyme EcoR124I cleaves DNA following extensive line ar translocation dependent upon ATP hydrolysis. Using protein-directed disp lacement of a DNA triples, we have determined the kinetics of one-dimension al motion without the necessity of measuring DNA or ATP hydrolysis. The tri plex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I si te, and then incubated with endonuclease, Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displace d with exponential kinetics. iis the distance between type I and triples si tes was shortened, the lag time decreased whilst the displacement reaction remained exponential, This is indicative of processive DNA translocation fo llowed by collision with the tripler and oligonucleotide displacement. A li near relationship between lag duration and intersite distance gives a trans location velocity of 400 +/- 32 bp/s at 20 degrees C, Furthermore, the data can only be explained by hi-directional translocation, An endonuclease wit h only one of the two HsdR subunits responsible for motion could still cata lyse translocation. The reaction is less processive. but can 'reset' in eit her direction whenever the DNA is released.