Measuring motion on DNA by the type I restriction endonuclease EcoR124I 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 triplex, 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. As the distance between type I and triplex 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 triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400+/-32 bp/s at 20 degrees C. Furthermore, the data can only be explained by bi-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.
Firman
K
K
Biophysics Laboratories, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT, UK.
Szczelkun
M D
MD
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D015280:DNA Restriction-Modification Enzymes / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D009038:Motion; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D015342:DNA Probes; D015280:DNA Restriction-Modification Enzymes; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D000255:Adenosine Triphosphate; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific; C109830:HsdR protein, E coli