Continuous assays for DNA translocation using fluorescent triplex dissociation: application to type I restriction endonucleases.
Fluorescent assays and accompanying kinetic models are described for the analysis of DNA translocation independent of duplex unwinding. A triplex binding site (TBS) was introduced into DNA substrates at precise loci downstream of recognition sequences for type IA, IB and IC restriction endonucleases (EcoKI, EcoAI and EcoR124I, respectively). Each endonuclease was incubated (without ATP) with substrates on which a hexachlorofluoroscein-labelled triplex-forming oligonucleotide (HEX-TFO) was pre-bound. Following addition of ATP, 1-D enzyme motion resulted in collision with, and displacement of, the HEX-TFO, producing a >twofold increase in fluorescent intensity. Alternatively, a decrease in anisotropy following displacement of a rhodamine-labelled TFO was monitored. Using rapid mixing in a stopped-flow fluorimeter, continuous kinetic profiles were produced in which displacement is preceded by a lag-phase, directly proportional to the distance moved. For each enzyme, we obtained not only the translocation rate but also information on slow isomerisation step(s) at initiation. Furthermore, we demonstrated that enzymes deficient in DNA cleavage but with maximal ATPase activity showed initiation and translocation rates identical to wild-type, confirming that DNA strand breaks are not a pre-requisite of motion.
McClelland
Sarah E
SE
DNA-Protein Interactions Group, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
Dryden
David T F
DT
Szczelkun
Mark D
MD
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism*; D005454:Fluorescence Polarization; D017930:Genes, Reporter / Q000235:genetics; D007700:Kinetics; D009068:Movement; D009154:Mutation / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
C051802:triplex DNA; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific