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- W2034669051 abstract "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." @default.
- W2034669051 created "2016-06-24" @default.
- W2034669051 creator A5041589194 @default.
- W2034669051 creator A5047949310 @default.
- W2034669051 creator A5069818913 @default.
- W2034669051 date "2005-05-01" @default.
- W2034669051 modified "2023-10-06" @default.
- W2034669051 title "Continuous Assays for DNA Translocation Using Fluorescent Triplex Dissociation: Application to Type I Restriction Endonucleases" @default.
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- W2034669051 doi "https://doi.org/10.1016/j.jmb.2005.03.018" @default.
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