REACTIONS OF THE ECORV RESTRICTION-ENDONUCLEASE WITH FLUORESCENT OLIGODEOXYNUCLEOTIDES - IDENTICAL EQUILIBRIUM-CONSTANTS FOR BINDING TO SPECIFIC AND NONSPECIFIC DNA

The EcoRV restriction endonuclease cleaves DNA specifically at its rec ognition sequence in the presence of magnesium ions, but several studi es have indicated that it binds to DNA in the absence of Mg2+ without any preference for its recognition site. However, specific binding to the recognition site has also been reported. To distinguish between th ese reports, oligodeoxynucleotides were tagged with either dansyl or e osin fluorophores at their 5' termini and annealed to form duplexes of 12 to 16 base-pairs. For each length of duplex, one derivative had th e EcoRV recognition sequence while another lacked this sequence. For t he duplexes with the recognition site, the fluorophores had no effect on DNA cleavage rates by EcoRV in the presence of Mg2+. The binding of the specific and non-specific duplexes to EcoRV in the absence of Mg2 + was measured by fluorescence resonance energy transfer and by fluore scence depolarization. In both procedures, the signal from the specifi c complex differed from the complex with non-specific DNA, with the de polarization data indicating that non-specific DNA bound to EcoRV reta ins a higher rotational freedom than specific DNA. Even so, the equili brium constant for the binding of specific DNA was identical, within e rror Limits, to that for non-specific DNA. (C) 1998 Academic Press Lim ited.