Fluorescent substrates for the EcoRV restriction endonuclease.

In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at the sequence GAT'ATC (where denotes the point of scission) [ I ] . Even the change of a single base pair within this sequence will lead to a million fold reduction of EcoRV activity [2]. Paradoxically, gel retardation experiments in the absence of Mg2+ show that, unlike EcoRl and many other type 11 restriction endonucleases, EcoRV binds to DNA without any sequence preference [3,4]. The specificity of EcoRV is in fact dependent on the production of a high affinity binding site for Mg2+ between the protein and the cognate DNA [5]. X-ray crystal structures show that the cognate DNA adopts a highly distorted, kinked conformation in its complex with EcoRV, in contrast to noncognate DNA which retains a B-like conformation [6]. The difference between the AGO for the binding of cognate and noncognate sequences is near to zero and hence the energy from the additional contacts with the specific DNA appears to be neutralised by the unfavourable energy change from the distortion of the DNA. In addition, the crystal structures shows that the protein itself must undergo a conformational change before it can bind DNA. Therefore, during one cycle of DNA cleavage, the EcoRV protein will undergo several conformational changes: due first to binding nonspecific DNA and then specific DNA, and later by the sequence of events leading to cleavage and product release. To further investigate the kinetics of the cleavage and binding evcnts and to show how EcoRV discriminates between cognate and noncognate sequences, a series of self-complementary oligodeoxynucleotides were synthesized, that either contained or lacked the EcoRV recognition sequence. The oligodeoxynucleotides were between 12 and 16 bases long and they contained at their 5' termini a primary amino group on a C, alkyl linker. The oligodeoxynucleotides could thus be labelled with a fluorophore such as Dansyl chloride (5[ Dimethy lamino] napthaleneI -sulphonyl chloride). The use of a fluorescent group allows the binding of DNA to protein to be followed by fluorescence resonance energy transfer (FRET). EcoRV contains eight tryptophan residues per protein dimer and thus one can monitor the non-radiative transfer from the tryptophans to the Dansyl label. From Foster's theory of dipole-dipole energy transfer 171, the efficiency of non-radiative transfer between donor and acceptor fluorophores is inversely proportional to the sixth power of their spatial separation. Thus any change in either the separation or the relative orientation of donor and acceptor dipoles, within the range from 10 to 80A, can be sensitively determined [8, 91. .4lternatively, binding can also be followed by monitoring changes in the fluorescence depolarisation and quantum yield of the Dansyl label itself. The amino group was introduced by the method of Connolly [lo] as an FMOC protected Bcyanoethyl phosphoramidite at the final cycle of an automated DNA synthesis [ 1 I]. After deblocking with ammonia, the oligodeoxynucleotides were reacted overnight at room temperature with a 100 fold excess of Dansyl chloride in a bicarbonate buffer at pH 10.00. The labelled oligodeoxynucleotides were then purified by reverse phase HPLC on a C18 column, using a linear gradient in acetonitrile, and dialysed against 50 mM Tris-HCI (pH 7.5), 100 mM NaCI, 0.1 mM EDTA The self-complimentary substrates were then converted to duplexes by heating to 70 C and cooling to room temperature overnight. The concentrations of oligodeoxynucleotides were determined from their extinction at 254 nm. CD analysis revealed characteristic spectra for B-form DNA. The fluorescence excitation and emission spectra for the labelled substrates were typical for the Dansyl fluorophore. implying that the probe has not stacked onto the DNA. In the presence of the EcoRV enzyme, the fluorescence from the Dansyl group was not significantly perturbed with respect to either the quantum yield or positions of the fluorescence maxima. However, FRET experiments revealed the binding of the labelled oligodeoxynucleotides to the EcoRV nuclease. The FRET experiments were done in the absence of Mg2+, so as to prevent DNA cleavage, with excitation at 295 nm and emission at 525 nm. Binding resulted in a fluorescence transfer from the tryptophans in EcoRV to the Dansyl group. This fluorescence signal was found to be sensitive to the length of the oligodeoxynucleotide: it was maximal with a 12 bp substrate but it decreased to zero with a 16 bp substrate. This is consistent with the distance between the 5'-termini of the DNA and the surface of the protein increasing with the increasing length of DNA.