NUCLEAR-MAGNETIC-RESONANCE STUDY OF THE INTERACTION OF T4 ENDONUCLEASE-V WITH DNA

T4 endonuclease V catalyzes the DNA strand cleavage in the vicinity of a thymine dimer. In order to obtain insight into the specific recognition mechanism of this enzyme with a thymine photodimer within DNA, the conformations of five different DNA duplexes, DNA I, d(GCGGATGGCG). d(CGCCTACCGC), DNA II, d(GCGGTTGGCG).d(CGCCAACCGC), DNA III, d(GCGGTTGGCG) box with bottom missing. d(CGCCAACCGC), DNA IV, d(GCGGGCGGCG).d(CGCCCGCCGC), and DNA V, d(GCGGCCGGCG).d(CGCCGGCCGC), with which the enzyme can interact, were studied by H-1 NMR. DNA I, DNA IV, and DNA V do not contain the TT sequence or a thymine dimer and hence, are expected to bind the enzyme only in a nonspecific manner. DNA II includes a single TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodimer. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure and that the overall structure of B-type DNA is retained. The photodimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The effects of T4 endonuclease V binding on these DNA duplexes were also investigated by H-1 NMR. The binding of this enzyme to DNA I, DNA IV, and DNA V causes no alternation in chemical shift values of the imino proton resonances, but the binding to DNA II induces a small downfield shift in the imino proton resonance of GC7. The binding of a T4 endonuclease V mutant, which lacks only the cleavage activity but retains the substrate-binding ability, to DNA III causes a strikingly large downfield shift in the imino proton resonance of GC7. Therefore, in addition to the TT box with bottom missing moiety, this position should be either the crucial point for T4 endonuclease V recognition or the particular site of a conformational change which occurs by T4 endonuclease V binding. Photo-CIDNP experiments showed that three tyrosine residues, including tyrosine 129 located in the aromatic segment (WYKYY) near the C-terminus, lie on the molecular surface of T4 endonuclease V and that the access of the dye to these residues is completely obstructed when the enzyme binds, respectively, to the three DNA duplexes (I, II, III). These findings reveal that this aromatic segment is involved in the interaction with DNA in a TT box with bottom missing specific or nonspecific manner. Using the wild-type and another mutant enzyme whose Arg 26 was replaced by Gln, the nonspecific binding of the enzyme with the three DNA duplexes (I-III) was investigated by observing the signal broadening of the NepsilonH signals of arginine side chains. The results reveal that Arg 26 is involved in nonspecific binding with the DNA duplexes presumably by electrostatic force.