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 recogni tion mechanism of this enzyme with a thymine photodimer within DNA, th e conformations of five different DNA duplexes, DNA I, d(GCGGATGGCG). d(CGCCTACCGC), DNA II, d(GCGGTTGGCG).d(CGCCAACCGC), DNA III, d(GCGGTTG GCG) box with bottom missing. d(CGCCAACCGC), DNA IV, d(GCGGGCGGCG).d(C GCCCGCCGC), and DNA V, d(GCGGCCGGCG).d(CGCCGGCCGC), with which the enz yme 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 expecte d to bind the enzyme only in a nonspecific manner. DNA II includes a s ingle TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodi mer. 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 D NA induces only a minor distortion in the structure and that the overa ll structure of B-type DNA is retained. The photodimer formation is fo und to cause a large change in chemical shifts at the GC7 base pair, w hich is located at the 3'-side of the thymine dimer, accompanied by th e major conformational change at the thymine dimer site. The effects o f T4 endonuclease V binding on these DNA duplexes were also investigat ed 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 res onances, 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 substra te-binding ability, to DNA III causes a strikingly large downfield shi ft 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 sit e of a conformational change which occurs by T4 endonuclease V binding . Photo-CIDNP experiments showed that three tyrosine residues, includi ng tyrosine 129 located in the aromatic segment (WYKYY) near the C-ter minus, 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). Th ese findings reveal that this aromatic segment is involved in the inte raction with DNA in a TT box with bottom missing specific or nonspecif ic manner. Using the wild-type and another mutant enzyme whose Arg 26 was replaced by Gln, the nonspecific binding of the enzyme with the th ree DNA duplexes (I-III) was investigated by observing the signal broa dening of the NepsilonH signals of arginine side chains. The results r eveal that Arg 26 is involved in nonspecific binding with the DNA dupl exes presumably by electrostatic force.