Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes.

摘要

The reaction mechanism of bacteriophage T4 endonuclease V was investigated using modified oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. For the pyrimidine dimer glycosylase step, the formation of a covalent intermediate has been proposed. A fluorine atom was attached to the 2'-position of the 5'-component of the thymine dimer site, which could stabilize the covalent complex and prevent the ring opening of the sugar moiety. The strand cleavage of the 12 base pair substrate analog did not occur, although the glycosyl bond was cleaved by this enzyme. A covalent enzyme--substrate complex was separated by gel electrophoresis under denaturing conditions. It was shown that the enzyme molecules were completely converted to a stable complex in the reaction mixture. Two mechanisms have been proposed for the beta-elimination step. A 12-mer containing a phosphorothioate linkage between adjacent thymidines was prepared. The diastereomers were separated, and the absolute configurations were determined. After formation of the thymine dimer and 32P-labeling of the 5'-terminus, these oligonucleotides were annealed to the complementary 12-mer, and the reaction rates of the pyrimidine dimer glycosylase step and the overall reaction for each duplex were measured under the substrate-saturation conditions. The rate constants indicated that the chemical reaction at the beta-elimination step was rate-limiting. Since no difference was observed in the rate constants for the Rp- and Sp-phosphorothioate substrates, it is concluded that the beta-elimination reaction is catalyzed, not by the internucleotide phosphate, but by an amino acid residue of the enzyme.