Melanoplus sanguinipes Entomopoxvirus DNA Topoisomerase: Site-Specific DNA Transesterification and Effects of 5′-Bridging Phosphorothiolates

Abstract

Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at ∼0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were kcl = 0.3 s−1 and Kcl = 0.25. The introduction of a 5′-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to ≥30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a ∼10−4 decrement in the rate of topoisomerase-catalyzed attack of 5′-SH DNA on the DNA-(3′-phosphotyrosyl)-enzyme intermediate. 5′-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on kcl.