Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates.
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 approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 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 approximately 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 k(cl).
Krogh
B O
BO
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, 10021, USA.
Cheng
C
C
Burgin
A
A
Shuman
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded; D017996:Entomopoxvirinae / Q000201:enzymology*; D004951:Esterification; D015870:Gene Expression; D006110:Grasshoppers / Q000821:virology*; D008969:Molecular Sequence Data; D010710:Phosphates; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013873:Thionucleotides / Q000378:metabolism
D004277:DNA, Single-Stranded; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I