Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: Site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates Journal Article

  

Authors:	Krogh, B. O.; Cheng, C.; Burgin Jr, A.; Shuman, S.
Article Title:	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 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 ~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).
Keywords:	binding affinity; animals; gene expression; enzyme activity; dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; kinetics; recombinant fusion proteins; base sequence; esterification; catalysis; dna, single-stranded; enzyme binding; vertebrate; phosphates; ion exchange chromatography; dna topoisomerase; dna topoisomerases, type i; virus dna; insect; entomopoxvirinae; entomopoxvirus; thionucleotides; priority journal; article; sedimentation rate; phosphorothioic acid derivative; grasshoppers
Journal Title:	Virology
Volume:	264
Issue:	2
ISSN:	0042-6822
Publisher:	Elsevier Inc.
Date Published:	1999-11-25
Start Page:	441
End Page:	451
Language:	English
DOI:	10.1006/viro.1999.0022
PUBMED:	10562506
PROVIDER:	scopus
DOI/URL:	https://www.scopus.com/inward/record.uri?eid=2-s2.0-0033604641&partnerID=40&md5=f8c78e2c8b33cec26ab878cf034bcbb4
Notes:	Article -- Export Date: 16 August 2016 -- Source: Scopus

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