SFV topoisomerase: sequence specificity in a genetically mapped interval.
Poxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination. To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase. SFV topoisomerase was expressed in Escherichia coli and purified as a glutathione S-transferase (GST) or (his)6-tagged fusion protein. The recombinant Leporipox-virus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase. In particular SFV topoisomerase recognizes the same pentanucleotide motif [5'-(C/T)CCTT-3'] and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme. The SFV enzyme can also efficiently cleave DNA 3' of the variant site 5'-CCCTG-3' in certain sequence contexts. These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites. We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (< 2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter. This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells.
Palaniyar
N
N
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Fisher
C
C
Parks
R
R
Evans
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral; D012776:Fibroma Virus, Rabbit / Q000201:enzymology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D004264:DNA Topoisomerases, Type I