Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli.
We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I. A family of plasmids expressing the yeast TOP1 gene or 5'-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E. coli topA mutant. These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling. Functional complementation of a conditional-lethal mutation in topA, which encodes E. coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA. We also show that approximately 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E. coli, however, is more sensitive to such deletions.
Bjornsti
M A
MA
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Wang
J C
JC
D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I