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 co...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1987-12, Vol.84 (24), p.8971-8975
Main Authors: Bjornsti, Mary-Ann, Wang, James C.
Format: Article
Language:English
Subjects:
Bacteriology
Biological and medical sciences
Biotechnology
Cell extracts
DNA
DNA Topoisomerases, Type I - genetics
DNA Topoisomerases, Type I - metabolism
DNA, Bacterial - metabolism
DNA, Superhelical - metabolism
Electrophoresis
Enzymes
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Gels
Genetic Complementation Test
Genetic engineering
Genetic mutation
Genetic technics
Genetics
Methods. Procedures. Technologies
Microbiology
Miscellaneous
Phenotypes
Plasmids
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Structure-Activity Relationship
Substrate Specificity
Synthetic digonucleotides and genes. Sequencing
Viability
Yeasts
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Summary: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 ≈ 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.84.24.8971