Genetic analysis of the gyrase A-like domain of DNA topoisomerase II of Saccharomyces cerevisiae.
We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3' end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein. Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tos1; tos1 is not allelic to any genes predicted to encode type I topoisomerase-related proteins. The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles. These top2 mutations may have compensatory effects on the general stability of the protein.
Thomas
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Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Spell
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Ming
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Holm
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D001483:Base Sequence; D004252:DNA Mutational Analysis; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000502:physiology; D005800:Genes, Fungal / Q000235:genetics*; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010641:Phenotype; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
D004250:DNA Topoisomerases, Type II