A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II.
BACKGROUND
Type II topoisomerases are a highly conserved class of enzymes which transport one double-stranded DNA segment through a transient break in another. Whereas the eukaryotic enzymes are homodimers of a single polypeptide, their bacterial homologues are homodimers of two independently coded protein subunits. Unlike eukaryotic topoisomerase II and bacterial topoisomerase IV, DNA gyrase is a bacterial type II topoisomerase which specializes in intramolecular DNA transport.


RESULTS
We have fused the Escherichia coli coding sequences for the proteins GyrB and GyrA, which comprise DNA gyrase. This fusion was expressed in yeast cells and yielded the expected full-length protein product. When it was expressed in Deltatop1- top2-4 yeast cells, the fusion protein compensated their slow growth and reverted their elevated chromosomal excision of ribosomal genes. Furthermore, it removed DNA positive supercoils. The fusion protein, however, was unable to complement the temperature-dependent lethality of top2-4 cells.


CONCLUSIONS
Fusion of the E. coli GyrB and GyrA proteins leads to a catalytically active topoisomerase which compensates several phenotypic traits attributed to unconstrained DNA supercoiling in topoisomerase-deficient cells. However, since the fusion protein cannot substitute for topoisomerase II, it may be efficient in intramolecular but not intermolecular DNA passage, resembling the catalytic properties of DNA gyrase.
Trigueros
Sonia
S
Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain.
Roca
Joaquim
J
D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013696:Temperature
D004269:DNA, Bacterial; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II