Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol - PubMed (original) (raw)
Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol
D Gadelle et al. Nucleic Acids Res. 2005.
Abstract
Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI 'Bergerat fold', whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.
Figures
Figure 1
Agarose gel electrophoresis showing the effect of radicicol (a) and geldanamycin (b) on decatenation of kDNA by the DNA topoisomerase VI. Lane 1, linear kDNA marker (L); lane 2, decatenated kDNA marker: nicked, open circular (N) and relaxed DNA (R); lane 3, catenated kDNA (C); lane 4, kDNA with Topo VI; lane 5, kDNA with Topo VI and DMSO; and lanes 6–11, kDNA with Topo VI in the presence of radicicol or geldanamycin (250, 200, 125, 100, 50 and 25 μM, respectively).
Figure 2
Agarose gel electrophoresis showing the effect of radicicol on relaxation of the plasmid pBR322. Lane 1, supercoiled pBR322; lane 2, supercoiled pBR322 in the presence of radicicol (250 μM); lane 3, supercoiled pBR322 with Topo VI; lane 4, supercoiled pBR322 with Topo VI and DMSO; and lanes 5–10, supercoiled pBR322 with Topo VI in the presence of radicicol (25, 50, 100, 125, 200 and 250 μM, respectively). SC: negatively supercoiled pBR322 DNA; R: relaxed DNA; OC: open circular (nicked) pBR322 DNA.
Figure 3
Agarose gel electrophoresis showing the effect of radicicol on supercoiling of the plasmid pBR322 by the DNA gyrase. Lane 1, control pBR322 negatively supercoiled; lane 2, relaxed pBR322 and 500 μM radicicol without gyrase; lane 3, relaxed pBR322 with gyrase; lanes 4 and 5, relaxed pBR322 with gyrase in the presence of 100 or 500 μM radicicol; and lane 6, relaxed pBR322 with gyrase in the presence of 100 μM novobiocin.
Figure 4
In vivo inhibition of radicicol on Haloferax and Sulfolobus growths. Effect of different drug concentrations of radicicol measured by monitoring the cultures' absorbances at 600 nm.
Figure 5
Stability of radicicol at 74°C in the Sulfolobus medium. Five flasks with 10 ml of Sulfolobus culture medium were incubated at 74°C, one for control without drug (flask T), and the four others with 100 μM of radicicol (flasks R:0, R:2, R:4 and R:6). At time = 0 h, 1 ml of a Sulfolobus culture (OD of 0.62) was added to flasks T and R:0, at time = 2, 4 and 6 h, 1 ml of the same Sulfolobus culture (OD of 0.62) was added, respectively, to flasks R:2, 4 and 6.
Figure 6
Effect of radicicol treatment (60 μM) on the morphology of H.volcanii. Microscopic observations ×100. (a) Cells before radicicol addition; (b) cells at time 24 h after radicicol addition (the culture has been concentrated by centrifugation for microscopic observation); (c) cells at time 24 h after removal of radicicol and resuspension; and (d) growth curves before and after removal of radicicol.
Figure 7
Effect of different drugs on E.coli growth measured by monitoring the cultures' absorbances at 600 nm.
Figure 8
Sliced surface representation of the S.shibatae DNA topoisomerase VI, S.cerivisiae Hsp90 and T.maritima histidine kinase ATP-binding sites complexed with ATP, radicicol and geldanamycin. The ligands are shown in sticks. Opaque regions correspond to the interior of the enzymes molecules.
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