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. 2004 May 11;32(8):2578-85.
doi: 10.1093/nar/gkh582. Print 2004.

Functions of yeast helicase Ssl2p that are essential for viability are also involved in protection from the toxicity of adriamycin

Takemitsu Furuchi  1 , Tsutomu Takahashi, Shogo Tanaka, Katsushi Nitta, Akira Naganuma
Affiliations

Functions of yeast helicase Ssl2p that are essential for viability are also involved in protection from the toxicity of adriamycin

Takemitsu Furuchi et al. Nucleic Acids Res. .

Abstract

We have found that, in the yeast Saccharomyces cerevisiae, overexpression of the DNA helicase Ssl2p confers resistance to adriamycin. Ssl2p is involved, as a subunit of the basic transcription factor TFIIH, in the initiation of transcription and in nucleotide-excision repair (NER), and this helicase is essential for the survival of yeast cells. An examination of the relationship between the known functions of Ssl2p and adriamycin resistance indicated that overexpression of Ssl2p caused little or no increase in the rate of RNA synthesis and in NER. The absence of any involvement of NER in adriamycin resistance was supported by the finding that yeast cells that overexpressed the mutant form of Ssl2p that lacked the carboxy-terminal region, which is necessary for NER, remained resistant to adriamycin. When we examined the effects of overexpression in yeast of other mutant forms of Ssl2p with various deletions, we found that, of the 843 amino acids of Ssl2p, the entire amino acid sequence from position 81 to position 750 was necessary for adriamycin resistance. This region is identical to the region of Ssl2p that is necessary for the survival of yeast cells. Although this region contains helicase motifs, the overexpression of other yeast helicases, such as Rad3 and Sgs1, had little or no effect on adriamycin resistance, indicating that a mere increase in the intracellular level of helicases does not result in adriamycin resistance. Our results suggest that the functions of Ssl2p that are essential for yeast survival are also required for protection against adriamycin toxicity.

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Figures

Figure 1
Figure 1
Effects of overexpression of Ssl2p on the adriamycin-induced inhibition of transcription in yeast. Yeast cells (1 × 107 cells) were incubated in the presence of [5,6-3H]uracil for 90 min. Incorporation of [5,6-3H]uracil into total RNA (A) and into mRNA (B) in yeast cells was measured by liquid scintillation counting. Each column and bar represent the mean value and S.D. of results from three cultures. Differences between respective levels of [5,6-3H]uracil in total RNA and in mRNA were not statistically significant (Student’s t-test). See text for details.
Figure 2
Figure 2
Sensitivity of yeast cells that overexpressed Ssl2p to adriamycin, cisplatin and to UV-B. (A and B) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were suspended in liquid-SD (–Leu) medium at 1 × 104, 1 × 103, 1 × 102 and 1 × 10 cells/µl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Leu) medium prepared with and without adriamycin (80 µM) or cisplatin (150 µM). Plates were photographed after incubation for 48 h at 30°C. (C) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were spread on agar-solidified SD (–Leu) medium as indicated above and irradiated with UV-B (302 nm) for the indicated times. After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
Figure 3
Figure 3
Sensitivity to adriamycin of yeast cells that harbored plasmids that encoded Rad3, Ssl2p or Sgs1. Yeast cells (1 × 104 cells/200 µl) that harbored pRS314, pRS314-RAD3, pRS314-SSL2 or pRS314-SGS1 were grown in SD (–Trp) medium supplemented with the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. Each point represents the mean value and S.D. of results from three cultures. The absence of a bar indicates that the S.D. falls within the symbol.
Figure 4
Figure 4
Effects of adriamycin on the helicase activity of GST-Ssl2 or GST-Rad3. Reaction mixtures containing GST-Ssl2 or GST-Rad3 protein (300 µg), a partial duplex of circular single-stranded M13mp18 DNA as helicase substrate and a 32P-labeled 24-base complementary fragment were incubated in the presence of adriamycin at 37°C for 45 min. The displacement of the 24-base fragment from the single-stranded circle was determined by nondenaturing polyacrylamide gel electrophoresis and autoradiography.
Figure 5
Figure 5
Schematic representation of the structural domains of Ssl2p and of mutant proteins. The amino acids of Ssl2p are indicated by a scale below the Arabic numerals. The Roman numerals below the diagram of Ssl2p indicate the numbering of the helicase domains. Small numbers above the diagrams of Ssl2p-deletion mutants indicate the positions at which BamHI sites were created by site-directed mutagenesis. A summary of the results shown in Figures 6 and 7 is given on the right. ‘ADRr’ indicates the effect of overexpression of each mutant form of Ssl2p on the sensitivity of wild-type yeast to adriamycin (+, conferred resistance; –, did not confer resistance). ‘Survival’ indicates the ability of each mutant form of Ssl2p to support the survival of Ssl2p-null cells (ssl2Δ). ‘NERa’ indicates the ability of each mutant form of Ssl2p to restore NER in ssl2Δ cells (+, exhibited NER activity; –, did not exhibit NER activity; N.D., not determined).
Figure 6
Figure 6
Effects of overexpression of mutant forms of Ssl2p on the sensitivity of wild-type yeast to adriamycin. Yeast cells (1 × 104 cells/200 µl) harboring pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6 (A), pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 (B) were grown in SD (–Trp) medium that contained the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. For other details, see legend to Figure 3. For structures of mutant proteins, see Figure 5.
Figure 7
Figure 7
Effects of expression of mutant forms of Ssl2p on the viability and sensitivity of Ssl2p-null yeast cells. (A) An overnight culture of Ssl2p-null yeast cells (ssl2Δ) harboring pRS316-SSL2 plus pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6, pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 was diluted in SD (–Trp) medium (1 × 105 cells/µl), and 5 µl of each suspension of cells were spotted onto agar-solidified SD (–Trp) medium that contained 5-FOA, to eliminate pRS316-SSL2, and cultured for 72 h. (B) Ssl2p-null yeast cells (ssl2Δ) harboring pRS314-SSL2, pRS314-N1, pRS314-N2, or pRS314-C2 were examined. An overnight culture of each yeast strain was diluted in SD (–Trp) medium at 1 × 104, 1 × 103, 1 × 102 and 1 × 10 cells/µl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Trp) medium and exposed to UV-B for the indicated times. To examine the sensitivity to cisplatin and adriamycin, the yeast cells were also grown on plates of agar-solidified SD (–Trp) medium that contained cisplatin (100 µM) or adriamycin (80 µM). After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
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