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. 1997 Jan 21;94(2):581-6.
doi: 10.1073/pnas.94.2.581.

Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae

Y Ho  1 , S Mason, R Kobayashi, M Hoekstra, B Andrews
Affiliations

Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae

Y Ho et al. Proc Natl Acad Sci U S A. .

Abstract

In the budding yeast, Saccharomyces cerevisiae, DNA damage or ribonucleotide depletion causes the transcriptional induction of an array of genes with known or putative roles in DNA repair. The ATM-like kinase, Mec1, and the serine/threonine protein kinases, Rad53 and Dun1, are required for this transcriptional response. In this paper, we provide evidence suggesting that another kinase, Hrr25, is also involved in the transcriptional response to DNA damage through its interaction with the transcription factor, Swi6. The Swi6 protein interacts with Swi4 to form the SBF complex and with Mbp1 to form the MBF complex. SBF and MBF are required for the G1-specific expression of G1 cyclins and genes required for S-phase. We show that Swi6 associates with and is phosphorylated by Hrr25 in vitro. We find that swi4, swi6, and hrr25 mutants, but not mbp1 mutants, are sensitive to hydroxyurea and the DNA-damaging agent methyl methane-sulfonate and are defective in the transcriptional induction of a subset of DNA damage-inducible genes. Both the sensitivity of swi6 mutants to methyl methanesulfonate and hydroxyurea and the transcriptional defect of hrr25 mutants are rescued by overexpression of SWI4, implicating the SBF complex in the Hrr25/Swi6-dependent response to DNA damage.

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Figures

Figure 1
Figure 1
Binding of p54(Hrr25) to Swi6 protein affinity columns. (A) Yeast extracts from BJ2168 were loaded onto either a control column (no coupled protein, lane 1) or a Swi6-coupled column (lane 2) and eluted with 1 M NaCl. (B) Extracts made from either a wild-type (lanes 1 and 2) or an hrr25Δ strain (lanes 3 and 4) were loaded onto either a control column (lanes 1 and 3) or Swi6-coupled columns (lanes 2 and 4) and eluted with 1 M NaCl. Protein molecular weight markers are indicated to the left of the gel photographs and the p54 (Hrr25) is indicated by the arrow on the right.
Figure 2
Figure 2
In vitro phosphorylation of Swi6 by Hrr25. (A) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or hrr25Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. (B) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.
Figure 3
Figure 3
Transcriptional induction of genes after treatment with 200 mM HU in swi4Δ, swi6Δ, and mbp1Δ strains. (A) Yeast strains (indicated at the top of each panel) were grown in minimal media to early logarithmic phase and a 0 time point was taken before HU was added to 200 mM. Aliquots of cells were taken after HU addition at the time points specified (in minutes). Total RNA was extracted and Northern hybridization analysis was performed with the probes indicated to the left of each panel. All time points shown for each probe are from the same exposure of the Northern blot. Blots were sequentially hybridized with the different probes. (B) PhosphorImager analysis of the Northern blots shown in A. The RNA levels of RNR2, RNR3, and UBI4 relative to ACT1 were determined and plotted versus time after HU addition. ACT1 encodes actin and served as a loading control. swi6 (SWI4) indicates an swi6 deletion strain transformed with a high-copy SWI4 plasmid.
Figure 4
Figure 4
Transcriptional induction of RNR2, RNR3, and UBI4 after treatment with 200 mM HU in an hrr25Δ strain. (A) RNA was isolated from the strains indicated at the top of each panel after treatment with HU as described in the legend to Fig. 3. Total RNA was extracted and Northern hybridization analysis was performed with the probes indicated to the left. (B) PhosphorImager analysis of the Northern blots shown in A. The RNA levels of RNR2, RNR3, and UBI4 relative to ACT1 were determined and plotted versus time after HU addition.
Figure 5
Figure 5
Model for the role of Hrr25 and SBF (Swi4/Swi6) in the transcriptional response to DNA damage. The Hrr25 protein kinase is proposed to phosphorylate Swi6 in response to DNA damage. An Hrr25-independent pathway may also function through Swi4. These two pathways serve to activate SBF to promote the transcriptional induction of repair genes. See text for details.
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