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Role of the Casein Kinase I Isoform, Hrr25, and the Cell Cycle-Regulatory

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Role of the Casein Kinase I Isoform, Hrr25, and the Cell Cycle-Regulatory Proc. Natl. Acad. Sci. USA Vol. 94, pp. 581–586, January 1997 Genetics 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 (DNA repairytranscriptionycell cycle) YUEN HO*, STEPHEN MASON*†,RYUJI KOBAYASHI‡,MERL HOEKSTRA§, AND BRENDA ANDREWS*¶ *Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON Canada M5S 1A8; ‡Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and §ICOS Corporation, 22021-20th Avenue SE, Bothell, WA 22021 Communicated by Ira Herskowitz, University of California, San Francisco, CA, November 1, 1996 (received for review July 31, 1996) ABSTRACT In the budding yeast, Saccharomyces cerevi- agents such as methyl methanesulfonate (MMS) and UV siae, DNA damage or ribonucleotide depletion causes the irradiation. Recent studies have delineated a pathway by which transcriptional induction of an array of genes with known or the damage signal is transduced to the checkpoint and tran- putative roles in DNA repair. The ATM-like kinase, Mec1, and scriptional response apparatus. The kinases, Mec1 (4, 17) and the serineythreonine protein kinases, Rad53 and Dun1, are Rad53 (6, 17), are required for both responses, whereas the required for this transcriptional response. In this paper, we Dun1 kinase, believed to act downstream of Mec1 and Rad53, provide evidence suggesting that another kinase, Hrr25, is is only required for the transcriptional induction response (16). also involved in the transcriptional response to DNA damage Mutations in another kinase, Hrr25, were identified as causing through its interaction with the transcription factor, Swi6. hypersensitivity to double-stranded DNA breaks induced by The Swi6 protein interacts with Swi4 to form the SBF complex endonuclease expression, x-irradiation, or continuous exposure and with Mbp1 to form the MBF complex. SBF and MBF are to MMS (18). Hrr25 is a casein kinase I (CKI) isoform that has required for the G1-specific expression of G1 cyclins and genes dual-specificity protein kinase activity in vitro (19). In addition to required for S-phase. We show that Swi6 associates with and having defects in DNA double-strand break repair, hrr25 mutant is phosphorylated by Hrr25 in vitro. We find that swi4, swi6, cells sporulate poorly, grow very slowly, and show a cell cycle and hrr25 mutants, but not mbp1 mutants, are sensitive to delay in G2 (18). Kinase assays carried out with Hrr25 immuno- hydroxyurea and the DNA-damaging agent methyl methane- precipitates from yeast extracts show phosphorylation of Hrr25 sulfonate and are defective in the transcriptional induction of itself as well as many coimmunoprecipitated proteins (20), sug- a subset of DNA damage-inducible genes. Both the sensitivity gesting that Hrr25 may have multiple substrates in vivo. The of swi6 mutants to methyl methanesulfonate and hydroxyurea potential role of Hrr25 in the transcriptional or checkpoint and the transcriptional defect of hrr25 mutants are rescued by response to DNA damage has not been investigated. overexpression of SWI4, implicating the SBF complex in the In this study, we show that Hrr25 interacts with and phosphor- Hrr25ySwi6-dependent response to DNA damage. ylates the Swi6 protein in vitro. Swi6 is a cell cycle-regulatory transcription factor that activates gene expression late in the G1 In budding yeast and other eukaryotic cells, exposure to phase of the cell cycle at START (reviewed in refs. 21 and 22). DNA-damaging agents invokes both a checkpoint response Swi6 does not bind DNA specifically (23), but functions as a and a repair response. Checkpoint pathways delay cell division transcription factor through its interaction with different DNA- to allow the repair of damaged DNA prior to proceeding binding partners (23–27). Swi6 interacts with the Swi4 protein to through the cell cycle; in general, checkpoints serve to ensure form the SBF complex (SCB-binding factor), which activates the integrity of the genome (reviewed in refs. 1 and 2). Three transcription of some G1 cyclin genes and the HO gene through checkpoint responses to DNA damage have been defined in a cis-acting element called the SCB (SWI4y6 cell cycle box; consensus CACGAAA). When bound to the Mbp1 protein, Swi6 yeast. First, a G2yM checkpoint acts to prevent mitosis in the presence of broken or damaged chromosomes (3–5). Second, forms a second transcription factor, MBF (MCB-binding factor, an S-phase checkpoint prevents entry into mitosis in the also known as DSC1), which acts through a distinct upstream sequence element, the MCB [MluI cell cycle box, consensus presence of unreplicated DNA (4, 6–8). Finally, a G1 check- point acts to delay S-phase entry in response to DNA lesions ACGCGTNA (see refs. 21 and 22)]. The SCB and MCB elements incurred early in the cell cycle (6, 9). are each sufficient to confer START-specific transcription on During checkpoint-induced cell cycle arrest, some genes heterologous promoters (28–30). MCB elements are found in the involved in DNA repair are transcriptionally induced. These promoters of many cell cycle-regulated genes involved in DNA genes include RNR1, RNR3 (large subunit of ribonucleotide replication such as CDC9, POL1, and the RNR genes (reviewed reductase, ref. 10), RNR2 (small subunit of ribonucleotide in ref. 31). In addition to being cell cycle regulated, the expression reductase, ref. 11), RAD54 (recombinational repair, ref. 12), of some MCB-controlled genes is also induced by DNA damage POL1 (DNA polymerase 1, ref. 13), and CDC9 (DNA ligase, (e.g., CDC9, POL1, RNR1, RAD51, RAD54, UNG1; refs. 10, 12, refs. 14 and 15). The importance of the transcriptional acti- and 32–34). Although a role for MCB elements in controlling cell vation of repair genes became evident with the isolation of a cycle-regulated transcription has been established, their role in DNA damage-induced transcription is unclear. mutant, dun1 (16), which is defective for DNA damage- We find that hrr25 mutants are defective in the transcriptional induced transcription and is hypersensitive to DNA damaging induction of the RNR2 and RNR3 genes in response to ribonu- cleotide depletion caused by HU (hydroxyurea) treatment. In The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: MMS, methyl methanesulfonate; HU, hydroxyurea; CKI, casein kinase I; HA, hemagglutinin. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA †Present address: German Cancer Research Center, D-69120, Hei- 0027-8424y97y94581-6$2.00y0 delberg, Germany. PNAS is available online at http:yywww.pnas.org. ¶To whom reprint requests should be addressed. 581 Downloaded by guest on September 25, 2021 582 Genetics: Ho et al. Proc. Natl. Acad. Sci. USA 94 (1997) addition to defining a biochemical interaction between Hrr25 and contained 100 ng Swi6 protein (see above), myelin basic Swi6, we show that, like hrr25 mutants, both swi6 and swi4 protein (Sigma), histone H1 (Boehringer Mannheim), or ca- mutants are sensitive to DNA-damaging agents and defective in sein (Sigma), as indicated. Reactions were stopped after 15 the damage-induced transcription of RNR2 and RNR3. Our min at 308C with SDS sample buffer and boiled before observations lead us to propose a novel role for the SBF complex electrophoresis on SDSypolyacrylamide gels. The gels were (Swi4ySwi6), through its interaction with the Hrr25 protein dried and exposed to XAR-5 film (Kodak). kinase, in the transcriptional response to DNA damage. HA–Hrr25 Immunoprecipitation Kinase Assay. Immunopre- MATERIALS AND METHODS cipitation kinase assays with hemagglutinin (HA)-tagged Hrr25 were done essentially as described (38), with modifications. Strain Yeast Strains. All yeast strains used for plating assays and JO34 (wild type) was transformed with a plasmid containing Northern blot analysis were isogenic to strain JO34 (S288C HRR25 tagged at the C terminus with a single HA epitope tag derivative, MATa, ura3-52, lys2-801a, ade2-1070, his3D200, (38) or vector pRS316 (39) and grown in selective medium (36) leu2-D1, SCB-LacZ) with the exceptions noted. The swi4D (swi4DHIS3, JO57-6B) and swi6D (swi6DHIS3, JO42-7C) to maintain the plasmid. Cells were harvested in early logarithmic strains have been described (35). The mbp1D (mbp1DTRP1) phase and lysed in IPK buffer (50 mM Tris, pH 7.5y1% Nonidet allele was constructed using PCR to replace the entire MBP1 P-40y0.05% SDSy0.05% sodium deoxycholatey5 mM EDTAy5 coding sequence with the TRP1 gene. The mbp1DTRP1 dis- mM DTTy100 mM NaCl with proteaseyphosphatase inhibitors ruption cassette was used to transform strain BY263 (trp1D63, as in lysis buffer). HA–Hrr25 was immunoprecipitated from the GAL21, otherwise isogenic to JO34). The hrr25D deletion extracts with monoclonal antibody 12CA5, washed twice in IPK strain was made by transformation of a diploid derivative of buffer, twice in IPK buffer with 1 M NaCl without inhibitors, and strain BY263 with an hrr25DURA3 disruption allele (18). The twice in kinase buffer (38). Where indicated, Swi6 and casein diploid was sporulated and meiotic progeny deleted for HRR25 were added to 100 ng per kinase reaction. recovered by tetrad dissection. For plating assays and Northern Plating Assay for Sensitivity to DNA-Damaging Agents. For blot analyses, yeast strains were transformed with either vector viability assays, cells were grown to early logarithmic phase in Yep24 or with a high-copy SWI4 plasmid, pBA314. Other yeast minimal medium. The cells were harvested, washed twice, and strains are described in the relevant sections below. resuspended in 100 mM KH2PO4 (pH 7.5). The cell suspension Protein Affinity Chromatography and Microsequencing of was then briefly sonicated and counted using a hemacytom- p54. Swi6 protein was expressed and purified essentially as eter. Cells were plated at densities of 100, 500, 5,000, 50,000, described (23).
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