Enhanced UV sensitivity of yeast cells induced by overexpression of Mg2+-dependent protein phosphatase α (type 2C α)

DNA Repair

ELSEVIER

Mutation Research 362 (1996) 213-217

Enhanced UV sensitivity of yeast cells induced by overexpression of MgZ+-dependent protein phosphatase (type 2Ca) Takayasu Kobayashi a, Akira Yasui b, Motoko Ohnishi a, Shunsuke Kato a, Yoji Sasahara a, Kazuyuki Kusuda a, Naoki Chida a, Yuchio Yanagawa ~, Akira Hiraga a, Shinri Tamura a,* Department of Biochemistry, Institute of Derelopment. Aging and Cancer, Tohoku Uniz'ersity, 4-1 seiryo-machi, Sendai 980, Japan b Department ofNeurochemistr3" and Molecular Biology, Institute of Decelopment, Aging and Cancer, Tohoku Unit'ersit3. 4-1 seiD,o-machi, Sendai 980, Japan

Received 10 May 1995; revised 10 July 1995; accepted 14 JuDy 1995

Abstract

The UV sensitivity of wild-type Saccharomyces ceret:isiae cells was increased 2-fold when rat Mg2%dependent protein phosphatase a (protein phosphatase type 2C a ) was overexpressed in the cells. The overexpression of this enzyme rendered the tad 18 mutant (defective in postreplication repair) more UV-sensitive than was observed in the wild-type ceils. However, this increase in UV sensitivity disappeared when the host cells had a rad I mutation (defective in excision repair). These results suggest that the Mg 2 +-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system. Keywords: Ultraviolet sensitivity; Mg2+-dependent protein phosphatase; Overexpression; Saccharomyces cererisiae

1. Introduction

All living organisms have complex DNA repair systems for maintaining their genetic information, and a great number of gene products are required for these repair systems (Cleaver, 1994). Recent investigation have revealed that protein phosphorylation plays important roles in the response of cells to

Abbreviations: MPP, Mg2+-dependent protein phosphatase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophopresis; UV, ultraviolet * Corresponding author, Tel.: 81-22-273-9498; Fax: 81-22-2733188.

DNA-damaging agent. For example, Walworth et al. (1993) have reported that in fission yeast chk 1 protein kinase links the rad check point pathway to cdc2, and that loss of chk 1 kinase activity produces sensitivity to UV irradiation. Zhou and Elledge (1993) also reported the involvement of protein serine/threonine kinase in regulation of the eukaryotic SOS response and showed that mutation in this gene produced much higher UV sensitivity. However, there is no evidence that demonstrates that protein kinases play actually essential roles in regulation of the DNA repair system. We, therefore, examined whether protein phosphorylation is involved in regulation of the DNA repair system in yeast strains by introducing and expressing a protein phosphatase

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gene. Mg2+-dependent protein phosphatase (MPP), also known as protein phosphatase type 2C, is one of the four major protein serine/threonine phosphatases (types 1, 2A, 2B and 2C) and exists as a monomeric 43-48-kDa enzyme (Hiraga et al., 1981). We have demonstrated that rat MPPc~, one of the three isotypes (c~, /3-1 and /3-2) isolated so far (Tamura et al., 1989; Terasawa et al., 1993), is expressed as an active enzyme in Saccharomyces cerevisiae cells (Kobayashi et al., 1993). We present here evidence that overexpression of this enzyme induces enhanced UV sensitivity in wild-type as well as in postreplicative repair-deficient yeast cells, but not in mutant cells with excision repair deficiency.

2.3. Assay o f protein phosphatase actiHty

Protein phosphatase activity was assayed by the release of [32P]phosphate from [32P]phosphohistone, essentially as described previously (Kobayashi et al., 1993). 2.4. Survit:al assay after U V irradiation

Anti-rabbit IgG/alkaline phosphatase conjugate was purchased from Promega (Madison, WI). Yeast nitrogen base without amino acids was from Difco (Detroit, MI). All other reagents were purchased from Wako Pure Chemical (Osaka, Japan).

Yeast cells were grown for 18 h at 30°C in 10 ml of minimal glucose medium (0.7% yeast nitrogen base without amino acids and 2% glucose) supplemented with amino acids. The cells were then harvested, washed three times in water and inoculated in minimal galactose medium (0.7% yeast nitrogen base without amino acids, 5% galactose and 0.2% sucrose). After shaking for 3 h at 30°C, the cells were diluted with water and exposed to UV light (FUNAUV-LINKER FS-800, Funakoshi, Japan) in a 3-cm dish with stirring, and then plated out on minimal galactose medium supplemented with 2% agar. After the plates had been incubated for 3 days at 30°C, the colonies were counted. All experiments were done under yellow light to prevent photoreactivation.

2.2. Plasmid and yeast strains

2.5. hnmunoblotting

The construction of plasmid pJDBMPP was described previously (Kobayashi et al., 1993). The S. cereHsiae strains used in this study are listed in Table 1. Transformation of the yeast cells with the plasmid was carried out by the method of Hinnen et al. (1978).

A polyclonal antibody, AB 103, directed against a purified recombinant rat MPPc~ was raised in rabbit. The cell extracts prepared using glass beads were subjected to SDS-PAGE, and immunostaining was performed using antibody ABI03 as described previously (Kobayashi et al., 1993).

Table 1 Description of S. eereHsiae strains Strains Genotype AI002 MAT a, ade 8, leu 2, met 14, trp l, ura 3, aro 7 YPC101 AI002 [pJDBMPP(expressionplasmid)] YPC102 A1002 [pJDB207(vector)] Yal8-1 MAT o~, leu 2,1ys l, ade 2, rad 18 YPC103 Yal8-1 [pJDBMPP] YPC104 Yal8-1 [pJDB207] Ya1032 MAT a, leu 2, ade 2, rad I YPC105 Ya1032 [pJDBMPP] YPC106 Ya1032 [pJDB207]

3. Results and discussion

2. Materials and methods 2.1. Materials

3.1. Effect o f oL'erexpression o f rat MPPol on U V sensitic'i O' o f yeast cells

In order to investigate the effect of rat MPPc~ on the yeast DNA repair system, the UV sensitivities of YPC101 (wild-type cells harboring the expression plasmid for MPPc~) and YPC102 (wild-type cells harboring the vector plasmid only) were determined. When YPCI01 was grown in galactose minimal

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medium, a 28-fold increase in Mg2+-dependent, okadaic acid-insensitive histone phosphatase activity was observed in the cell extract as compared with that observed in YPCI02 grown in galactose medium. Furthermore, protein bands of 46 kDa and 40 kDa were stained by anti-MPP~ antibody only when the cells were transformed with the expression plasmid (Fig. 1, lanes 1 and 2). It had been demonstrated previously that the 46-kDa band corresponds to the full length MPPa (Kobayashi et al., 1993). The 40-kDa band was found to be the MPPc~ partially digested by yeast proteases, since no band was detected in the lane of the extracts of the cells transformed with the vector plasmid (Fig. 1, lane 1). Under these conditions, YPC101 was twice more sensitive to killing by UV irradiation than YPC102, as shown in Fig. 2A. Yeast cells contain a protein phosphatase with enzymatic properties similar to those of mammalian MPP (Cohen, 1989; Cohen et al., 1989), and the gene encoding yeast MPP has sequence similarity with mammalian MPP (Maeda et al., 1993). Therefore, it is reasonable to assume that we did not introduce a unique enzyme of mammalian origin into yeast cells, but we rather increased the Mg2+-dependent protein phosphatase activity over the normal level of endogenous activity. Increased UV sensitivity is surely due to overproduction of this enzyme, because no difference in UV sensitivity was evident when YPCI01 (transformed with expression

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plasmid) and YPC102 (vector alone) were grown in glucose-containing medium (Fig. 2B). The growth rate of the cells harboring the expression construct of MPPa gene was decreased about 50% in comparison with that of the cells harboring the vector alone when they were incubated in galactose-containing medium (Kobayashi et al,, 1993). Slowly growing cells are generally more resistant to DNA-damaging agents, because DNA repair systems have more time to work before DNA replication. Therefore, the UV sensitivity of the transformed cells expressing MPPc~ may be still underestimated, because in such cells residual repair activity may be enhanced. These results thus suggest that the observed UV sensitivity is not due to the difference in growth rate, but to dephosphorylation of certain phosphoprotein(s) by the expressed MPPc~. 3.2. Effect of overexpression of rat MPPa on UV sensitivi~ of the DNA repair-defective mutants

In order to determine whether the sensitizing action of overexpression of MPPa is related to the yeast DNA repair system, we used DNA repair-defective mutants. The radiation sensitivity of S. cere-

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cisiae is influenced by many genes, which are arranged in three epistasis groups, RAD 3, RAD 6 and RAD 52, reflecting three repair pathways (Friedberg, 1988). The genes in group RAD 3 act in excision repair, and thus mutations in these genes confer a high degree of sensitivity to UV irradiation. Group PAD 6 is required for resistance to both UV- and X-rays, and the genes in this group are thought to act in the pathway of postreplication repair. Group RAD 52 contains the genes related to recombinational repair and the mutation in one of these genes causes only slightly enhanced UV sensitivity (Game and Mortimer, 1974), We, therefore, chose the rad 1 (RAD 3 epistasis group) and rad 18 (RAD 6 epistasis group) mutant strains as hosts, because these two groups are thought to be involved mainly in the repair of DNA damage induced by UV irradiation. Ya 1032 (rad 1, a mutant in epistasis group RAD 3 group) and Ya 18-1 (rad 18, a mutant in epistasis group RAD 6) were transformed with the expression plasmid and vector (Table 1). GAL 7-induced expression of rat MPPc~ was monitored by immunoblotting of whole-cell extracts using rabbit antibody AB103 (Fig. l). The transformants YPCI03 and YPCI05 gave immunoreactive bands at positions corresponding to 46 kDa and 40 kDa (lanes 4 and 6), whereas the isogenic controls (YPCI04 and YPC 106) showed no detectable bands (lanes 3 and 5). The survival curves of these transformants after UV irradiation are shown in Fig. 3. As was the case for the wild-type strain, overexpression of rat MPPc~ rendered the tad 18 mutant (defective in postreplicative repair) more sensitive to UV irradiation (Fig. 3A). In contrast, the effect of rat MPPc~ on UV sensitivity shown in Fig. 3A disappeared when the rad 1 mutant (defective in excision repair) was used as a host (Fig. 3B). These results suggested that the MPPa expressed in yeast cells inhibits the excision repair pathway which is functional both in wild-type and in the tad 18 mutant. This is the first report suggesting the involvement of protein phosphatase in the regulation of DNA repair system. The molecular mechanism responsible for inhibition of the excision repair pathway is unknown. In general it is rather difficult to identify which gene product is the target of the introduced protein phosphatase, because MPP has a broad substrate specificity. However, Teiz et al. (1990) reported that

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Fig, 3. UV sensitivities of Ya 18-1 (A) and Ya1032 (B) cells harboring the vector plasmid (YPCI04 and YPC106, O ) and expression vector (YPCI03 and YPCI05, 0 ) . The data represents one of two reproducible experiments.

expression of the cDNA for the /3 subunit of human casein kinase II conferred partial UV resistance on xeroderma pigmentosum (XP-C and XP-D) cells. They considered it unlikely that either the XP-C or XP-D DNA repair deficiency is associated directly with a defect in the /3 subunit of casein kinase II, but suggested that the response of cells to DNA damage might be modulated by phosphorylation by casein kinase If. Therefore, it may be reasonable to assume that rat MPPa dephosphorylates the substrate(s) identical or analogous to those recognized by casein kinase II and renders the cells sensitive to UV irradiation.

Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (TK and ST), a grant from the Uehara Memorial Bioscience Foundation (ST) and a grant from the Kato Memorial Bioscience Foundation (TK).

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