Purification and characterization of Saccharomyces cerevisiae DNA damage-responsive protein 48 (DDRP 48).

A yeast protein was purified from wild type Saccharomyces cerevisiae (S. cerevisiae) to near homogeneity using an ethanolamine affinity chromatography procedure. The N-terminal amino acid sequencing and the amino acid composition analyses identified this protein as the product of the second open reading frame of S. cerevisiae DNA Damage-responsive gene 48 (DDR48) (Treger, J.M., and McEntee, K. (1990) Mol. Cell. Biol. 10, 3174-3184) The first methionine residue encoded by the translation starting codon was not present in the mature protein which is designated as DDRP 48. DDRP 48 was found to be a negatively charged and highly hydrophilic glycoprotein. The glycosidase cleavage analyses suggested that DDRP 48 was mainly N-link-glycosylated. The apparent molecular mass of DDRP 48 was estimated to be approximately 65 kilodaltons. DDRP 48 was found able to hydrolyze ATP and GTP yielding PPi. The Km values for ATP and GTP are 0.29 mM and 0.58 mM, respectively. The Western blot analysis demonstrated that DDRP 48 was expressed to various concentrations in different S. cerevisiae strains. Increased DDRP 48 abundance was observed after yeast cells carrying the wild type RAD 52 gene were exposed to either ethylmethane sulfonate or heat shock treatments. After similar DNA-damaging treatments, however, no significant inductions of DDRP 48 were found in a rad 52 mutant strain. These observations are consistent with the predictions resulting from previous studies on transcriptional regulation of the DDR 48 gene (Maga, J.A., McClanahan, T.A., and McEntee, K. (1986) Mol. & Gen. Genet. 205, 276-284; McClanahan, T., and McEntee, K. (1986) Mol. Cell. Biol. 6, 90-96).

The nucleotide sequence(s) reported in this paper has been submitted

M36110.
to the GenBankTM/EMBL Data Bank with accession number(s) 4 To whom correspondence and reprint requests should be addressed. Tel.: 904-392-8408; Fax: 904-392-8598. DNA repair (5, 6), mutagenesis (7), and inhibition of cell division (8), etc. When the ascomycete Neurospora crassa was exposed to sublethal doses of UV, x-ray, or nitrous acid, they showed an enhanced ability to rescue lethally irradiated cells when fused into a heterokaryon (9). This observation is considered to be the first evidence of eukaryotic inducible responses to environmental stresses. Since then, more evidence has been obtained that similar inducible responses also exist in other eukaryotic systems. Several classes of eukaryotic inducible genes have been identified. Yet, in comparison with the well established E. coli inducible response pathways, the regulation of the eukaryotic inducible response system is still a mystery, and the functions of those inducible genes and their products remain unclear.
Saccharomyces cerevisiae has been used as a model for studying the regulation of eukaryotic inducible responses to environmental stresses. Several groups of inducible genes in S. cerevisiae have been isolated and characterized. Among them are heat shock genes, DNA damage-inducible ( D I N ) genes, and DNA damage-responsive (DDR) genes (10-16). The results of previous studies demonstrate that the transcriptions of some inducible genes are preferentially elevated by certain kinds of environmental stresses. For example, heat shock genes YG102 and YGlOO are activated by heat shock, but not by 4-nitroquinoline-1-oxide (NQO)' treatment, and DNA damage-inducible gene DIN 1 can be induced by chemical mutagen, but not by thermal stress. More complex transcriptional regulation patterns have been observed with two DDR genes: DDR A2 and DDR 48. Ten-to fifteen-fold more transcripts of DDR A2 and DDR 48 are obtained when the cells are exposed to either heat shock or NQO treatment (17).
In addition to environmental stresses, some S. cerevisiae genomic elements, their products, or both, are involved in the regulation of stress-inducible gene expressions. RAD genes, required to repair UV-irradiated DNA in vivo, play some indirect roles in the transcriptional regulations of DDR A2 and DDR 48 genes (18). Normally, untreated wild type S. cerevisiae cells do not produce a detectable amount of DDR A2 transcripts. The induction of DDR A2 transcription by NQO or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) seems to require the presence of a wild type RAD 3 gene. A higher abundance of DDR A2 transcripts is observed in untreated S. cerevisiae rad 6 and rad 52 mutants. When these two mutants are exposed to NQO or MNNG, the increase of DDR A2 transcripts demonstrates a normal dose dependence. to NQO of MNNG, the transcription of DDR 48 increases in both wild type and rad 3 mutant cells. Although similar stress treatments also activate DDR 48 transcription in rad 6 and rad 52 mutant S. cerevisiae, the induction is severely reduced compared to that in the wild type strains.
In 1990, the detailed structure of the DDR 48 gene was determined (19 Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly where X is either Asn or Asp. Since similar repeated sequences are found throughout the primary sequence, the protein is, therefore, predicted to be extremely hydrophilic. The function of the protein encoded by DDR 48 (DDRP 48) remains unclear, although the viability studies of diploid yeast cells containing disrupted DDR 48 genes (ddr 48) suggest that DDRP 48 may be involved in S. cerevisiae spontaneous mutations or recovery from mutations.
The purification of DDRP 48 was an unexpected outcome from our effort to purify recombinant human asparagine synthetase expressed in S. cerevisiae. Initially, a yeast protein with an apparent molecular mass of approximately 75 kDa was often co-purified with the recombinant enzyme by a monoclonal antibody affinity chromatography procedure (20). This protein was identified as yeast DDRP 48. In this paper, the purification and enzymatic characterizations of DDRP 48 are described. The expression of DDRP 48 in several S. cerevisiae strains cultured under different conditions are also reported.

EXPERIMENTAL PROCEDURES
Chemicals and Reagents-Yeast culture media was purchased from Difco. Cyanogen bromide-activated Sepharose 4B resin and PD-10 desalting columns were purchased from Pharmacia LKB N-glycosidase F' (PNGase F) and 0-glycosidase* (POGase) were purchased from Boehringer Mannheim. Protein concentration assay dye and protein electrophoresis reagents were purchased from Bio-Rad. The polyclonal antisera against both DDRP 48 and recombinant human asparagine synthetase was produced by Kel Farm (Alachua, FL). Pyrophosphate assay reagents and other chemicals were purchased from Sigma.
Cell Cultures-The names, genotypes, and sources of the S. cereuisiae strains used in this study are listed in Table I. The growth condition for yeast cells in liquid media was 30 "C with vigorous shaking. In the DDRP 48 expression studies, single colonies of yeast cells on solid media were picked to inoculate 10 ml of YPD or yeast synthetic selective media. Cells were harvested when the absorbance of the culture at 600 nm (ODm) reached 2.5-3.0. In DDRP 48 induction experiments, S. cereuiine cells were grown in 20 ml of YPD media. When the culture OD, reached approximately 3.0, each 20-ml culture was aliquotted into three fractions. One set of the aliquots was heat-shocked in a 42 "C water bath for 2 min, incubated at 37 "C for another 30 min, followed immediately by the cell harvesting. Another set of aliquots was treated with chemical mutagen ethylmethane sulfonate (EMS). A total of 200 pl of EMS was mixed well with each aliquot by vigorous shaking in a fume hood. The cells were harvested after another 30 min of incubation at 30 "C. While the first 2 aliquots of yeast cultures were being treated by heat shock or EMS, cells in the third set of aliquots were allowed to grow for another 30 min at 30 "C before the cells were harvested. The protein extraction was performed as follows (24): the cell pellets were washed with 1 ml of deionized distilled water. After 30 s of centrifugation at 3000 X g and 4 "C, the cell pellets were resuspended in 0.5 ml of cell lysis buffer (50 mM Tris-HC1 (pH 8.0), 0.5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM B-aminobenzamidine) and mixed with 0.5 ml of ice-cold, acid-washed glass beads (0.45-0.5 mm in diameter). The cell disruption was complete after 6 cycles of vortexing (15 s) and cooling (5 min on ice). Clear cell extracts were collected after a 15-min centrifugation at 10,000 X g and 4 "C.
For DDRP 48 purification, 1-liter of YPD media was inoculated with S. cereuisiae BJ2168 from a 10-ml culture obtained as described above. Cells were harvested at the late log phase of growth (ODm 5.5-6.0). The protein extractions were performed as follows (20): the cells were centrifuged at 3000 X g and 4 "C for 5 min and washed once with cell lysis buffer. The cell pellets were resuspended in 35 ml of cell lysis buffer, and the entire volume was transferred into the 70ml chamber of a Bead-Beater (Biospec Products, Bartlesville, OK). Cold, acid-washed glass beads (0.45-0.50 mm in diameter) were added to fill the rest of the space, and the chamber was installed in a melting ice bath. After a short burst of 2 s, a 15-s blending was applied to the cell suspension followed by 5 min of cooling. The burst-blendingcooling process was repeated five more times. The glass beads were separated from the homogenate and rinsed twice with a total of 150 ml of fresh cell lysis buffer. All the portions of the yeast homogenate were combined and centrifuged at 30,000 X g and 4 "C for 90 min. The clear cell extract supernatant fluid was carefully collected and immediately loaded onto the affinity column. Ethanolamine Affinity Resin-One gram of cyanogen bromideactivated Sepharose 4B resin was soaked at room temperature in 50 ml of 1 mM HCl for 15 min with occasional gentle stirring. Within the next 20 min, the swollen resin was washed with approximately 200 ml of ice-cold 1 mM HCl and rinsed briefly with 5 ml of 1 M ethanolamine (pH 8.0) before being mixed with 8 ml of 1 M ethanolamine (pH 8.0) solution. The resin-ethanolamine mixture was gently shaken in a rotatory manner for 3 h at room temperature and packed into a Bio-Rad Econo column (1.5 cm in diameter). The resin was then washed with 500 ml of 0.1 M NaHC03 (containing 0.5 M NaCl (pH 8.3)) and 500 ml of 0.1 M NaOAc (containing 0.5 M NaCl (pH 4.0)). The resin thus obtained was stored at 4 "C and equilibrated with 100 ml of ice-cold phosphate-buffered saline (pH 7.2) prior to the affinity purification of DDRP 48.
Affinity Chromatography Purification of DDRP 48-The following procedure was performed at 4 "C. A total of 150 ml of cell extract from 1 liter of S. cereuisiae BJ2168 culture was loaded onto the ethanolamine resin at a flow rate of approximately 0.3 -0.5 ml/min. The resin was then washed with phosphate-buffered saline buffer (pH 7.2) until the protein concentration of the run-through phos- Available in authors' laboratory rived from wild type strain C1278b phate-buffered saline buffer was lower than 0.1 pg/ml as detected by the method of Bradford (28). DDRP 48 was eluted with 0.1 M NalCOn (pH 10.6) a t a flow rate of 0.25-0.30 ml/min. The eluate was collected in 3-ml fractions. Fractions with protein concentrations higher than 50 mg/ml were pooled together, and solid ammonium sulfate was added to a final saturation of 60%. Following centrifugation a t 25,000 X g for 30 min, the precipitated protein was redissolved in a minimum volume of enzyme buffer (50 mM Tris-HCI (pH 8.0), 0.5 mM EDTA (pH 8.0), 1 mM dithiothreitol, and 20% (v/v) glycerol). Excess ammonium sulfate was removed by chromatography through a PD-10 column, and the desalted protein was finally stored in the enzyme buffer a t a concentration of 1.0-1.5 mg/ml. DDRP 48 Protein N-Terminal Sequencing and Amino Acid Composition Analysis-Ethanolamine affinity-purified DDRP48 was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (25), and electroblotted onto polyvinylidene difluoride membranes with 10 mM MES buffer (pH 6.0, containing 20% methanol) a t 340 mA and 4 "C for 90 min. Two layers of polyvinylidene difluoride membranes were used to prevent the loss of protein due to overtransfer. The proteins blotted on the membranes were stained with Coomassie Brilliant Blue R-250 (26), and the DDRP 48 band was excised for sequencing. The protein N-terminal sequencing was performed by the Protein Chemistry Core Facility of the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida, using an Applied Biosystems 473A Protein Sequencer and 610A software for data analysis.
DDRP 48 amino acid composition analysis was also performed by the Protein Chemistry Core Facility of ICBR. Purified DDRP 48 was decomposed by a standard 6 N HCI hydrolysis procedure. The quantities of the resulting amino acids were determined using a Beckman 6300 amino acid analysis system.
Deglycosylation of DDRP 48-Thirty micrograms of purified and desalted DDRP 48 (0.14 mg/ml) was denatured by boiling for 2 min in the presence of 1% SDS. Denatured DDRP 48 was cooled to room temperature and diluted with the deglycosylation buffer (20 mM sodium phosphate (pH 7.6), 10 mM sodium azide, 0.5% (v/v) Nonidet P-40) to a final concentration of 0.1 mg/ml. The resulting deglycosylation mixture was boiled for 2 min, cooled to room temperature, and then divided into three aliquots. DDRP 48 deglycosylation reactions were initiated by adding 4 units of PNGase F, 10 milliunits of POGase, and a mixture of 4 units of PNGase F and 10 milliunits of POGase into each aliquot, respectively. The reaction mixtures were incubated a t 37 "C for 50 h. SDS-PAGE sample buffer was used to terminate the reactions. One-third of each resulting mixture was used for SDS-PAGE analysis.
Coupled Enzyme Activity Assay-Sigma PPi assay reagents containing the enzymatic coupling system that utilizes PPi as a substrate and subsequently converts NAD' to NADH (27) was used to determine the PP, production catalyzed by DDRP 48. A vial of PP, assay reagents was reconstituted with 1 ml of deionized distilled water. The final DDRP 48 reaction mixtures of 160 pl contained 50 mM Tris-HCI (pH 7.0). 50 mM NaCI, 20 pl of reconstituted PP, assay reagent, 5 pg of purified DDRP 48, and substrates at various concentrations as indicated under "Results." Upon the addition of DDRP 48 and quick vortex-mixing, the reaction mixtures were immediately transferred into quartz microcuvettes, and their absorbances a t 340 nm (A%,,) a t 37 "C were monitored on a Beckman D v -6 4 spectrophotometer for 20 min. The blanks were performed in a similar manner except that MR2+ was excluded from the reaction mixtures. For each vial of PPI assay reagents, a standard curve was obtained using the rates of Awo decrease for pure PPI in DDRP 48 substrate mixtures. The productions of PPI in DDRP 48 reactions were calculated accordingly.
Protein concentrations were determined by the method of Bradford (28) using pure mouse immunoglobulin G to construct the standard curve. Protein molecular mass estimations by SDS-PAGE and protein Western blotting by polyclonal antisera were performed as described earlier (24,29).

RESULTS
DDRP48 was first isolated from yeast S. cereuisiae BJ2168/ pVTXASl as a contaminant of recombinant human asparagine synthetase (20). When human asparagine synthetase was purified from the total cell-extractable protein by immunoaffinity chromatography, a yeast protein of an apparent molecular mass of approximately 75 kDa (previously designated as "YP 75") was often co-purified. Such contamination could be eliminated by using an ethanolamine affinity pre-column procedure. Later, it was found that YP 75 could be specifically purified from crude yeast extract to near homogeneity (see Fig. 1, a and b) by one-step ethanolamine affinity chromatography. Approximately 500-800 pg of pure YP 75 was obtained from each liter culture of S. cereuisiue BJ2168. Purified YP 75 was highly hydrophilic and negatively charged. Isoelectric focusing gel electrophoresis of YP 75 indicated that the protein had an isoelectric point below 3.0 (data not shown). When it was electroblotted from SDS-PAGE gel onto polyvinylidene difluoride membranes for protein N-terminal sequencing, two layers of membranes were used. After a 90-min transfer a t 340 mA a t 4 "C, approximately 90% of YP 75 was blotted onto the second layer membrane. The sequence of the first 30 N-terminal amino acid residues of YP 75 was obtained by HPLC analysis (see Fig. 2) and compared to the known protein sequences in the genEMBL database. Except for the absence of the N-terminal methionine residue, this sequence was identical with that deduced from the nucleotide sequence of the second open reading frame of S. cereuisiup DNA damage-responsive 48 gene, DDR 48 (1). Further evidence confirming that YP 75 is DDR 48 gene product DDRP 48 was obtained from amino acid composition analyses. As demonstrated in Table 11, the amino acid composition of YP 75 is in good agreement with that of DDRP 48.
As illustrated in Fig. 1, a and b, the apparent molecular mass of DDRP 48 varied from 75 kDa to 65 kDa as the concentration of the acrylamide in SDS-PAGE was increased from 10% to 15%. No further significant changes in the apparent molecular mass of DDRP 48 were observed when the acrylamide concentration was higher than 15%. Such anomalous mobility in SDS-PAGE is considered to be typically characteristic of glycoproteins (30). Given the fact that the abundances of asparagine and serine residues in DDRP 48 are 23% and 26%, respectively, both N-link or 0-link glycosylations seemed possible. In order to study the proteincarbohydrate linkages in DDRP48, it was digested by PNGase

DDRp48
: quence predicted for DDRP 48. types of asparagine-bound N-glycans when both the amino group and the carboxyl group are present in a peptide linkage (31). After DDRP 48 was treated with POGase (160 kDa), which liberates the disaccharide GalP( 1-3)GalNAc from serine-or threonine-attached glycans (32), only one common band of approximately 33 kDa was observed (lane 4  in the deglycosidases could not be ruled out, the fact that the PNGase F treatment resulted in a fragment of approximately 44 kDa, which coincides with the molecular mass of DDRP 48 protein predicted from the protein primary sequence, may suggest that DDRP 48 protein was N-linked to carbohydrates. According to the method of Segrest and Jackson (33), 65 kDa, the minimum apparent molecular mass of intact DDRP 48 on SDS-PAGE, is considered to be the real molecular mass of DDRP 48 whole molecule, 30.77% of which is glycan.

-AspSer-Gly-Asn-Asn-Asn-Gln-Gly-AspTyr-Val-Thr-Lys-Y P 75": -AspSer-Gly-Asn-Asn-Asn-Gln-Gly-AspTyr-Val-Thr-Lys-
The polypeptide sequence of DDRP 48 protein core does  48 acting as R yeast intrinsic asparagine synthetase was investigated, hecause it was co-purified with recombinant human asparagine synthetase by immunoaffinity chromatography, and therefore had been suspected to share some structural and functional similarities with that enzyme. DI)RP 48 was added into the substrate mixtures optimized for asparagine synthetase (36). Asparagine synthetase activities were determined either hv measuring the production of I,-asparagine by HPLC on a Applied Riosystems 420 Derivatizer/l:lOA Separation Svstem, or by measuring the production of PP, by a coupled enzyme activity assay. The coupled PP, assav was derived from the method of O'Rrian (27). Asparagine synthetase substrates had no effect on the counlinn enzvmatic reactions. When Dure not have any homology with that of human asparagine syn-PPi was added as described under&"Experimental Proceduk," thetase (EC 6.3.1.1) (34) which catalyzes the biosynthesis of a linear detection range of 0-125 p~ P P , was obtained. The L-asparagine as follows (35) Fig. 4). When ATP hydrolysis activities of DDRP 48 were measured by coupled PPi assay with additional 10 mM concentrations of AMP or ADP, the DDRP 48 specific activities were reduced 18% and 20%, respectively. In a parallel experiment, the addition of 10 mM CAMP resulted in a nearly 40% decrease of DDRP 48 specific activity (see Fig. 5).
The DDR 48 gene was so named because its transcripts were elevated more than 10-fold after S. cerevisiae cells were exposed to heat shock or chemical mutagenesis (19). While little is known about the role, if any, of DDRP 48 in yeast cellular responses against DNA-damaging treatments, it is important to find out whether the DDRP 48 protein concentrations were also elevated in response to the above stimuli. When DDRP 48 was first co-purified with recombinant human asparagine synthetase, rabbit polyclonal antisera against a mixture of these two proteins was produced. The polyclonal antisera thus obtained was used in DDRP 48 immunoblotting analysis. The quantities of DDRP 48 were estimated by a secondary antibody-linked alkaline phosphatase reaction (24). As expected, the polyclonal antisera showed cross-reactivities with human asparagine synthetase (see Fig. 6). In addition, the polyclonal antisera bound to a protein band of 55 kDa, which could be a major degradation product of DDRP 48 (see Fig. 3). A detectable DDRP 48 signal was obtained from every untreated S. cerevisiae strain tested. This result is consistent with a previous observation that low abundances of DDR 48 transcripts in untreated yeast cells were always detectable (19). The intensities of DDRP 48 signals in Fig. 6    performed. Yet, some evidence suggests that the transcription of the DDR 48 gene was indirectly regulated by yeast RAD genes. In order to investigate the roles of RAD genes in DDRP 48 expression, a wild type S. cerevisiae strain, BJ2168, and a rad 52 mutant strain, JN934, were chosen for use in a DDRP 48 induction experiment. After the cells were either heatshocked or treated with EMS, equal amounts of cell-extractable protein from each culture were subjected to Western blotting with polyclonal antisera. As shown in Fig. 7 (lanes 5-7). The difference in the DDRP 48 induction pattern between BJ2168 and JN934 may imply that RAD 52 gene was somehow involved in the regulation of DDRP 48 expression. Since the high abundance of DDRP 48 was also found in other untreated yeast S. cerevisiae strains, e.g. AB116/pBS24GASl (lane 8 in Fig. 6) carrying the wild type RAD 52 gene, the role of RAD genes in DDRP 48 expression may not be as important as previously proposed.

DISCUSSION
Since 1984, when it was first found that the transcripts of S. cerevisiae DNA damage-responsive gene DDR 48 and DDR  (10). It, is wort,h noting t.hat t.he DIIR 48 sequence has no homology with any known yeast heat shock genes. The possihility that the DDR 48 gene and its product, IIDRP 48, may he involved in a yeast SOS-like pathway (10) has heen investigated. I t seems unlikely that, D1)111'4R is directly associated with DNArepair act,ivitv. Some evidence suggests that the wild t?rpe DDR 48 gene is required for maintaining the rate of spontaneous mutagenesis in yeast, (19). Current understanding of in the reaction mixtures were separated hy l)l<AE-anion exchange paper chromatographv. Our preliminary results suggested that small amounts of radinactivr CAMMI' and d;\lI' were produced (data not shown). Further investigation will he necessary to determine whether the production o f C A M P and cGMP was caused hv the contaminating cyclase nctivity in the 1)I)NP 48 preparat ion.
A T P hvdrolvsis activitv has heen relatrd t o several strrssinduced proteins in hoth prokaryotes and eukaryotes. T h e l a , ' .
coli DnaK, which is involved in the initiation of DNA rrplication, has heen found t o hind tightly t o ATP and catalyze a weak DNA-independent A T P hydrolysis in (,itro, protlucing ADP and inorganic phosphate (37, 34). SSAI. ;I merntw of' the S. cereuisiae hsp 70 family, is involved in the posttranslational transport of proteins across mitochodrial membranes (39). It is known that an early step of such protein import into mitochondria is ATP-dependent. In fact, almost all hsp 70 and related proteins in eukaryotes have high affinities for ATP and are often found in association with other proteins (40). Interestingly, our experiments with purified DDRP 48 demonstrated that DDRP 48 could use both ATP and GTP in uitro. ATP appears to bind to DDRP 48 more tightly than GTP. It is not clear whether ATP/GTP hydrolysis is the main activity or only a side reaction of DDRP 48, i.e. DDRP 48 may primarily catalyze an ATP (or GTP)dependent reaction and, when other substrates are absent in the assay mixture, it could slowly hydrolyze ATP (or GTP). If ATP/GTP hydrolysis is the main function of DDRP 48, a thorough understanding of the mechanism of this enzyme activity would provide more insight into the pathways of yeast-inducible responses to environmental stresses. It would be also important to find out whether some possible in vivo association of DDRP 48 with other proteins could affect the selectivity of nucleotide bindings and whether such different bindings are important for regulating the biological activity of DDRP 48 in yeast-inducible responses to environmental stresses.
The differences between DDRP 48 and some better characterized yeast heat shock proteins that have ATPase activities are obvious. Firstly, the DDRP 48 protein sequence is not homologous to any known sequences of yeast heat shock proteins. Secondly, as demonstrated in this paper, DDRP 48 is abundant in yeast cells under normal growth conditions and can be induced by more than one kind of DNA-damaging treatments. The DDRP 48 induction patterns in the RAD 52 wild type strain and the rad 52 mutant strain (shown in Fig.  7) were found to be consistent with corresponding DDR 48 gene transcription induction patterns (18). The fact that the DDRP 48 basal concentration varies among RAD 52 wild type S. cerevisiae strains (Fig. 6) suggested that RAD 52 may not be the only gene involved in the regulation of DDRP 48 expression. The substantial amounts of DDRP 48 found in untreated S.cereuisiae, as well as the fact that DDRP 48 transcripts can be elevated by many kinds of stresses, also imply that DDRP 48 expression is subject to a more complicated regulation. In order that yeast could better adapt to frequent environmental changes, it is possible that not only the expression but also the biological activities of DDRP 48 are subject to delicate regulation. As an example of the possibilities, it should be noted that the DDRP 48 characterized in this study was purified from wild type S. cereuisiae cells that had not been treated with any DNA-damaging agents. It would be interesting to find out whether the DDRP 48 from different mutant strains or from cells treated with DNAdamaging agents have the same enzymatic activity and subcellular distribution.