MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 3256-3257

Vol. 10, No. 6

0270-7306/90/063256-02$02.00/0 Copyright C) 1990, American Society for Microbiology

The Saccharomyces cerevisiae DNA Repair Gene RAD2 Is Regulated in Meiosis but Not during the Mitotic Cell Cycle KIRAN MADURAt AND SATYA PRAKASH* Department of Biology, University of Rochester, Rochester, New York 14627 Received 9 October 1989/Accepted 22 February 1990

The expression of the RAD2 gene of Saccharomyces cerevisiae is elevated upon DNA damage. Here, we show that RAD2 transcript levels also rise approximately eightfold during meiosis but remain constant during the mitotic cell cycle. The period of maximal RAD2 mRNA accumulation during meiosis is consistent with a possible role of RAD2 in a late stage of recombination, in mismatch repair of heteroduplexes, or both.

In the yeast Saccharomyces cerevisiae, DNA excision repair requires the products of at least ten genes (8, 9, 11, 14). Mutations in five of the genes, RADI, RAD2, RAD3, RAD4, and RADIO, result in the complete loss of incision activity (8, 11, 14). It is likely that the products of these five genes constitute the incision enzyme complex, whereas the other five genes encode accessory proteins. Our approach to the study of excision repair in yeast has involved the cloning of these genes and the characterization of their encoded proteins. We have also begun a systematic study of the regulation of these genes. Of the excision repair genes, RAD2 is the only gene that has been reported to be regulated by DNA damage (7, 12). By Northern (RNA) hybridizations, we have examined whether the RAD2 gene is expressed periodically during the mitotic cell cycle and during meiosis. For the analysis of cell cycle regulation, we used the yeast strain 4910-3-3a (MATa his7 ural cdc74 barl-1). This strain can be easily synchronized during the cell cycle by very low concentrations of the mating pheromone a-factor, and synchronous divisions are maintained for at least two generations (10). Cells were grown at 23°C in YPD medium to a density of -1.5 x 107 cells per ml. Treatment with 10 ng of a-factor per ml for 3 h arrested more than 95% of cells in the Gl stage of the cell cycle. The pheromone was removed by filtration, and cells were suspended in fresh YPD medium lacking a-factor and incubated at 23°C. Upon release from a-factor arrest, the cells underwent synchronous divisions. The first peak in the fraction of budding cells was observed at 60 min, and the second peak was observed at 180 min; each peak of budding was followed by a doubling in cell number. Total RNA was isolated at various times and electrophoresed in a 1% agarose gel (7). The resolved RNA was transferred by electroblotting to GeneScreen nylon membranes and hybridized both to a nick-translated 1.96kilobase (kb) EcoRI-BglII internal fragment of the RAD2 gene (7) and to nick-translated plasmid pTRT2, which hybridizes to histone H2B mRNA (3). The DNA probes were radiolabeled to a specific activity of approximately 2 x 108 cpm/,Lg of DNA. The levels of RAD2 and H2B mRNAs present during various stages in the cell cycle are shown in Fig. 1. The H2B mRNA levels fluctuate during the cell cycle, reaching a maximum during the S phase (4). During a-factor treatment, H2B mRNA levels remained very low (Fig. 1, lanes 2 through 4), whereas upon release from the a-factor Corresponding author. t Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. *

arrest (lanes 5 through 15), three distinct cycles of peak accumulation of H2B mRNA were observed at intervals of

-2 h. As expected, the magnitude of fluctuation in the levels of H2B mRNA exceeded 20-fold (4). In sharp contrast to the cyclic fluctuation of H2B mRNA levels, RAD2 mRNA levels remained nearly constant during the cell cycle. RAD2 mRNA levels in the Northern blot (Fig. 1) were quantitated by densitometry with an LKB laser densitometer. These results indicated at most a twofold variation in the level of RAD2 mRNA at different times in the cell cycle; however, this minor fluctuation lacked a consistent pattern of periodicity in different cell cycles. Results of another experiment also gave no evidence of periodic changes in RAD2 transcription during the cell cycle. To further verify the constancy of RAD2 expression during the cell cycle, we examined RAD2 protein levels in samples from an experiment identical to that shown in Fig. 1 by Western (immuno-) blotting with affinity-purified anti-RAD2 antibodies. As expected, we found that RAD2 protein levels also remained constant during the cell cycle (data not shown). We next examined RAD2 mRNA levels during meiosis in the MATaIMATa diploid strain g857, a derivative of the well-characterized yeast strain SKi (6). Haploid strains g833-1B (MATa leu2 canl hisl-l trp2) and g833-2D (MATa hom3-10 hisl-7 ade2) were mated, and the resulting diploid, g857, was selected on synthetic minimal medium supplemented with histidine. After growth of strain g857 in presporulation medium for 18 h to a density of 4 x 107 cells per ml, cells were collected by filtration and resuspended in sporulation medium at 2 x 107 cells per ml. Strain g857 undergoes rapid, very efficient (>95% asci), and synchronous sporulation. In sporulation medium, commitment to recombination began at about 2 h and maximal recovery of recombinants occurred by 5 to 6 h, whereas spore formation began after 6 h and was complete by 10 h. Total RNA was isolated from yeast cells grown in presporulation and sporulation medium, and the results of Northern blot analysis are shown in Fig. 2. An initial increase in RAD2 mRNA levels was observed immediately upon transfer of cells from presporulation to sporulation medium (compare lanes 2 and 3). RAD2 mRNA levels then remained nearly constant during the first 2 h in sporulation medium (lanes 3 through 7), whereas between 3 and 6 h in sporulation medium, a substantial increase in RAD2 mRNA levels was observed (lanes 8 through 11). By 8 h in sporulation medium, RAD2 mRNA levels had fallen to basal levels (lane 13). The kinetics of histone mRNA accumulation observed during meiosis by us are in agreement with previous observations 3256

NOTES

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FIG. 1. Constant levels of RAD2 mRNA during the mitotic cell cycle. A logarithmic culture of strain 4910-3-3a was synchronized in the cell cycle by treatment with a-factor (Sigma Chemical Co.) for 3 h. Portions of the culture were withdrawn before, during, and after release from a-factor treatment, and total RNA was isolated. A 100-jLg portion of total RNA was electrophoresed in each lane. The resolved RNA was transferred to a nylon membrane and hybridized to RAD2 and histone H2B DNA probes. Lanes: 1, total RNA from untreated, exponentially growing cells; 2 to 4, total RNA from cells treated with a-factor for 2, 2.5, and 3 h, respectively; 5 to 15, total RNA isolated at 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min, respectively, after release from the a-factor arrest. The equality of RNA concentrations was confirmed by staining a parallel gel with ethidium bromide and by probing a parallel Northern blot for URA3 mRNA, which does not vary during the cell cycle.

(5). Quantitation of the Northern blot by densitometry indicated that, compared with the level in presporulation medium, an approximately eightfold increase occurred in the RAD2 mRNA levels by 5 to 6 h in sporulation medium. These observations of meiotic induction of RAD2 transcription were confirmed in two other experiments. To establish that the elevation of RAD2 mRNA was specific to cells undergoing meiosis and not caused by the culture medium (glucose or nitrogen deprivation), or by diploidy per se, we examined the levels of RAD2 mRNA in a closely related asporogenous MATaIMATa diploid strain, g721-2 (MATal MATa leu2-JILEU2 canlrICANJS hom3-JOIHOM3 his] -l/ hisl-7 trp2ITRP2 ade2IADE2). In this strain, RAD2 mRNA levels remained constant for 8 h in sporulation medium (results not shown). Thus, these observations show that the expression of the RAD2 gene is regulated during meiosis.

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FIG. 2. Elevated levels of RAD2 mRNA during meiosis. Diploid MATa/MATTa strain g857 was transferred from the presporulation medium to the sporulation medium, and cells were incubated at 30°C. Portions of the culture were withdrawn at various times, and total RNA was prepared. A 40-t±g portion of total RNA was electrophoresed in each lane and Northern blot hybridized to a radiolabeled RAD2 probe and a histone H2B probe. Lanes: 1, total RNA from strain KM57 (rad2A) grown in YPD medium; 2, total RNA from g857 cells grown in presporulation medium; 3 to 13, total RNA isolated at 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 h, respectively, after transfer of g857 cells to sporulation medium. The accuracy of RNA concentrations was confirmed by staining a parallel gel with ethidium bromide. The faint band between the RAD2 and H2B transcripts is an rRNA-associated artifact.

3257

The period of maximal accumulation of RAD2 mRNA during meiosis is consistent with a possible role(s) of RAD2 either in a late stage of recombination or in mismatch repair of heteroduplexes arising from recombination or both. Although neither meiotic recombination nor postmeiotic segregation is affected in rad2 mutants (1, 13), a role for RAD2 in meiosis is indicated by the results of studies of sporulation in rad2 radl8 double mutants. Spore viability is markedly reduced in the rad2 radl8 double mutant but not in either of the single mutants (2), indicating that RAD2 and RAD18 participate in alternative pathways that ensure a high level of spore viability. Whether recombination or mismatch repair is affected in the double mutant is not known. In summary, the expression of RAD2, a DNA damageinducible gene, is also regulated during meiosis but not during the mitotic cell cycle. It will be of interest to determine whether the same or different upstream-activating sequences control the transcriptional activation of RAD2 in response to DNA damage and in meiosis. We thank Janet Ives for expert technical assistance. This work was supported by Public Health Service grant CA 35035 from the National Cancer Institute. LITERATURE CITED 1. di Caprio, L., and P. J. Hastings. 1976. Post-meiotic segregation in strains of Saccharomyces cerevisiae unable to excise pyrimidine dimers. Mutat. Res. 37:137-140. 2. Dowling, E. L., D. H. Maloney, and S. Fogel. 1985. Meiotic recombination and sporulation in repair-deficient strains of yeast. Genetics 109:283-302. 3. Hereford, L., K. Fahrner, J. Woolford, Jr., M. Rosbash, and D. B. Kaback. 1979. Isolation of yeast histone genes H2A and H2B. Cell 18:1261-1271. 4. Hereford, L. M., M. A. Osley, J. R. Ludwig, and C. S. McLaughlin. 1981. Cell-cycle regulation of yeast histone mRNA. Cell 24:367-375. 5. Kaback, D. B., and L. R. Feldberg. 1985. Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation. Mol. Cell. Biol. 5:751-761. 6. Kane, S. M., and R. Roth. 1974. Carbohydrate metabolism during ascospore development in yeast. J. Bacteriol. 118:8-14. 7. Madura, K., and S. Prakash. 1986. Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae. J. Bacteriol. 166:914-923. 8. Miller, R., L. Prakash, and S. Prakash. 1982. Genetic control of excision of Saccharomyces cerevisiae interstrand DNA crosslinks induced by psoralen plus near-UV light. Mol. Cell. Biol. 2:939-948. 9. Miller, R,, L. Prakash, and S. Prakash. 1982. Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 188:235-239. L0. Potashkin, J. A., and J. A. Huberman. 1986. Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae. Exp. Cell Res. 165:29-40. 11. Reynolds, R. J., and E. C. Friedberg. 1981. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo. J. Bacteriol. 146:692-704. 12. Robinson, G. W., C. M. Nicolet, D. Kalainov, and E. C. Friedberg. 1986. A yeast excision-repair gene is inducible by DNA damaging agents. Proc. Natl. Acad. Sci. USA 83:18421846. 13. Snow, R. 1968. Recombination in ultraviolet-sensitive strains of Saccharomyces cerevisiae. Mutat. Res. 6:409-418. 14. Wilcox, D. R., and L. Prakash. 1981. Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae. J. Bacteriol. 148:618-623.

The Saccharomyces cerevisiae DNA repair gene RAD2 is regulated in meiosis but not during the mitotic cell cycle.

The expression of the RAD2 gene of Saccharomyces cerevisiae is elevated upon DNA damage. Here, we show that RAD2 transcript levels also rise approxima...
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