Vol. 10, No. 9
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4678-4684 0270-7306/90/094678-07$02.00/0
Copyright C 1990, American Society for Microbiology
Differential Repair of UV Damage in rad Mutants of Saccharomyces cerevisiae: a Possible Function of G2 Arrest upon UV Irradiation CARROL TERLETH, PAUL SCHENK, RAYMOND POOT, JAAP BROUWER,* AND PIETER VAN DE PUTTE Laboratory of Molecular Genetics, Department of Biochemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received 9 January 1990/Accepted 23 April 1990 After UV irradiation, the transcriptionally active MATa locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HMLa locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, radl6, and rad24, were impaired in the removal of UV dimers from the inactive HMLa locus, whereas they had generally normal repair of the active MATa locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HMLe locus.
The method outlined above makes it possible to determine whether any of the radiation-sensitive rad mutants of S. cerevisiae has a defect in differential repair. The rad mutants have been assigned to the epistasis groups RAD3, RAD6, and RAD52 (reviewed in reference 7). Analysis of rad mutants has indicated that loci in the RAD3 epistasis group are involved in nucleotide excision repair, whereas those in the RAD6 epistasis group are required for mutagenesis. Loci in the RAD52 epistasis group are probably involved in postreplication repair. Several mutants from the RAD3 epistasis group (radi, rad2, rad3, rad4, and radiO) have been shown to be completely incision deficient, whereas rad7, radl4, radl6, and rad23 possess residual incision activity (22, 28). Mutants from the RAD6 and RAD52 epistasis groups are proficient for the repair of pyrimidine dimers. In this study, we screened rad mutants from all three epistasis groups for the removal of UV damage from the transcriptionally active MATa and the inactive HMLa loci. The results indicate that rad9, radl6, and rad24 have a deficiency in the repair of HMLa and could therefore have a distinct role in making chromatin accessible for excision
Quick recovery of RNA synthesis upon UV irradiation through preferential repair of transcriptionally active DNA has been suggested to provide eucaryotic cells a better chance for survival (1, 11, 13). In addition, it has been shown that not all cyclobutane dimers have to be removed from the genome overall in order for human cells to survive (4). The existence of preferential repair of active genes was first shown to occur in CHO and human cells (for reviews, see references 3 and 8). Recently we reported that differential repair occurs after UV irradiation in the lower eucaryote Saccharomyces cerevisiae (25). Differential repair was shown by comparing the removal of T4 endonuclease V-sensitive sites (ESS) (2) from the MATa and HLMa loci. These loci together with the HMRa locus form the mating type system, which is situated on chromosome III and determines the mating type of the haploid yeast cell. When present in the MAT locus, a or a information is expressed and so determines the a or a phenotype of the haploid yeast cell. The HMLa and HMRa loci contain, respectively, the a and a information, but as a result of SIR-mediated repression, these loci are not expressed. These silent loci enable the haploid yeast cell to change the information present in the MAT locus to the opposite mating type by a mechanism of unidirectional gene conversion, the so-called mating type switch (for reviews, see references 9 and 18). We studied differential repair by comparing the removal of UV damage from the two identical HMLa and MATa loci which differ only in transcriptional expression and concomitant chromatin structure. It was shown that the transcriptionally active MATa locus is repaired preferentially to the inactive HMLa locus. In addition, nondifferential repair was observed in a strain in which the silent loci HMLa and HMRa are derepressed as a result of a sir3 mutation. These results indicate that either transcription or chromatin structure or both are related to differential repair (25). Eucaryotic cells may have a specific capacity for opening up the genome to render DNA damage accessible for repair (7). Some of the many proteins involved in the nucleotide excision repair process in yeast cells could have this specific function. Mutants defective for such a function will display anomalous differential repair. *
repair.
MATERIALS AND METHODS Yeast strains. The stains used are listed in Table 1. Culture medium. Cells were grown in complete medium (YEPD) as previously described by Yang and Friedberg (29). DNA isolation. DNA was isolated according to Sherman et al. (24) and purified on CsCl gradients (12). UV irradiation. Exponentially growing yeast cells diluted in ice-cold phosphate-buffered saline were irradiated with 254-nm UV (Philips T UV 30W) at a rate of 2 JIm2 per s. Cells were then collected by centrifugation, resuspended in YEPD, and incubated for various times in the dark at 28°C before DNA isolation. Quantitative UV survival of S. cerevisiae strains was measured as described previously (19). For the determination of G2 arrest, cells were incubated for several hours after irradiation. Every 15 min, a sample was taken and examined by light microscopy. Cells in the different stages of the cell cycle are recognized as follows: G, cells are unbudded, S cells have buds that are less than two-thirds the size of the mother, and G2 cells have buds larger than two-thirds the size of the mother. T4 endoV isolation. T4 endoV was isolated from Echerichia coli cells containing a plasmid with the denV gene that
Corresponding author. 4678
REPAIR OF UV DAMAGE IN S. CEREVISIAE rad MUTANTS
VOL. 10, 1990
TABLE 1. Strains useda Strain
Relevant genotype
S Survival1
a
35 A. Klar RAD 34 K. Tatchell RAD
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4678-4684 0270-7306/90/094678-07$02.00/0
Copyright C 1990, American Society for Microbiology
Differential Repair of UV Damage in rad Mutants of Saccharomyces cerevisiae: a Possible Function of G2 Arrest upon UV Irradiation CARROL TERLETH, PAUL SCHENK, RAYMOND POOT, JAAP BROUWER,* AND PIETER VAN DE PUTTE Laboratory of Molecular Genetics, Department of Biochemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received 9 January 1990/Accepted 23 April 1990 After UV irradiation, the transcriptionally active MATa locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HMLa locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, radl6, and rad24, were impaired in the removal of UV dimers from the inactive HMLa locus, whereas they had generally normal repair of the active MATa locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HMLe locus.
The method outlined above makes it possible to determine whether any of the radiation-sensitive rad mutants of S. cerevisiae has a defect in differential repair. The rad mutants have been assigned to the epistasis groups RAD3, RAD6, and RAD52 (reviewed in reference 7). Analysis of rad mutants has indicated that loci in the RAD3 epistasis group are involved in nucleotide excision repair, whereas those in the RAD6 epistasis group are required for mutagenesis. Loci in the RAD52 epistasis group are probably involved in postreplication repair. Several mutants from the RAD3 epistasis group (radi, rad2, rad3, rad4, and radiO) have been shown to be completely incision deficient, whereas rad7, radl4, radl6, and rad23 possess residual incision activity (22, 28). Mutants from the RAD6 and RAD52 epistasis groups are proficient for the repair of pyrimidine dimers. In this study, we screened rad mutants from all three epistasis groups for the removal of UV damage from the transcriptionally active MATa and the inactive HMLa loci. The results indicate that rad9, radl6, and rad24 have a deficiency in the repair of HMLa and could therefore have a distinct role in making chromatin accessible for excision
Quick recovery of RNA synthesis upon UV irradiation through preferential repair of transcriptionally active DNA has been suggested to provide eucaryotic cells a better chance for survival (1, 11, 13). In addition, it has been shown that not all cyclobutane dimers have to be removed from the genome overall in order for human cells to survive (4). The existence of preferential repair of active genes was first shown to occur in CHO and human cells (for reviews, see references 3 and 8). Recently we reported that differential repair occurs after UV irradiation in the lower eucaryote Saccharomyces cerevisiae (25). Differential repair was shown by comparing the removal of T4 endonuclease V-sensitive sites (ESS) (2) from the MATa and HLMa loci. These loci together with the HMRa locus form the mating type system, which is situated on chromosome III and determines the mating type of the haploid yeast cell. When present in the MAT locus, a or a information is expressed and so determines the a or a phenotype of the haploid yeast cell. The HMLa and HMRa loci contain, respectively, the a and a information, but as a result of SIR-mediated repression, these loci are not expressed. These silent loci enable the haploid yeast cell to change the information present in the MAT locus to the opposite mating type by a mechanism of unidirectional gene conversion, the so-called mating type switch (for reviews, see references 9 and 18). We studied differential repair by comparing the removal of UV damage from the two identical HMLa and MATa loci which differ only in transcriptional expression and concomitant chromatin structure. It was shown that the transcriptionally active MATa locus is repaired preferentially to the inactive HMLa locus. In addition, nondifferential repair was observed in a strain in which the silent loci HMLa and HMRa are derepressed as a result of a sir3 mutation. These results indicate that either transcription or chromatin structure or both are related to differential repair (25). Eucaryotic cells may have a specific capacity for opening up the genome to render DNA damage accessible for repair (7). Some of the many proteins involved in the nucleotide excision repair process in yeast cells could have this specific function. Mutants defective for such a function will display anomalous differential repair. *
repair.
MATERIALS AND METHODS Yeast strains. The stains used are listed in Table 1. Culture medium. Cells were grown in complete medium (YEPD) as previously described by Yang and Friedberg (29). DNA isolation. DNA was isolated according to Sherman et al. (24) and purified on CsCl gradients (12). UV irradiation. Exponentially growing yeast cells diluted in ice-cold phosphate-buffered saline were irradiated with 254-nm UV (Philips T UV 30W) at a rate of 2 JIm2 per s. Cells were then collected by centrifugation, resuspended in YEPD, and incubated for various times in the dark at 28°C before DNA isolation. Quantitative UV survival of S. cerevisiae strains was measured as described previously (19). For the determination of G2 arrest, cells were incubated for several hours after irradiation. Every 15 min, a sample was taken and examined by light microscopy. Cells in the different stages of the cell cycle are recognized as follows: G, cells are unbudded, S cells have buds that are less than two-thirds the size of the mother, and G2 cells have buds larger than two-thirds the size of the mother. T4 endoV isolation. T4 endoV was isolated from Echerichia coli cells containing a plasmid with the denV gene that
Corresponding author. 4678
REPAIR OF UV DAMAGE IN S. CEREVISIAE rad MUTANTS
VOL. 10, 1990
TABLE 1. Strains useda Strain
Relevant genotype
S Survival1
a
35 A. Klar RAD 34 K. Tatchell RAD
