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Homologous recombination is required for the viability of rad27 mutants
Introduction
Materials And Methods
Media, growth conditions and genetic methods
Yeast strains and plasmids
Mutagenesis
MMS sensitivity assays
Measurement of recombination rates
Results
Identification of four alleles of RAD27 as hyper-recombination mutants
Characterization of the rad27 mutants
Recombination is required for the viability of rad27 mutants
Discussion
Acknowledgements
References
Homologous recombination is required for the viability of rad27 mutants
ABSTRACT
INTRODUCTION
The completion of DNA synthesis requires the activity of an exonuclease to remove RNA primers from Okazaki fragments prior to ligation of the nascent strands. In prokaryotes, this function is fulfilled by the combined activities of RNase H and DNA polymerase I (Pol I). Mutation of the gene encoding Pol I, polA, confers a pleiotropic phenotype that includes temperature-sensitive (ts) growth, UV sensitivity, sensitivity to DNA methylating agents, elevated rates of recombination and synthetic lethality when combined with mutations in recombination genes, such as recA (1-3).
In mammalian cells, a 5[prime]-3[prime] exonuclease activity required for the completion of lagging strand DNA synthesis in vitro has been identified in several laboratories (4-7). This activity, like the 5[prime]-3[prime] exonuclease activity of Pol I, acts to remove the terminal ribonucleotide of the RNA primer of Okazaki fragments (5,6). The same activity was discovered as a flap endonuclease, FEN-1, capable of removing a non-base paired single-stranded region from a branched duplex structure (8,9). The sequence of the mouse FEN-1 protein revealed significant homology to the Schizosaccharomyces pombe rad2 protein (10) and to the predicted product of an open reading frame on chromosome XI of Saccharomyces cerevisiae that has been named RAD27, RTH1 or ERC11 (11-13). The FEN-1/Rad27 family of proteins is also related to S.cerevisiae Rad2 (S.pombe rad13; human XPG), an endonuclease essential for nucleotide excision repair (9,11,12), and ExoI, a 5[prime]-3[prime] exonuclease implicated in mismatch repair and genetic recombination (14-17). The RAD27 gene product was purified and shown to exhibit flap endonuclease and 5[prime]-3[prime] exonuclease activities in vitro (9). Genetic analysis of yeast strains deleted for RAD27 has revealed a pleiotropic phenotype similar to that exhibited by Escherichia coli polA mutants (11,12). This includes a reduced growth rate at 30°C, inability to grow at 37°C (ts growth defect), sensitivity to methyl methane sulfonate (MMS), and elevated rates of mitotic recombination and spontaneous mutation. The spectrum of spontaneous mutations generated in rad27 strains is unusual, resulting from duplications between short repeated sequences (18). Because of the enhanced rate of duplicative mutations, rad27 mutants also show greatly elevated rates of di- and trinucleotide tract instability (19-21). Several models have been proposed to account for the formation of duplications in rad27 strains. These include slip mispairing by displacement of the unrepaired primer of one Okazaki fragment by displacement synthesis from the preceding one, and strand breakage to channel lesions into the recombination pathway (18,21). The latter hypothesis is consistent with the observed hyper-recombination phenotype of rad27 mutants and also with the synthetic lethality of rad27 rad51 double mutants (10,12,13,18).
In bacteria, yeast and humans, a number of genes have been identified that when mutated confer a hyper-recombination phenotype (3,22-28). These generally define proteins directly involved in DNA metabolism, e.g. DNA ligase (29), DNA polymerases (3,22,23), DNA helicases (28,30-32) and DNA topoisomerases (24,33). It is assumed that defects in DNA metabolism lead to the accumulation of recombinogenic lesions, hence the hyper-recombination phenotype, and, in some cases, lethality when combined with mutations in recombination genes (10,18,30,34). In this report we describe a yeast strain that was used to identify hyper-recombination mutants by a simple colony color-sectoring assay. Of the mutants identified, four were shown to contain alleles of RAD27. Characterization of the different alleles is presented.
MATERIALS AND METHODS
Media, growth conditions and genetic methods
Rich medium (YPD), synthetic complete (SC) medium lacking the appropriate amino acid or nucleic acid base, and sporulation medium were prepared as described previously (35). Rich medium plates containing MMS were used within 1 day of preparation. Yeast cells were grown at 30°C unless otherwise stated. Transformations were performed by the lithium acetate method (36). Sporulation and tetrad dissection was carried out as described previously (35). For crosses of rad27 to recombination mutants, at least two independently derived diploids were used for tetrad dissection.
Yeast strains and plasmids
All of the strains used for this study are derivatives of W303-1A or W303-1B and are listed in Table 1. The strain used to identify recombination mutants contains an inverted repeat of two mutant copies of the ade2 gene (37) and a direct repeat of two mutant copies of leu2 separated by LYS2 sequences (Fig.
Table 1.
| Name | Relevant genotype or descriptiona | Source |
| W303-1A | MATa leu2-3, 112 trp1-1 can1-100 ade2-1 his3-11, 15 rad5-535 | R. Rothstein |
| W303-1B | MAT[alpha] leu2-3, 112 trp1-1 can1-100 ade2-1 his3-11, 15 rad5-535 | R. Rothstein |
| W838-24D | MATa rad1::LEU2 rad52::TRP1 | R. Rothstein |
| R877 | MAT[alpha] hdf1::LEU2 | R. Rothstein |
| YAR71 | MATa ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | 37 |
| yAR91 | MAT[alpha] ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | 37 |
| B360-1A | MATa rad59::LEU2 | 49 |
| HKY579-9A | MAT[alpha] RAD5 LEU2 | 39 |
| HKY595-1C | MAT[alpha] RAD5 rad51::LEU2 | H. Klein |
| HKY604-17A | MAT[alpha] RAD5 rad50::hisG-URA3-hisG | H. Klein |
| YBL2 | MAT[alpha] rad27::TRP1 | This study |
| YBL3 | MATa rad27::TRP1 | This study |
| LSY114-6C | MATa LEU2 lys2 | This study |
| LSY409 | MATa leu2-bst::LYS2::leu2-112 derivative of LSY114-6C (integration of pLS119) | This study |
| LSY412-7C | MAT[alpha] leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY412-19A | MATa leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG ade2-5[prime][Delta]-TRP1-ade2-n (spore from LSY409 X yAR91 diploid) | This study |
| LSY459 | MATa leu2-bst::LYS2::leu2-112 ade2::hisG ade2-5[prime][Delta]-TRP1-ade2-n (5-FOA resistant derivative of LSY412-19A) | This study |
| LSY470-2C | MATa rad27-3 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY470-4A | MAT[alpha] rad27-3 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY471-1D | MAT[alpha] rad27-4 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY471-2D | MATa rad27-4 leu2-bst::LYS2::leu2-112 ade2::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY472-2C | MATa rad27-5 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY472-7A | MAT[alpha] rad27-5 leu2-bst::LYS2::leu2-112 ade2::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY473-1B | MATa rad27-6 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY473-2D | MAT[alpha] rad27-6 leu2-bst::LYS2::leu2-112 ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n | This study |
| LSY486-2A | MAT[alpha] rad27::URA3 | 16 |
| LSY502-15C | MATa rad27::URA3 ade2::hisG-URA3-hisG spo13::hisG ade2-5[prime][Delta]-TRP1-ade2-n (derived from yAR71 X LSY486-2A) | This study |
| LSY702-6B | MAT[alpha] rad27::TRP1 RAD5 (derived from YBL3 X HKY579-9A) | This study |
| LSY703-9D | MATa rad27-3 LEU2 RAD5 (derived from LSY470-2C X HKY579-9A) | This study |
| LSY704-6C | MAT[alpha] rad27-4 LEU2 RAD5 (derived from LSY471-2D X HKY579-9A) | This study |
| LSY705-4B | MATa rad27-5 LEU2 RAD5 (derived from LSY472-2C X HKY579-9A) | This study |
| LSY706-3A | MATa rad27-6 LEU2 RAD5 (derived from LSY473-1B X HKY579-9A) | This study |
Plasmid pLS119 that was used to construct the chromosomal leu2 recombination reporter was made as follows. A SpeI/EcoRI fragment from plasmid pLS32, containing the leu2-bst allele (40), was substituted for the SpeI/EcoRI fragment of pUC19::leu2-112 to generate a leu2 gene containing two mutant alleles (pLS97). A 1.1 kb HindIII fragment containing the URA3 gene was cloned into the HindIII site within the multiple cloning site of pLS97, generating plasmid pLS98. A 5 kb EcoRI/HindIII fragment containing the LYS2 gene was inserted into the StuI site within the URA3 gene of pLS98 to generate plasmid pLS119.
Mutagenesis
Mutagenesis of strain LSY412-7C was as described (41). In brief, cells were grown to saturation in rich medium, harvested and resuspended at a density of 2 × 107 ml in 50 mM potassium phosphate, pH 7.0. Ethyl methane sulfonate (EMS) was added to 3% and cells were incubated at 30°C, with shaking, for 30 min. An equal volume of 10% sodium thiosulfate was added to the culture. The cells were harvested, washed twice with water and then suspended in rich medium. The culture was incubated at 30°C, with shaking, for 3 h and then harvested, washed with water and stored at 4°C for up to 1 week. Cells were diluted with water and plated on YPD plates at a density of ~200 colonies/plate to screen for recombination mutants.
MMS sensitivity assays
Stationary phase cultures were harvested and resuspended in 0.1 M potassium phosphate, pH 7.0, at a density of 4 × 107/ml. MMS was added to a final concentration of 0.2% and samples were incubated at 30°C with shaking. Samples (0.1 ml) were removed at various times after MMS addition, mixed with an equal volume of 10% sodium thiosulfate, and then serially diluted with water. Dilutions were plated on solid YPD medium and counted after 3 days at 30°C. To assay for MMS sensitivity on solid medium, serial dilutions of late log-phase cultures were spotted onto YPD medium containing 0, 0.01 or 0.015% MMS. The plates were incubated at 23, 30 or 35°C for 3-4 days.
Measurement of recombination rates
For rapid rescreening of putative mutants, 1 cm patches of cells grown on YPD were replica plated to SC medium lacking adenine and leucine. The double selection for recombination events at both reporters provided a more stringent test for hyper-recombination. Strain LSY412-7C yielded 1-10 papillae by this assay whereas hyper-recombination mutants exhibited >40 papillae/patch of cells. Minifluctuation tests were performed by suspending individual colonies that had been grown on YPD plates for 3 days in 1 ml water and then plating 0.1 ml on SC-Ade or SC-Leu plates. Recombination rates were determined as described previously (37). For each genotype rates were determined for at least two independent spores.
Figure 1. Recombination reporters used to detect mitotic recombination. (A) The ade2 inverted repeat reporter contains one copy of ade2 with a deletion of the 5[prime] coding region; the other copy of ade2 contains a frameshift mutation at the unique NdeI site. Recombination events can occur by gene conversion (shown) and/or crossing over (not shown) to generate a wild type copy of the ADE2 gene, which in turn results in the formation of white-colored Ade+ sectors among red-colored Ade- cells (37). (B) The leu2 recombination reporter contains heteroalleles of leu2 separated by plasmid sequences and the LYS2 gene. Recombination events can occur by a variety of mechanisms to produce a functional copy of LEU2. Only one type of recombination event, gene conversion, is shown. A colony color sectoring assay based on recombination between inverted repeats of mutant alleles of the ade2 gene was developed to identify recombination mutants in S.cerevisiae (37). Recombination events are detected by the formation of a white sector within a red colony providing a simple visual screen for mutants with elevated or reduced levels of recombination. This strain was further modified to include a direct repeat of mutant alleles of the leu2 gene separated by a copy of the LYS2 gene. Thus the strain used for mutagenesis contained two recombination reporters to monitor the rate of mitotic recombination (Fig. The rate of recombination for each of the strains, including a rad27[Delta] mutant, was determined (Table 2). Each of the mutants showed a >10-fold higher rate of recombination than the wild type strain, but among the rad27 mutant strains there was only a 2-fold difference. The mutants were tested for defects in meiotic recombination by generation of homozygous diploids and determination of sporulation efficiency and spore viability. Defects in meiotic recombination are manifested by sporulation deficiency or reduced spore viability. The rad27[Delta] diploid exhibited 70% spore viability, whereas the other diploids showed between 80 and 96% spore viability, compared with 98% for the wild type strain. The reduction in spore viability observed for the rad27[Delta] strain is most likely due to the accumulation of recessive mutations during vegetative growth of the diploids prior to sporulation rather than a defect in meiotic recombination, because extended vegetative growth led to much higher spore inviability. Table 2. The doubling time in rich medium was determined for each of the mutants at both 30 and 37°C (Table 3). The growth rate of the rad27-4 strain was similar to the rad27[Delta] strain, whereas the other strains had less severe growth defects. The strain containing the rad27-6 allele showed the least severe growth defect at 30°C, but had a doubling time of 4.5 h, similar to the rad27-4 strain, at 37°C. The rad27-3 mutant, which was originally characterized as non-ts for growth, had the least severe growth defect at 37°C. Table 3. 
RESULTS
Identification of four alleles of RAD27 as hyper-recombination mutants
Characterization of the rad27 mutants
Strain
Rate Ade+ × 10-3
Relative rate
RAD27
0.2
1.0
rad27[Delta]
3.9
21.0
rad27-3
2.4
12.7
rad27-4
3.2
16.9
rad27-5
2.4
12.4
rad27-6
2.1
10.9
Strain genotype
Doubling time (min) at 30°C
Doubling time (min) at 37°C
RAD27
105
138
rad27[Delta]
173
318
rad27-3
132
186
rad27-4
165
306
rad27-5
132
207
rad27-6
123
270
Because deletion alleles of RAD27 confer sensitivity to the DNA damaging agent MMS, each of the rad27 mutants was tested for survival following exposure to MMS. W303, the common laboratory strain that was used for mutagenesis, was discovered later to contain a weak allele of RAD5, rad5-535, that confers partial sensitivity to high concentrations of MMS (39). To eliminate effects of the rad5-535 allele, all of the strains were crossed to a RAD5 derivative of W303 to obtain haploid segregants that were RAD5 rad27. Initially, the tests for MMS sensitivity were done in liquid using stationary phase cultures. Under these conditions, mutants rad27-3 and rad27-4 were almost as sensitive to MMS as the rad27 null mutant, and rad27-6 was the most resistant (Fig.
Figure 2. Sensitivity of rad27 mutants to MMS. Stationary phase cultures were treated with MMS as described in Materials and Methods. Figure 3. Effects of temperature on the MMS sensitivity of rad27 mutants. Cultures of each strain were serially diluted and 4 µl aliquots were spotted onto YPD plates containing the indicated amount of MMS. Duplicate plates were incubated at 23 and 35°C. To determine whether the viability of rad27 strains is dependent on recombination, a strain containing a null allele of RAD27 was crossed to strains containing a mutation in either the RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 or XRS2 gene. For all of the crosses at least 22% of the spores were inviable. The viable spores were genotyped according to the biosynthetic markers used to make the gene disruptions. No viable spore products were recovered that contained both mutations, even when germinated at 23°C (Fig. Figure 4. Inviability of rad27 with mutants of the rad52 epistasis group. (A) Spores derived from a diploid heterozygous for RAD27, RAD1 and RAD52. (B) Spores derived from a diploid heterozygous for RAD27 and RAD59. (C) Spores derived from a diploid heterozygous for RAD27 and RAD50. (D) Spores derived from a diploid heterozygous for RAD27 and HDF1. In each case the grid below the tetrad dissection indicates the genotype of each spore. +, wild type locus; -, mutant locus; O, dead spore. In all of the above crosses, strains containing a rad27[Delta] allele were used. Since the rad27-6 allele confers a ts phenotype for growth and MMS sensitivity it was of interest to determine whether a strain could be made containing the rad51 and rad27-6 mutations. When a rad27-6 strain was crossed to a rad51[Delta] strain and the dissected spores germinated at 23°C, most were found to be viable. The rad27-6 rad51 double mutants exhibited reduced growth at 23°C and showed no growth at 35°C, whereas the single mutants grew at both temperatures verifying the conditional nature of the rad27-6 allele.
Recombination is required for the viability of rad27 mutants
DISCUSSION
A yeast strain is described with general utility for the identification of mutants with increased rates of recombination. This strain has several advantages over those described previously: (i) the strain exhibits high rates of spontaneous mitotic recombination;(ii) recombination events can be visualized by a colony color sectoring assay; (iii) a second reporter ensures that defects are general rather than specific to the ade2 reporter; and (iv) the use of an inverted repeat substrate avoids events that can occur by non-conservative mechanisms, such as single-strand annealing. Using this strain, four alleles of RAD27 were identified, as well as cdc2 and cdc17 mutants. The identification of mutants defective in three different replication functions verifies the utility of this strain in screens for hyper-recombination defects. The most likely explanation for the recovery of four rad27 mutants in this screen is that the strains grow quite well at 30°C, whereas most cdc mutants that exhibit hyper-recombination grow poorly at the semi-permissive temperature.
The rad27 strains all show a similar elevation of the rate of recombination (Table 2), but differ in the severity of other phenotypes. For example, the rad27-6 allele shows the least severe growth defect and MMS sensitivity at 30°C, but at 37°C has growth characteristics and MMS sensitivity similar to the null mutant (Figure
It has previously been reported that rad27 rad51 double mutants of S.pombe and S.cerevisiae are inviable (10,18). However, Sommers et al. (12) reported that rad52 rad27 double mutants are viable, but grow slowly. To determine the requirement for genes of the RAD52 epistasis group for viability of rad27 strains, diploids were constructed that were heterozygous for RAD27 and for each of the members of the RAD52 group genes and then dissected to determine the percent spore viability. From each cross, 22-32% of the spores were inviable as would be expected for lethality of two unlinked mutations. The observed lethality of the rad27 rad52 double mutant conflicts with the results of Sommers et al. One possible explanation is that the alleles used in the study by Sommers et al. are leaky, or that the strains used in their study contain a suppressor mutation.
The finding that rad27 rad59 double mutants are inviable was surprising because rad59 mutants show much greater resistance to ionizing radiation than other mutants of the rad52 group (49). The RAD59 gene was originally identified by its requirement for RAD51-independent mitotic recombination and subsequently shown to encode a homolog of Rad52 (49). Because rad59 mutants show no defect in recombination between homologs, or in meiosis, but are defective in recombination between inverted repeats, it was suggested that Rad59 might be involved in sister chromatid recombination. The observed lethality with rad27 is consistent with this hypothesis. Furthermore, it suggests that synthetic lethality with rad27 is a sensitive screen for recombination defects. Although rad1 and rad59 mutants show similar sensitivity to ionizing radiation, rad1 rad27 double mutants are viable. This result confirms that Rad1 function during mitotic recombination is limited to removal of non-homologous sequences (44).
We were also unable to generate double mutants of rad27 with rad50, xrs2 or mre11. These three mutants show poor vegetative growth and extreme sensitivity to ionizing radiation. The recent finding that these mutants are defective in the NHEJ pathway for double-strand break repair, show elevated rates of heteroallelic recombination in diploids and are proficient at mating type switching, has led to the suggestion that the primary function of these genes is in the end-joining pathway. The Ku heterodimer is essential for NHEJ and has no known role in homologous recombination. In this study, a rad27 hdf1 was shown to be viable indicating that the NHEJ pathway is not required for repair of lesions that occur in rad27 strains. From these experiments one can conclude that Rad50, Mre11 and Xrs2 are required for recombination events between sister chromatids, or that the nuclease activity of the Mre11/Rad50/Xrs2 complex is essential for processing Okazaki fragments in the absence of Rad27 (50).
In this study we have shown that yeast strains containing a null mutation of RAD27 are dependent on homologous recombination for survival. Homologous recombination is also required to process DNA lesions formed by defects in DNA ligase I and PCNA (34,51). All of these factors participate in lagging strand DNA synthesis. Mutation of RAD27 also causes inviability in combination with the pol3-01 mutation (an allele containing a mutation in the `proofreading' exonuclease domain of DNA polymerase [delta]), with an exo1 mutation (defective for a 5[prime]-3[prime] exonuclease), and with a ts allele of DNA2, which encodes a DNA helicase (17,21,52). These defects may define alternate modes of processing Okazaki fragments in the absence of the Rad27 nuclease.
ACKNOWLEDGEMENTS
I would like to thank E. Friedberg, H. Klein, A. Rattray and R. Rothstein for gifts of plasmids and yeast strains, and Y. Bai, J. Ferguson and W. K. Holloman for helpful comments on the manuscript. This work was supported by a grant from the NIH (GM41784).
REFERENCES
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R. Ayyagari, X. V. Gomes, D. A. Gordenin, and P. M. J. Burgers
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A. Sundararajan, B.-S. Lee, and D. J. Garfinkel
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Genetics,
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F. Fabre, A. Chan, W.-D. Heyer, and S. Gangloff
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PNAS,
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L. S. Symington
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T. Morishita, Y. Tsutsui, H. Iwasaki, and H. Shinagawa
The Schizosaccharomyces pombe rad60 Gene Is Essential for Repairing Double-Strand DNA Breaks Spontaneously Occurring during Replication and Induced by DNA-Damaging Agents
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