Sep 29, 1990 - Induction of RAD52 in Gl-arrested mutant cells also causes a .... the period of arrest, samples were plated to determine ... clear, Boston, Mass.
MOLECULAR AND CELLULAR BIOLOGY, Apr. 1991, p. 2013-2017 0270-7306/91/042013-05$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 11, No. 4
Effects of Controlled RAD52 Expression on Repair and Recombination in Saccharomyces cerevisiae KENNETH J. DORNFELD AND DENNIS M. LIVINGSTON* Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received 29 September 1990/Accepted 7 January 1991
We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO] promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GALl promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in Gl-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.
The yeast Saccharomyces cerevisiae is very proficient at homologous recombination during both mitotic and meiotic division. Although no single mutation which completely abolishes mitotic recombination in yeast cells has been found, perhaps the most drastic effects are seen in some rad52 mutants. rad52 mutants were originally identified by their sensitivity to X rays (12, 26). Further studies revealed that these mutants are defective in mitotic recombination (15, 18, 25), particularly gene conversion (18), with recombination rates generally reduced 100-fold. Mating-type switching (22, 33), the repair of double-strand DNA breaks (27), methyl methanesulfonate (MMS)- and ionizing-radiation-induced damage repair (31), and meiosis (5, 13) are all severely impaired in these rad52 mutants. Despite its apparent central role in repair and recombination, the activity of Rad52, the gene product of RAD52, is not well understood. To further examine its action, we have overexpressed the gene in order to determine whether its gene product is rate limiting in repair and recombination. Also, we have placed the gene under the control of an inducible promoter in order to temporally control its action in recombination. By temporally controlling RAD52 expression, we have been able to address the role of replication in mitotic recombination. On the basis of their analyses of recombination products, Esposito and Golin (8, 14) have proposed that mitotic recombination events are resolved primarily by replication. The arrangement of markers on recombined chromosomes and the assortment of recombined chromosomes during mitosis suggest that a majority of events are initiated in Gl and are resolved by replication during S phase. An alternative view of similar arrangements and assortments of recombined chromosomes is offered by Roman and Fabre (29). They argue that gene conversion events in Gl lead to a high probability of reciprocal exchange at the same site in G2. Recombination induced by DNA-damaging agents in Gl-arrested cells has been observed in a variety of systems by a number of investigators (9-11, 34). These *
Corresponding author. 2013
observations suggest that induced recombination may occur without replication. In this study, we present evidence concerning spontaneous mitotic recombination in the absence of replication. MATERIALS AND METHODS
Strains and plasmids. Three derivatives of S. cerevisiae SSL204 (MATTa his3A200 trpl leu2 ura3-52 ade2) (2) were made. First, the rad52-1 allele was crossed into this strain from strain g160/2b (Berkeley Stock Center), with three backcrosses to SSL204 to make strain SSL209. Second, the mating type of SSL204 was converted to MATa by controlled action of the HO endonuclease to yield SSL204A. Finally, the wild-type allele of RAD52 in SSL204A was replaced by a deletion disruption of RAD52 (rad52AHS) to yield SSL212A. The rad52AHS deletion lacks the sequence from the HpaII site 9 bp upstream from the initiation codon to the SphI site 14 bp from the stop codon (1). LEU2 (3) has been inserted into this deletion. David Schild (University of California) supplied the wild-type RAD52 sequence. pDML10 was constructed by placing one HindlIl linker at the HpaII site near the start of the RAD52 gene and one in the 3'-flanking sequence and then inserting the HindlIl fragment into a 2,um-based plasmid, pMAC101 (Michael Innis, Cetus Corporation). pMAC101 is related to pAC1 (17) and contains a HindlIl site distal to the yeast ENO] promoter. pDML5 was constructed by inserting the same HindIll fragment containing RAD52 into pBM272 (Mark Johnston, Washington University, St. Louis, Mo.). pBM272 is a CEN-based plasmid and a derivative of pBM150 (19) which has a HindlIl site downstream of the yeast GAL] promoter. Construction of pBYA819, our recombination reporter plasmid, has been described previously (2). Cell manipulation. MMS killing was performed on strains SSL204 (RAD52) and SSL209 (radS2-1) as described by Prakash and Prakash (24), except cells were grown to 5 x 106 to 10 x 106 cells per ml in minimal media to maintain RAD52-expressing plasmids. After MMS treatment, cells were plated on synthetic complete media lacking leucine.
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DORNFELD AND LIVINGSTON
Plasmid recombination was carried out in strains SSL204A (RAD52) and SSL212A (rad52AHS). Events were scored, and the rate of recombination was determined by fluctuation analysis (20, 21) as previously described (2). In all experiments, the recombination reporter plasmid pBYA819 was held under tryptophan selection both during experimental manipulation and upon plating for His' cells. For the induction experiments, SSL212A transformed with both pDML5 and pBYA819 was grown to 1 x 106 to 5 x 106 cells per ml in minimal medium containing 2% raffinose and buffered to pH 4.0 with succinic acid. The culture was divided, and galactose was added to a portion to 2%. An additional amount of raffinose was added to the remaining portion to bring the total concentration to 4%. Samples of cells were removed from each portion at various times and were washed once with 0.9% NaCl, and an appropriate number of cells were plated to selective agar containing glucose. To repress the galactose induction, glucose was added to a sample already containing galactose to a final concentration of 2%. For cycloheximide inhibition, the drug was added to a final concentration of 100 ,ug/ml. The inhibitor was removed by washing. Platings showed that the inhibitor did not affect viability. To arrest cells in Gl, alpha factor (Sigma Chemical Co., St. Louis, Mo.) was added to a cell culture growing on raffinose to 15 ,ug/ml. The culture was incubated for a period equivalent to that of one cell doubling, as determined by hemacytometer counts of a parallel dividing culture, at which time the arrested culture was >95% unbudded. At this time, the arrested culture was divided, and galactose or raffinose was added as before. Throughout the period of arrest, samples were plated to determine whether any unwanted division had occurred. RNA manipulation. RNA was prepared and electrophoresed as described by Brill and Sternglanz (6) and then blotted to GeneScreen II membranes (New England Nuclear, Boston, Mass.) and treated per the manufacturer's specifications. The probe for the pDML10 Northern (RNA) blot was the 870-bp BglII-PvuII fragment of RAD52 (1). The probe for the dot blot analysis was the HpaII-SphI fragment of RAD52. The ARG4 probe used in the dot blot experiment was the 2.06-kbp HpaI fragment of ARG4 (4).
RESULTS To overexpress RAD52, we placed the RAD52 gene under the control of the constitutive ENO] promoter on pDML10 (see Materials and Methods) (23). Cells with this fusion produce approximately 10-fold more RAD52 mRNA than cells with vector (pMAC101) alone (Fig. 1). To assay the effects of overexpression, we first examined the MMS sensitivity of these cells (Fig. 2). We found that despite overexpression of RAD52, pDML10-transformed wild-type cells were just as sensitive to MMS as those cells expressing endogenous amounts of RAD52 mRNA. That the overexpression could, in fact, provide Rad52 activity was shown by the result that pDML10 restored MMS resistance to a rad52-1 mutant. The restoration was complete yet did not surpass the wild-type level. Our result quantitatively confirms the observation made during the cloning of RAD52 which showed that the expression of the gene on a multicopy plasmid from its own promoter did not increase resistance to MMS killing (1, 30). We next examined the effects of RAD52 overexpression on mitotic recombination. To assay recombination, we utilized plasmid pBYA819 (Fig. 3), which contains heteroalleles of the yeast HIS3 gene and undergoes recombination in
MOL. CELL. BIOL.
a 1 234
b
1 234
se
_
_...S {
:
_-
_-...
FIG. 1. Quantity of RAD52 mRNA from cells with ENOI-promoted RAD52. Duplicate samples of 5 mg of total cellular RNA from SSL204 (RAD52) with pDML10 (lane 1), SSL209 (rad52-1) with pDML10 (lane 2), SSL204 (RADS2) with pMAC101 (lane 3), and SSL209 (rad52-1) with pMAC101 (lane 4) were electrophoresed in the same gel. Half of the gel was blotted and probed for RAD52 RNA (a), and the other half was stained with ethidium bromide (b). The darkest band in lanes 1 and 2 and the fainter band of equivalent size in lanes 3 and 4 of the probed gel (a) are of the size expected for full-length RAD52 message (1).
mitotically dividing cells (2). The rate of plasmid recombination drops 100-fold in a radS2 mutant (Table 1) and is, therefore, a sensitive measure of RAD52 action on mitotic recombination. When RAD52 is overexpressed from pDML10 in wild-type cells also containing pBYA819, the rate of plasmid recombination does not exceed that measured in wild-type cells in the absence of overexpression. Thus, neither MMS resistance nor spontaneous plasmid recombination is enhanced by overexpression of RAD52. To examine how quickly expression of RAD52 could restore wild-type levels of recombination in mutant cells, we fused the gene to the controllable GAL] promoter. The GAL] promoter is inactive in cells growing on raffinose or glucose as the carbon source but is very active in cells utilizing galactose as the primary carbon source (19). Measurement of the amount of RAD52 mRNA in galactosegrown cells harboring pDML5 with the GALl-promoted gene shows that they contain approximately threefold more
exposure time (min) FIG. 2. Survival of strains SSL204 (RAD52) and SSL209 (rad52-1) transformed with either pDML10 or pMAC101 in the presence of 0.5% MMS. Symbols: 0, SSL204::pMAC101; 0, SSL204::pDML10; *, SSL209::pMAC101; 0, SSL209::pDML10.
RAD52 OVEREXPRESSION
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a time (hrs) after gal induction 0
0.5
2
5 5.75 5.75 (glc added at 5 hr) probe
RAD52 - e@ eO
*
ARG4
b 0-%
15
V-
FIG. 3. Recombination reporter plasmid pBYA819. We have placed in a CEN-based yeast-Escherichia coli shuttle vector two copies of the yeast HIS3 gene (arrows) along with flanking DNA sequences (open thick segments). Each allelic segment has been mutagenized with restriction site linkers (A, ClaI; A, Sacl; O, SmaI). Only one mutation occurs in each H153 coding region; the two mutations are approximately 200 bp apart. When transformed into yeast cells and held under selection for tryptophan, the plasmid undergoes recombination to yield a wild-type HIS3 allele. Most events occur by gene conversion. Independent events can be analyzed by examining the alteration of restriction sites within plasmids which have undergone recombination (2).
message than pDML10-transformed (ENOJ-promoted) cells (data not shown). Before carrying out a kinetic analysis of recombination induction, we first measured the rate of plasmid recombination in mutant cells catabolizing raffinose, glucose, or galactose (Table 1). In glucose- and raffinosegrown cells, the rate of plasmid recombination remains at the rad52 mutant level, 50- to 100-fold lower than the wild-type level (Table 1). However, when RAD52 is overexpressed from the GAL] promoter in galactose-grown cells, the rate of recombination is that measured for wild-type cells. Thus, as with ENOJ-promoted RAD52 on pDML10, high-level expression of RAD52 with the GAL] promoter brings a mutant to a wild-type level of spontaneous recombination but not to a higher level. To determine whether induction of plasmid recombination parallels GALl-promoted induction of RAD52, we measured both the amount of RADS2 mRNA and the fraction of cells with recombined plasmids. We found that within 30 min of addition of galactose to raffinose-grown rad52 cells with pDML5, the cells begin to amass large amounts of RAD52 mRNA (Fig. 4a). Samples of the culture taken as soon as 1 h after addition of galactose show a significant increase in the fraction of cells with recombined plasmids (Fig. 4b). (In other trials of the induction, we measured an increase by 30 min.) The rate at which recombined plasmids appear can be TABLE 1. pBYA819 recombination rates Allele
RAD52
Extrachromosomal source of RADS2 (promoter)
rad52AHS
None None
RAD52
pDML1O (ENOI)
rad52AHS rad52AHS
pDML5 pDML5 pDML5
rad52AHS
(GALl) (GALl) (GALl)
source
Recombination rate (1o-4 events/cell/ generation)
Glucose Glucose Glucose Glucose Raffinose Galactose
4.2 0.0039 4.0 0.0083 0.0072 2.6
Carbon
galactose
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+
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0
0
1
2
3
4
5
6
7
time (hr) after the addition of galactose FIG. 4. (a) Changes in amount of RAD52 mRNA in SSL212A (rad52AHS) transformed with pDML5 upon galactose induction and glucose repression. pDML5-transformed SSL212A was grown with raffinose as the carbon source. At 0 h, galactose was added to 2%. At the times indicated, samples of cells were removed and RNA was extracted from them. At 5 h after galactose addition, the culture was divided and glucose was added to one portion to 2%. Samples were removed 45 min later (5.75 h) from both portions, and RNA was extracted from them. The RNA was blotted and probed with RAD52 DNA, after which the filter was stripped and probed with ARG4 DNA. (b) Changes in the fraction of His' recombinants in a culture of SSL212A (rad52MHS) with recombination substrate pBYA819 and RAD52 source pDML5 upon galactose induction and glucose repression. A culture of doubly transformed SSL212A was grown on raffinose until 0 h, when it was divided into three portions and galactose was added to two portions to 2%. At 2 h, glucose was added to one of these. The third portion served as an uninduced control. Samples of cells were plated to determine the fraction of recombinants. The recombinant fraction in the control culture shows no change with time (see Fig. 5) and is not shown.
measured from the slope of the line. Using a generation time of 6 h, as determined by platings to determine total cell number, we calculated an initial rate of recombination of 4 x 10-4 events per cell per generation, very comparable to the wild-type rate determined by fluctuation analysis of cells in steady-state growth (Table 1). In several trials of this experiment, we found that recombinants consistently increase at a wild-type rate for at least 6 h, after which the fraction of recombinants is 10- to 15-fold greater than the initial fraction. After this time, recombinants do not form as rapidly. We have also checked the recombination products formed in galactose-induced cells and found that the spectrum of conversion and crossover products is very similar to that recovered from cells in steady-state growth (2, 7a). Having found that recombination ensues rapidly after RAD52 induction, we examined whether recombination would cease rapidly upon glucose repression of the GAL] promoter. RNA blot analysis shows that 45 min after addi-
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DORNFELD AND LIVINGSTON
MOL. CELL. BIOL.
1l5
6
7
8
9
10
11
12
time (hr) after alpha factor arrest FIG.
5.
Changes
galactose
in
induction
(radS2AvHS)
o)f His+
fraction
the
in
Gi-arrested
a
doubly transformed
with
recombinants
culture
p1BYA819
and
after
SSL212A
of
pDML5.
A
culture was grown on raffinose until the cells reached a density of 106 cells per ml, at which time it was divided into three portions and alpha factor was added to one. At 6.25 h later, galactose was added
to the
factor and to one of the remaining two the fraction of recombinants
portion with alpha
portions.
Samples
were removed, and
was determined.
tion
of
mRNA
galactose-induced culture, RAD52
glucose to a is reduced to
an
amount
comparable
to
that
in
cells (Fig. 4a). Similarly, when glucose is added 2 h after galactose induction of cells harboring both the GALl-promoted RADS2 gene and the recombination reporter plasmid, nascent recombinants no longer form (Fig. preinduced
4b).
That recombination must be quickly curtailed after is suggested by the absence of any increase in the
repression
recombinant
fraction after
glucose addition
(Fig. 4b). The
addition of cycloheximide in place of glucose after 2 h of
galactose induction
also quickly
inhibits
the
appearance of
recombinants (data not shown).
To examine
of
whether recombination occurs in the absence
plasmsid replication,
we induced
in cells arrested in Gi factor.
Plasmids
apr under cell cells
(35).
pBYA819
like our
rad52
recombination reporter
cells
with
on
the
raffinose
recombined
pBYA819
plasmids
pDML5
and then
arrested
(see Materials and Methods). When
the cells were unbudded, indicating Gi induced with
RADS2
cycle control and do not replicate in arrested
were grown
alpha factor
GALl-promoted
with the mating pheromone alpha
arrest, RAD52 was
galactose. Initially, the fraction
plasmids
increased
at
the
and
with >95% of
same
of cells with rate
as
that
measured for dividing cells (Fig. 5). However, after 6 h in
galactose, the fraction of recombinants is smaller in the arrested culture than in the unarrested culture. One reason for this could be that the amount of RAD52 mRNA produced in Gi-arrested cells is about half that produced in dividing cells (data not shown) (32). As before, examination of the recombination products produced in arrested cells shows the spectrum of events to be very similar to that of events occurring in dividing cells (2, 7a). DISCUSSION
We have shown that overexpression of RADS2 mRNA does not increase MMS resistance in RAD52 cells and raises the resistance of radS2-J cells to, but not beyond, RADS2 levels. Our observation, along with those of other workers (1, 30), suggests that the amount of Rad52 is not rate limiting
for DNA damage repair in wild-type cells. Another repair function must determine the extent of repair of MMSinduced damage. This result is interesting because a set of tetraploid yeast strains with from zero to four wild-type copies of RAD52 become progressively more resistant to X-radiation with the increasing numbers of copies (16). Possibly, the ratio of gene product to DNA content is crucial for resistance to DNA damage under limiting conditions. Interestingly, overexpressing cells do not become more susceptible to MMS. This could occur if Rad52 interacts with another repair factor and if overexpression sequesters this other factor, effectively lowering its concentration and limiting repair. Recombination rates, like MMS resistance, do not surpass the wild-type level in cells overproducing RADS2 mRNA. Either the production of DNA configurations which initiate recombination or a factor other than RAD52 determines the rate of mitotic recombination. In contrast to the ready availability of Rad52 in mitotic cells, limitations in the amount of the gene product could become important in meiotic cells. Cole et al. (7) have shown that RADS2 mRNA undergoes a 15-fold induction during meiosis, when a level of recombination 1,000-fold that found in mitosis occurs. This increase could signify that with the general elevation of all factors involved in recombination, possibly including the one(s) which are rate limiting in mitosis, the absence of an increase in RAD52 expression could impair the attainment of the high levels of recombination achieved during meiosis. We have also studied controlled induction of RADS2 in a rad52 mutant. Induction produces a rapid rise in the number of recombinants at a rate commensurate with recombination in steady-state growth, followed by a decline in rate. After a more than 10-fold increase in the fraction of recombinants within a 6-h induction, the fraction of recombinants increases more slowly. One explanation for this result is that the initial rate reflects the rate found for steady-state cells but the decline represents the depletion of other protein components which have been consumed by the initial rise. An alternative explanation is that the initial burst in recombination upon induction may represent nascent Rad52 acting on an accumulated pool of recombination substrates present in the formerly rad52 mutant cells. Once the pool is exhausted, new substrates for Rad52 action become limiting. When RADS2 expression is stopped by the action of glucose on the GALI promoter, the fraction of recombinants reaches a plateau, showing that no new recombinants are formed after RADS2 expression is repressed. Since the quantity of RAD52 mRNA quickly diminishes upon repression, this finding suggests that Rad52, the protein product, either has a short active half-life relative to the duration of a recombination event or must be continually synthesized during the process to be active. The similar cessation which is caused by the addition of cycloheximide confirms that a protein component of the recombination pathway must be very labile or synthesized contemporaneously. We have also shown that a burst of recombinants appears in alpha-factor-arrested rad52 cells upon RAD52 induction. This result argues that replication is not required for plasmid recombination. The caveat to this conclusion is that recombination intermediates may form in the Gl-arrested, galactose-induced cells and be suspended at an intermediary stage, only to be rescued by a round of DNA replication after the arrest is lifted in the cells plated in the absence of alpha factor. If such intermediates do accumulate, however, they must be stable because cells held under arrest for 5 h retain greater than 50% of the recombinants they possess at 2 h.
VOL . 1 l, 1991
Such long-lived intermediates have been postulated to occur in sporulating cultures of rad52 mutants which are returned to mitotic growth (28). In repeated trials, we find that the fraction of recombinants in arrested, induced cultures is always smaller than that in dividing cultures. This could reflect either the need for replication to resolve some of the recombination intermediates or the attenuated induction of RAD52 in arrested cells. Yet another possibility is that the process of replication causes some plasmids to enter into recombination. ACKNOWLEDGMENT This work was supported by grant MV-373B from the American Cancer Society. REFERENCES 1. Adzuma, K., T. Ogawa, and H. Ogawa. 1984. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2735-2744. 2. Ahn, B.-Y., and D. M. Livingston. 1986. Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerei'isiae plasmid system. Mol. Cell. Biol. 6:3685-3693. 3. Andreadis, A., Y.-P. Hsu, M. Hermodson, G. Kohlhaw, and P. Schimmel. 1984. Yeast LEU2. J. Biol. Chem. 259:8059-8062. 4. Beacham, I. R., B. W. Schweitzer, H. M. Warrick, and J. Carbon. 1984. The nucleotide sequence of the yeast ARG4 gene. Gene 29:271-279. 5. Borts, R. H., M. Lichten, and J. Haber. 1986. Analysis of meiosis-defective mutations in yeast by physical monitoring of recombination. Genetics 113:551-567. 6. Brili, S. J., and R. Sternglanz. 1988. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell 54:403-411. 7. Cole, G. M., D. Schild, and R. K. Mortimer. 1989. Two DNA repair and recombination genes in Saccharomyces cerei'isiae, RAD52 and RAD54, are induced during meiosis. Mol. Cell. Biol. 9:3101-3104. 7a.Dornfeld, K. Unpublished observation. 8. Esposito, M. S. 1978. Evidence that spontaneous mitotic recombination occurs at the two-strand stage. Proc. Natl. Acad. Sci. USA 75:4436 4440. 9. Esposito, R. E. 1968. Genetic recombination in synchronized cultures of Saccharomyces cerei'isiae. Genetics 59:191-210. 10. Fabre, F. 1978. Induced intragenic recombination can occur during the G1 mitotic phase. Nature (London) 272:795-798. 11. Fabre, F., A. Boulet, and H. Roman. 1984. Gene conversion at different points in the mitotic cycle of Saccharomyces cerev,isiae. Mol. Gen. Genet. 195:139-143. 12. Game, J. C., and R. K. Mortimer. 1974. A genetic study of X-ray sensitive mutants in yeast. Mutat. Res. 24:281-292. 13. Game, J. C., T. J. Zamb, R. J. Braun, M. Resnick, and R. M. Roth. 1980. The role of radiation (rad) genes in meiotic recombination in yeast. Genetics 94:51-68. 14. Golin, J. E., and M. S. Esposito. 1981. Mismatch correction and replicational resolution of Holliday structures formed at the two strand stage in Saccharomyces. Mol. Gen. Genet. 183:252-263. 15. Haber, J. E., and M. Hearn. 1985. RAD52-independent mitotic gene conversion in Saccharomyces cerevisiae frequently results
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in chromosome loss. Genetics 111:7-22. 16. Ho, K. S. Y. 1975. The gene dosage effect of the rad52 mutation on X-ray survival curves of tetraploid yeast strains. Mutat. Res. 33:165-172. 17. Innis, M. A., M. J. Holland, P. C. McCabe, G. E. Cole, V. P. Wittman, R. Tal, K. W. K. Watt, D. H. Gelfand, J. P. Holland, and J. H. Meade. 1985. Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces. Science 228:21-26. 18. Jackson, J. A., and G. R. Fink. 1981. Gene conversion between duplicated genetic elements in yeast. Nature (London) 292:306311. 19. Johnston, M., and R. W. Davis. 1984. Sequences that regulate the divergent GALI-GALIO promoter in Saccharomyces cerei'siae. Mol. Cell. Biol. 4:1440-1448. 20. Lea, D. E., and C. A. Coulson. 1949. The distribution of the numbers of mutants in bacterial populations. J. Genet. 49:264285. 21. Luria, S. E., and M. Delbruck. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491-511. 22. Malone, R. E., and R. E. Esposito. 1980. The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc. Natl. Acad. Sci. USA 77:9503-9507. 23. McAlister, L., and M. J. Holland. 1982. Targeted deletion of a yeast enolase structural gene. J. Biol. Chem. 257:7181-7188. 24. Prakash, L., and S. Prakash. 1977. Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae. Genetics 86:33-55. 25. Prakash, S., L. Prakash, W. Burke, and B. Montelone. 1980. Effects of the RAD52 gene on recombination in Saccharomyces cere0isiae. Genetics 94:31-50. 26. Resnick, M. A. 1969. Genetic control of radiation sensitivity in Saccharomyces cerevisiae. Genetics 62:519-531. 27. Resnick, M. A., and P. Martin. 1976. The repair of doublestrand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control. Mol. Gen. Genet. 143:119-129. 28. Resnick, M. A., J. Nitiss, C. Edwards, and R. E. Malone. 1986. Meiosis can induce recombination in rad52 mutants of Saccharomyces cerevisiae. Genetics 113:531-550. 29. Roman, H., and F. Fabre. 1983. Gene conversion and associated reciprocal recombination are separable events in vegetative cells of Saccharomyces cerei'isiae. Proc. Natl. Acad. Sci. USA 80:6912-6916. 30. Schild, D., B. Konforti, C. Perez, W. Gish, and R. Mortimer. 1983. Isolation and characterization of the yeast DNA repair genes. I. Cloning of the RAD52 gene. Curr. Genet. 7:85-92. 31. Strike, T. L. 1978. Characterization of mutants of yeast sensitive to X-rays. Ph.D. thesis. University of California, Davis, Davis. 32. Throm, E., and W. Duntze. 1970. Mating-type-dependent inhibition of deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 104:1388-1390. 33. White, C. I., and J. E. Haber. 1990. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 9:663-673. 34. Wildenberg, J. 1970. The relation of mitotic recombination to DNA replication in yeast pedigrees. Genetics 66:291-304. 35. Zakian, V. A., B. J. Brewer, and W. L. Fangman. 1979. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell 17:923-934.
