MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2485-2491

Vol. 10, No. 6

0270-7306/90/062485-07$02.00/0 Copyright ©) 1990, American Society for Microbiology

RADIO, an Excision Repair Gene of Saccharomyces cerevisiae, Is Involved in the RADI Pathway of Mitotic Recombination ROBERT H. SCHIESTL AND SATYA PRAKASH*

Department of Biology, University of Rochester, Rochester, New York 14627 Received 28 November 1989/Accepted 21 February 1990

The RADIO gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RADIO gene is also required for mitotic recombination. The radlOA mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3A3' his3A5'). The rate of formation of HIS3+ recombinants in the radlOA mutant was not affected by the radlA mutation but decreased synergistically in the presence of the radlOA mutation in combination with the rad52A mutation. These observations indicate that the RADI and RADIO genes function together in a mitotic recombination pathway that is distinct from the RADS2 recombination pathway. The radiOA mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RADI and RADIO gene products act in recombination after the formation of the recombinogenic substrate. The radiA and radlOA mutations did not affect meiotic intrachromosomal recombination of the his3A3' his3A5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes. In the yeast Saccharomyces cerevisiae, excision repair of DNA damaged by UV light and bulky adducts that distort the DNA helix is controlled by at least 10 different genes. Mutations in the RADI, RAD2, RAD3, RAD4, and RADIO genes abolish the incision of damaged DNA (17, 29, 43), whereas mutations in the RAD7, RAD14, RAD16, RAD23, and MMSJ9 genes affect the proficiency of excision repair (17, 18, 43). With the goal of purifying and characterizing the protein components required for excision repair, we have cloned many of these genes and have purified and characterized the RAD3-encoded protein (40-42). The cloned genes have also been used to construct genomic deletion mutations. Studies with these mutations have revealed that RAD3 is essential for cell viability (8, 20) and that RADI functions in mitotic recombination (12, 34). Thus, in contrast to Escherichia coli, in which the uvrA, uvrB, and uvrC genes are required only for the incision of UV-damaged DNA, some of the yeast excision repair genes affect other cellular

and have found that RADIO is also involved in mitotic recombination. The effects of the RADIO gene on mitotic recombination are very similar to those of the RADI gene. Like the radlA mutation (34), the radiOA mutation lowered mitotic recombination synergistically in combination with the radS2A mutation; in contrast, recombination was reduced to the same level in the radiA radJOA double mutant as in the radiA and radJOA single mutants. These observations indicate that the RADI and RADIO genes function in the same pathway of mitotic recombination and that this pathway constitutes an alternate and competing pathway to the RADS2 recombination pathway.

genes

MATERIALS AND METHODS Strains and media. E. coli HB101 (hsdR hsdM recAl supE44 lacZ4 leuB6 proA2 thi-i) was used for plasmid propagation. The yeast strains used are listed in Table 1. Growth, minimal, and sporulation media were prepared as described previously (36, 37). Sporulation media for some experiments (see Table 8) were prepared as described by Roth and Halvorson (31). Cells were incubated in presporulation medium for 24 h and in sporulation medium for 3 days. Genetic analyses. Standard procedures were used for sporulation and genetic analyses (37). Transformation and other procedures. Large-scale plasmid isolation from E. coli, E. coli transformation, and electrophoresis of DNA were performed as described by Maniatis et al. (15). Isolation of DNA fragments from agarose gels was carried out with the Geneclean Kit (BIO 101, La Jolla, Calif.) as recommended by the supplier. Small-scale plasmid preparations from E. coli were made by a modification of the boiling method (7). Transformation of yeasts was carried out by treating cells with lithium acetate (9) to promote DNA uptake. Plasmids. Plasmids YEplacl95, pDG95, and pSP56 have been described previously (34). Deletions in RAD genes. Genomic deletions in RAD genes (radA) were made by replacing the entire or most of the open

processes.

Previously, we showed that the radlA mutation lowers the rate of mitotic intrachromosomal recombination of a his3 duplication in which one of the his3 alleles has a deletion at the 3' end and the other has a deletion at the 5' end (34). The

RAD52 gene, required for double-strand break repair and recombination (23), is also involved in this recombination. However, the RADI and RADS2 genes affect mitotic recombination via alternate pathways, since the formation of HIS3+ recombinants is decreased synergistically in the radlA rad52A double mutant (34). Based on the observation that the radiA mutation reduces the efficiency of integration of linear plasmids and DNA fragments into homologous genomic sequences, we have suggested that the action of RADI in recombination occurs after the formation of the recombinogenic substrate (34). We have now examined the effect on recombination of deletion mutations in several other yeast excision repair *

Corresponding author. 2485

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SCHIESTL AND PRAKASH TABLE 1. Strains useda Genotype

Strain

Source or reference

MATa ura3-52 leu2-3,112 trpS-27 arg4-3 ade240 ilv1-92 HIS3::pRS6b MATaL lysl-l ura3-52 his4-864,1176 pBR313 his4-260,39 HIS4' derivative of LP2752-4B MATa leu2-3,112 ura3-52 hisl-] trp2 XS803-3A with rad52/ LEU2+ MATa arg4-3 or arg4-17 trpl-289 or TRPI+ trp5-27 ade240 ilvl-92 leu2-3,112 ura3-52 rad1OAURA3' radS2ATRPIJ HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or TRPI+ ade240 leu2-3,112 ura3-52 rad1OAURA3' radS2ATRP1 HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or TRPI+ leu2-3,112 ura3-52 radJOAURA3' rad52ATRPl+ HIS3::pRS6 MATa arg4-3 or arg4-17 ade240 ilvl-92 leu2-3,112 ura3-52 HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or trpS-27 ilvi-92 leu2-3,112 ura3-52 HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or TRP1+ trpS-27 ade240 leu2-3,112 ura3-52 rad52ATRPJ + HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or TRPI+ trpS-27 ilvl -92 leu2-3,112 ura3-52 rad52ATRPI+ HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or trp5-27 ilvi-92 leu2-3,112 ura3-52 rad]OAURA3' HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or trpS-27 ilvl-92 leu2-3,112 ura3-52 radlOAURA3' HIS3::pRS6 MATa arg4-3 or arg4-17 trpl-289 or trpS-27 ilv1-92 leu2-3,112 ura3-52 radlOAURA3' HIS3::pRS6 MATa ade240 leu2-3,112 ura3-52 rad1AURA3' HIS3::pRS6 MATa arg4-3 trpS-27 ilvl-92 lysl-J leu2-3,112 ura3-52 radJOAURA3' HIS3::pRS6 MATa arg4-3 trpS-27 ade240 ilv1-92 leu2-3,112 ura3-52 radJAURA3' rad1OAURA3' HIS3::pRS6 MATa arg4-3 ade240 ilv1-92 leu2-3,112 ura3-52 radJOAURA3+ HIS3::pRS6 MATa arg4-3 trpS-27 ade240 leu2-3,112 ura3-52 HIS3::pRS6 MATa arg4-3 ade240 leu2-3,112 ura3-52 HIS3::pRS6 MATa arg4-3 trp5-27 ade240 ilvl-92 leu2-3,112 ura3-52 radJAURA3+HIS3::pRS6 MATa arg4-3 ilv1-92 leu2-3,112 ura3-52 radJAURA3' rad1OAURA3' HIS3::pRS6 MATa arg4-3 trp5-27 ilv1-92 leu2-3,112 ura3-52 radJAURA3' radJOAURA3' HIS3::pRS6 MATa arg4-3 trpS-27 ade240 ilvi-92 leu2-3,112 ura3-52 radlOAURA3+ HIS3::pRS6 MATa arg4-3 trp5-27 ade240 leu2-3,112 ura3-52 radJAURA3' HIS3::pRS6 MATa trpl-289 lysJ-J ura3-52 his4-864,1176 pBR313 his4-260,39 MATa trpl -289 lysl -I ura3-52 his4-864,1176 pBR313 his4-260,39 rad52ATRPJ + MATa ade2-1 or ade240 trpl or trpS-27 leu2-3,112 ura3-52 HIS3::pRS6 MATa ade2-1 or ade240 trpl or trp5-27 leu2-3,112 ura3-52 cdc9-2 HIS3::pRS6 RS117-11B with radlOAURA3+

33 L. Prakash 34 D. Schild This study This study

RS112

MATa ura3-52 leu2-3,112 trpS-27 arg4-3 ade240 ilv1-92 LYS2 HIS3::pRS61 MATa ura3-52 leu2A&98 TRP5 ARG4 ade2-101 ILVI lys2-801 his3A200

34

RS114

MATa ura3-52 leu2-3,112 trpS-27 arg4-3 ade240 ilv1-92 LYS2 radJAURA3' HIS3::pRS6I MATa ura3-52 leu2A&98 TRP5 ARG4 ade2-101 ILVI lys2-801 radlAURA3' his3A200

34

RS177

MATa ura3-52 leu2-3,112 trp5-27 arg4-3 ade240 ilv1-92 LYS2 rad1OAURA3' HIS3::pRS61 MATa ura3-52 leu2A&98 TRP5 ARG4 ade2-101 ILVI lys2-801 rad1OAURA3+ his3A200

This study

RSY6 LP2752-4B LP2752-4B/H+ XS803-3A XS803-3A/52A RS143/1-7B

RS143/1-13C RS143/1-14B RS143/2-1A RS143/2-1D RS143/2-2B

RS143/2-2D RS143/2-3D RS143/2-9A RS143/2-15A RS144/1-1C RS144/1-5B RS144/1-12C RS144/2-2B RS144/2-3C RS144/2-4C RS144/2-6C RS144/2-8D RS144/2-9A RS144/2-9C RS144/2-19A RS22-8B RS22-8B/52A RS1174A RS117-11B TS117-11B/10A

This study

This study

This study This study This study This study

This This This This This This This This This This This This This This 34 This This This This

study study study study study study study study study study study study study study

study study study study

a The genes RADI, RAD2, RAD4, RAD7, RADIO, RAD23, and RADS2 were replaced in strain RSY6 and strain LP27524B with the URA3 gene (see Materials and Methods) to give rise to isogenic derivatives designated RSY6/1A, RSY6/4A, RSY6/7A, RSY6110A, RSY6/23A, RSY6/52A, and RSY6/7A/23A& and to isogenic derivatives designated LP2752-4B/1A, LP2752-4B/2A, LP27524B/4A, LP27524B/7A, LP27524B/10A, LP2752-4B/23A, and LP2752-4B/52A&. HIS3::pRS6 indicates the integration of plasmid pRS6 at the chromosomal HIS3 locus, generating a duplication of the his3 gene with 5'- and 3'-terminal deletions. b

reading frame (ORF) of these genes with the S. cerevisiae URA3 gene or another selectable gene by the one-step gene disruption method (32). The radlA and rad2A mutations were constructed as described previously (34). A 3.7-kilobase XbaI fragment from plasmid pDG38 was used to delete nucleotide positions +223 to +1559 in the ORF of the genomic RAD4 gene (4). pDG38 was constructed as follows. Plasmid pDG26 was obtained by cloning the 4.1-kilobase BamHI-HindIII fragment with the inactive rad4 gene from plasmid pR169 (30) into pUC9. The internal fragment of the RAD4 gene from positions +223 to +1559 was removed by digestion with Asp718 and MstII and replaced by the 3.8-kilobase BamHI-BglIl fragment containing the yeast URA3 gene flanked by direct repeats of the

Salmonella typhimurium hisG DNA from plasmid pNKY51

(1) to yield plasmid pDG38. A genomic deletion in the RAD7 gene was constructed by replacing RAD7 sequences from nucleotide positions +214 to + 1445 in the ORF (25) with the URA3 gene as described previously (35). The genomic RADIO gene was disrupted by insertion of the URA3 gene as described previously (26) into the XbaI site at position +472 in the RADIO ORF (28). A genomic deletion in the RAD23 gene (16) was constructed by replacing positions +47 to + 1110 in the ORF with the URA3 gene as described previously (35). The genomic RAD52 gene was replaced with LEU2 to produce rad52-7, with TRPJ to produce radS2-8, or with URA3 to produce rad52-9. rad52-7 and rad52-8 were constructed by digestion of plasmids

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TABLE 2. Effect of rad mutations on mitotic recombination of the his3A3' his3A5' duplication Straina

RSY6 RSY6/1A RSY6/10A RSY6/52A RSY6/4A RSY6/7A RSY6/23A RSY6/7A/23A

Genotype

RAD+b radlAb radJOA

rad52Ab rad4A

rad7A rad23A rad7A rad23A

No. of HIS3+ recombinants/104 viable cells in culture: 1 2 3

3.6 0.88 0.59 0.66 8.7 5.5 5.3 6.7

4.0 0.90 0.55 0.55 6.7 6.7 7.0 6.3

Mean ± SD recombination rate

3.3 0.74 0.64 0.47 10.5 6.9 7.0 5.6

3.6 0.84 0.59 0.56 8.63 6.4 6.4 6.2

re of RADt recombination

(1O-4)

± 0.35

100 23 16 15 240 178 178 172

± 0.078 ± 0.0045

± 0.095 ± 1.90 ± 0.75 ± 0.98 ± 0.56

a All strains are isogenic. b These data have been published previously (34) and are shown here for comparison.

pSM20 and pSM21, respectively (kindly provided by David Schild and R. Mortimer), with BamHI and transformation of yeast cells with the BamHI fragment in which the LEU2 or the TRPI gene had been inserted at the BgIII site in the RADS2 ORF. rad52-9 was constructed by digestion of plasmid pSM22 (kindly provided by David Schild and R. Mortimer) with BamHI and transformation of yeast cells with the BamHI fragment in which the Bglll-ClaI fragment in the ORF of the RADS2 gene had been replaced by a BamHI-ClaI fragment containing the URA3 gene. All radS2 deletions are henceforth termed rad52A in this paper. The deletion in each RAD gene was confirmed by complementation analyses with known rad mutations. Determination of rates and frequencies of recombination. Plasmid pRS6 contains an internal fragment of the HIS3 gene, the LEU2 gene, and pBR322 sequences. Integration of pRS6 at the genomic HIS3 site yields two copies of the his3 gene, one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end (his3A3' his3A5'), separated by the LEU2 gene and pBR322 sequences (33, 34). In cells carrying the his3A3' his3A5' duplication, over 99% of HIS3+ recombinants lose the LEU2 gene; therefore, to screen for HIS3+ recombinants, we grew three parallel cultures in synthetic complete (SC) medium lacking leucine to an approximate density of 107 cells per ml and plated the cells onto SC medium lacking histidine to score for recombinants and onto SC medium to determine the number of viable cells in the culture. Because HIS3+ recombinants do not divide in medium lacking leucine, the HIS3+ frequency is a direct measure of the recombination rate, and there is little variation in the recombination rates among different cultures. Heteroallelic recombination was examined in cells carrying an intrachromosomal duplication of the HIS4 gene. In this construct (10), the genes are arranged in the chromosome in the order his4C pBR313 his4A and there are two point mutations in the his4A or the his4C gene. To screen for HIS4+ recombinants, we grew five 5-ml parallel cultures in SC medium to a density of approximately 107 cells per ml and plated the cells onto SC medium lacking histidine to score for recombinants and onto SC medium to determine the number of viable cells in the culture. The recombination rates (see Table 6) were calculated by the method of the median (13). For the determination of meiotic recombination frequencies, five cultures were grown in YPAD medium (36, 37) and mitotic recombination frequencies were determined. The cultures were diluted 1/1,000 in presporulation medium and grown to 107 cells per ml. Thereafter, cells were inoculated in sporulation medium at a density of 107 cells per ml and

incubated at 30°C for 3 days. The sporulated culture was collected by centrifugation and washed with sterile distilled water, and different dilutions were plated onto SC medium to determine the plating efficiency, onto SC medium lacking histidine to determine the frequency of intrachromosomal recombination of the his3A3' his3A5' duplication, and onto SC medium lacking adenine to determine the frequency of interchromosomal recombination of ade2 heteroalleles. RESULTS Requirement of the RADIO gene for mitotic intrachromosomal recombination between his3 deletion alleles. We examined intrachromosomal recombination between two his3 genes, one of which has a deletion at the 3' end and the other of which has a deletion at the 5' end (his3A3' his3AS'). The two his3 genes share homology of about 400 base pairs and are separated by the LEU2 and pBR322 sequences (33). Table 2 shows the rate of formation of HIS3+ recombinants in the isogenic RAD+ strain and in various rad mutant strains. As has been reported previously (34) and shown in Table 2 for comparison, recombination between his3 deletion alleles was lowered about four- and sevenfold by the radlA and rad52A mutations, respectively. The radJOA mutation caused a sixfold decrease in the rate of formation of HIS3+ recombinants, whereas mutations in other excision repair genes led to a twofold increase in the rate of formation of HIS3+ recombinants. We also examined recombination in TABLE 3. Effect of the radlA mutation in combination with the radJOA mutation on mitotic recombination of the

his3A3' his3AS' duplication No. of HIS3+ recombinants/104 Straina

viable cells in culture:

Genotype

RS144/2-3C RAD+ RS144/2-4C RAD+ radlA RS144/1-1C RS144/2-6C radlA RS144/2-19A radlA RS144/1-SB radJOA RS144/2-2B

radIOA

RS144/2-9C RS144/1-12C RS144/2-8D RS144/2-9A

radJOA radlA radlOA

radlA radJOA radlA radJOA

Mean + SD recombination rate (1o-4)

1

2

3

7.8 4.7

8.5 3.5 1.1 0.49 0.52 0.40 0.37 0.42

3.5 9.1 1.6 0.39 0.52 0.62

1.7

1.3

1.4 ± 0.23

0.43 0.89

0.69 0.84

0.52 ± 0.15 0.84 ± 0.055

1.2 0.47 0.51 0.48 0.36 0.49 1.3 0.43 0.78

0.38

0.48

6.6 5.8 1.3 0.45 0.52 0.5 0.37 0.46

± 2.7 ± 2.9 ± ± ± ± ±

0.26 0.053 0.0058 0.11 0.01

± 0.038

aAll RS144 strains are congenic segregants from the same cross.

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SCHIESTL AND PRAKASH

TABLE 4. Effect of the rad52A mutation in combination with the radJOA mutation on mitotic recombination of the his3A3' his3A5' duplication No. of HIS3+ recombinants/104 viable cells in culture:

Straina

RS143/2-1A RS143/2-1D RS143/2-3D RS143/2-9A RS143/2-15A RS143/2-2B RS143/2-2D RS143/1-7B RS143/1-13C RS143/1-14B

Genotype

RADW RADW radJOA radJOA radJOA rad52A rad52A radJOA radS2A radlOA rad52A radJOA radS2A

1

2

3

5.6 2.7 0.47 0.30 0.41 0.49 0.20 0.0078 0.035 0.018

5.7 2.6 0.45 0.39 0.44 0.58 0.20 0.0067 0.0019 0.026

3.7 4.0 0.58 0.38 0.47 0.69 0.24 0.0075 0.016 0.016

Mean t SD recombination rate

(1O-4) 5.0 t 3.1 t 0.50 + 0.36 t 0.44 t

0.59 0.21 0.0073 0.018 0.02

± t t t t

1.1 0.78 0.07 0.049 0.03 0.10 0.023 0.00057 0.017 0.0053

a All RS143 strains are congenic segregants from the same cross.

the rad7A rad23A double mutant, since this mutant has a greater sensitivity to DNA-damaging agents and a higher

level of deficiency in excision repair than has the rad7A or rad23A single mutant (18). However, HIS3+ recombinants occurred to the same extent in the rad7A, rad23A, and rad7A rad23A mutants (Table 2). RADIO acts in the RADI pathway of mitotic recombination. To determine whether RADIO acts in the RADI recombination pathway, we examined the rate of intrachromosomal recombination of the his3 duplication in the RAD+, radiA, radJOA, and radlA radJOA congenic strains (Table 3). The occurrence of HIS3+ recombinants was reduced to about the same extent in the radlA and radJOA single mutants and in the radlA radiOA double mutant. This epistatic interaction between the radiA and radJOA mutations indicates that the RADI and RADIO genes act in the same recombination pathway. Since the radiA mutation lowers the incidence of HIS3+ recombinants synergistically when present in combination with the rad52A mutation (34), we expected the radJO4 mutation to have a similar effect. Table 4 shows the rate of formation of HIS3+ recombinants in the RAD+, radJOA, radS2A, and radJOA rad52A congenic strains. The rate of formation of HIS3+ recombinants fell -30-fold in the radIOA rad52A mutant strain as compared with the radJOA or radS2A mutant strain. The extreme synergism of the radJOA and rad52A mutations indicates that the RADIO and RAD52 genes provide alternate routes for HIS3+ recombinant formation. Requirement of RADIO for the elevated level of mitotic recombination in cdc9 mutants. The CDC9 gene of S. cerevisiae encodes DNA ligase (2). At the restrictive temperature of 36°C, cdc9 mutants are defective in joining Okazaki fragments (11) and arrest in cell division before mitosis. Because of defective DNA ligase activity, cdc9 mutants show enhanced levels of recombination even at the permis-

sive temperature (6, 19). Table 5 shows the rate of HIS3+ recombinants arising at the permissive temperature (25°C) in RAD+ CDC9+, radJOA, cdc9, and radJOA cdc9 strains carrying the his3A3' his3AS' duplication. The cdc9 mutation causes a 30-fold increase in HIS3+ recombinants (34), whereas coupling of the radJOA mutation with the cdc9 mutation resulted in approximately a 7-fold decrease in the rate of recombination (Table 5). We have previously shown a similar effect of the radlA mutation on recombination in cdc9 mutants (34). Thus, both the RADI and RADIO genes are involved in mediating the high level of recombination in cdc9 mutants. Intrachromosomal mitotic recombination between the duplicated his4 heteroalleles is not affected by the radlOA mutation. We next examined whether the radlOA mutation affects the recombination of a his4 duplication. In this duplication, the his4 genes are arranged on chromosome III in the order his4C pBR313 his4A LEU2 (10), and there are two point mutations in either the his4A or the his4C gene. In RAD+ strains carrying the duplication, about 75% of HIS4+ recombinants arise by gene conversion and about 25% arise by reciprocal recombination (10). The rad52 mutation abolishes gene conversion and has no effect on reciprocal recombination (10), whereas the radiA mutation has no effect on the formation of HIS4+ recombinants (34). Table 6 shows the effect of various excision repair-defective mutations and of the rad52A mutation on recombination of the his4 duplication. In the radS2A mutant, HIS4+ recombinants were formed at 40% the rate in the RAD+ strain. In contrast, the radJOA mutant or other excision repair-defective mutants showed some elevation in the rate of formation of HIS4+ recombinants. These observations are similar to those in a previous study which examined heteroallelic recombination in various excision repair-defective mutants (39).

TABLE 5. Effect of the radJOA mutation on mitotic recombination of the his3A3' his3A5' duplication in cdc9 mutant strains at 25°C No. of HIS3' recombinants/104 viable cells in culture: Mean ± SD recombiStrain Genotypea nation rate (1o-4)

RS117-4A RS144/1-SB

RADW CDC9+b

RS117-11B RS117-11B/iOA

cdc9-2b radJOA cdc9-2

radJOA

1

2

3

7.2 0.35 207 21

6.0 0.65 188 24

6.2 0.95 199 46

6.5 0.65 198 30

± 0.64 ± 0.30 ± 9.54 ± 13.7

a The radJOA cdc9-2 strain was constructed by disrupting the RADIO gene in the RS117-11B strain; therefore, the cdc9-2 and radJOA cdc9-2 strains are isogenic. b Data are from Schiestl and Prakash (34).

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TABLE 6. HIS4+ recombinants in RAD+ and rad strains carrying the his4C pBR313 his4A duplication Strain

Genotype

LP2752-4B LP2752-4B/11A LP2752-4B/10A LP2752-4B/2A LP2752-4B/4A

RADW radlA

LP2752-4B/7A LP2752-4B/23A RS22-8B/52A a

b

radlOA rad2A rad4A rad7A rad23A rad52A

Mean no. of viable cells/culture

3.9 7.0 9.8 5.6 9.2 1.27 9.0 3.96

Median no. of recombinants/culture

Mean no. of recombinants arising/culturea

Recombination rateb (10-6)

836 2,320 3,955 1,960 5,060 3,480 3,198 295

136 330 527 284 660 470 438 55.7

4.7 5.4 5.1 7.2 3.7 4.9

x 107

x x x x x x x

107 107 107 107 108 107 107

3.5

1.4

Determined by the method of the median (13). Mean number of recombinants arising per culture/mean number of viable cells per culture.

Integration of linear DNA molecules and circular plasmids into homologous genomic sequences is impaired in the radlOA mutant. Plasmids lacking S. cerevisiae origin of replication sequences transform by integration into homologous chromosome sequences; however, such integration events occur quite infrequently. Introduction of a double-strand cut in the yeast DNA sequence on the plasmid increases transformation frequency by several orders of magnitude, and integrations occur in the region of homology in the yeast chromosome (24). The study of Orr-Weaver et al. (24) and another study (32) with linear DNA molecules have clearly indicated that double-strand breaks are highly recombinogenic in S. cerevisiae.

Table 7 shows the transformation frequencies observed in the RAD1, radlA, radJOA, and rad52A strains with the integrating circular plasmid, the linearized plasmid, and a double-stranded DNA fragment. The rad52A mutation almost abolished transformation with all of these DNA substrates (34; Table 7). The radJOA mutation lowered the transformation efficiency ninefold with the circular uncut plasmid and about fivefold when this plasmid was cut with a restriction enzyme to target homologous integration in the yeast genome. The efficiency of targeted integrations by a linear DNA fragment was reduced 40-fold by the radJOA mutation. The effects of the radJOA mutation on integrative recombination were very similar to those reported previously for the radlA mutation (34; Table 7). Meiotic recombination is not affected by the RADi and RADIO genes. To determine whether the RADl and RADIO genes affect meiotic recombination, we examined intrachromosomal recombination of the his3A3' his3Ai' duplication and recombination of ade2 heteroalleles located on homologous chromosomes in isogenic RAD+IRAD+, radlAlradlA,

and radlOA&radJOzA diploid strains following sporulation. The frequencies of recombinants formed in the mitotic and meiotic cells are shown in Table 8. In diploids, as in haploids, the radJOA and radlA mutations lowered the frequencies of mitotic HIS3' recombinants three- to fivefold. The frequencies of meiotic HIS3+ recombinants, however, were not affected by the radlA and radJOA mutations. The radlA and radJOA mutations also did not affect the recombination of ade2 heteroalleles in mitosis or meiosis.

DISCUSSION The principal finding in this paper is that the excision repair gene RADIO is required for several types of mitotic recombination events. The radJOA mutation caused a decline in the rate of formation of HIS3+ recombinants in cells carrying an intrachromosomal duplication of the his3 gene in which each his3 gene bears a terminal deletion at the 3' or the 5' end (33). For the excision repair genes examined, only the radiA and radJOA mutations lowered recombination of the his3 duplication, whereas deletion mutations in the RAD2 gene (34) and in the RAD4, RAD7, and RAD23 genes (Table 2) resulted in a twofold elevation in the incidence of HIS3+ recombinants. Like the radlA mutation (34), the radJOA mutation lowered recombination of the his3 duplication synergistically when present in combination with the radS2A mutation, indicating that RADIO and RAD52 affect recombination via different pathways. The similar rates of formation of HIS3+ recombinants in the radiA, radiOA, and radiA radJOA mutants indicate that the RADI and RADIO genes function in the same recombination pathway. The RADI and RADIO genes appear almost identical in their effects on mitotic recombination. Deletion mutations in

TABLE 7. Transformation of RAD+, radlA&, radlOA, and rad52A strains with circular and linear plasmids and DNA fragmentsa No. of transformants/,ug of DNA (% with respect to 2,um value) for strain:

DNA

YEplacl95 (2p.m)b pSP56 (uncut plasmid)c pSP56 (ds cut plasmid)d pDG95 (ds DNA fragment)e

LP2752-4B/H+ (RAD+)

LP2752-4B/H+ (radlA)

LP2752-4B/H+ (radJOA)

XS803-3A (rad52A)

11,000 (100) 18 (0.16) 4,500 (41) 1,098 (10)

21,400 (100) 2.6 (0.012) 974 (4.6) 44 (0.21)

24,750 (100) 4.6 (0.018) 1,852 (7.5) 61 (0.24)

4,700 (100) 0.3 (0.006) 0.2 (0.004)