Current Genetics

Current Genetics (1983) 7:85-92

© Springer-Verlag 1983

Isolation and Characterization of Yeast DNA Repair Genes I. Cloning o f the R A D 5 2 G e n e

David Schild 1, Boyana Konforti 1,3, Carl Perez 1, Warren Gish 2, and Robert Mortimer 1 1 Department of Biophysicsand MedicalPhysicsand Donner Laboratory 2 Department of MolecularBiology,Universityof California,Berkeley,CA 94720, USA

Summary. The RAD52 gene of Saccharomyces cerevisiae has previously been shown to be involved in both recombination and DNA repair. Here we report on the cloning of this gene. A plasmid containing a 5.9 kb yeast DNA fragment inserted into the BamH1 site of the YEpl3 vector has been isolated and shown to complement the X-ray sensitive phenotype of the rad52-1 mutation. The rad52-1 cells containing the plasmid form larger colonies than similar cells having lost the plasmid. This plasmid has been shown not to complement either the U.V. sensitivity or the recombination defect of the E. coli recA mutation. From the insert various fragments have been subcloned into the YRp7 and YIp5 vectors. Integration events of two of the subclones have been genetically mapped to the chromosomal location of RAD52, indicating that the structural gene has been cloned. A 1.97 kb BamH1 fragment subcloned into YRp7 in one orientation complements the rad52-1 mutation, while the same fragment in the opposite orientation fails to complement. Various other subclones indicate that a BgllI site, within the BamH1 fragment, is in the RAD52 gene. This BgllI site has been deleted by Sl-nuclease digestion and the resulting deletion inactivates the RAD52 gene. BAL31 deletions from one end of a 1.9 kb Sall.BamH1 fragment have been isolated; up to 0.9 kb can be deleted without loss of RAD52 activity, indicating that the RAD52 gene is approximately 1 kb or less in length. Key words: Yeast - RAD52 - Cloning - S1 and BAL31 Deletions

Offprint requests to: D. Schild 3 Current address: Department of Biology, York University,

Downsview, Canada

Introduction

Several different DNA repair pathways have been identified in the yeast Saccharomyces cerevisiae, including excision repair, error-prone associated repair, photoreactivation and a presumptive recombinational repair pathway (reviewed by Lemmont 1980; Haynes and Kunz 1981). The genes in the the RAD50 to RAD5 7 epistasis group are thought to be involved in recombinational repair, since mutations in some of these genes decrease both recombination and repair (Game and Mortimer 1974; Haynes and Kunz 1981). The best characterized gene in this group is RAD52. The rad52-l mutation was originally identified as causing extreme sensitivity to X-rays and slight sensitivity to ultraviolet light (U.V.) (Resnick 1969). Strains carrying this mutation have also been shown to be partially or completely defective in meiosis and sporulation (Game and Mortimer 1974; Strike 1978; Prakash et al. 1980; Game et al. 1980), meiotic recombination (Strike 1978; Game et al. 1980; Prakash et al. 1980), mitotic gene conversion (Strike 1978; Game et al. 1980; Prakash et al. 1980; Saeki et al. 1981 ; Jackson and Fink 1981), double strand break repair (Ho 1975; Resnick and Martin 1976), homothallic switching (Malone and Esposito 1980), maintenance of chromosome stability, resulting in chromosome loss (Mortimer et al. 1981; Weiffenbach and Haber 1981), and integration oflinearized plasmids (Orr-Weaver et al. 1981). In addition, vegetative cells with the rad52-1 mutation have been shown to have elevated rates of spontaneous mutation (Von Borstel et al. 1971) and rad52/rad52 diploids in meiosis have been shown to accumulate single strand breaks during premeiotic DNA synthesis (Resnick et al. 1981). If one assumes that double strand breaks and other X-ray induced DNA lesions are repaired by a recombinational mechanism (Resnick 1976), it is possible to account for

86

D. Schild et al.: Cloning of the Yeast RAD52 Gene

most of the phenotypes associated with the rad52-1 mutation b y its defect in recombination. Although many phenotypes of the rad52-1 mutation have been observed, and similar phenotypes have been observed in mutations of several other genes of the R A D 5 0 - R A D 5 7 group, no protein has been identified as the product of any of these genes. In order to studythese genes at the molecular level, we have isolated plasmids containing them (Schild et al. 1982). The cloning of E. coli DNA repair genes, such as RECA and L E X A , has already been very important in isolating and characterizing the primary products of these E. coli genes and in studying their transcriptional regulation (reviewed by Rupp 1982). Several of the yeast genes involved in excision repair (J. F. Lemontt and F. W. Larimer, personal communication; Naumovski et al. 1982; Yasui and Chevalier 1982), and in error-prone DNA repair (Lemontt et al. 1982; Prakash et al. 1982) have recently been cloned and are being studied. Here we report on the cloning of the yeast R A D 5 2 gene and in a subsequent paper (Calderon et al. 1983) we report on cloning other genes of the R A D 5 0 group. The cloned genes will be used to determine whether these genes are transcriptionally regulated and if so, what their transcriptional inducers are. The cloned R A D 5 2 gene is currently being used to identify and isolate the R A D 5 2 gene product and to determine whether this gene might code for an essential function.

pH 5 and 10 mM EDTA) and 100 #1 ofzymolyase 60,000 (Kirin Brewery) at 1 mg/ml was added. Cells were incubated at 30 °C until 80-90% spheroplasting had occurred (1/2 to 11/2 h). Spheroplasting was monitored both microscopically and by loss of turbidity when spheroplasts were diluted into 10% SDS. Spheroplasts were washed 2 times in 1 M sorbitol and once with 1 M sorbitol, 10 mM CaC12 and 10 mM Tris-HClpH 7.4. Spheroplasts were resuspended in 0.5 ml of this buffer and divided into 100 #1 aliquots. 5-20 #g plasmid DNA was added to ceils and the mixture was incubated 15 min at room temperature. 1 ml of 20% polyethylene glycol (PEG-4,000, Sigma), 10 mM CaC12 and 10 mM Tris pH 7.4 was added to each sample and incubated 15 min at room temperature. After gently spinning the spheroplasts out of the PEG solution, they were resuspended in 150 ~zlof 1 M sorbitol, 33.5% YEPD and 50 mM CaC12. 50 #1 aliquots were plated in yeast regeneration agar onto minimal dropout plates as described previously (Hinnen et al. 1978).

Materials and Methods

Survival Curves. Logarithmically growing cultures in liquid synthetic complete media, lacking either leucine or tryptophan (depending on the plasmid used), were diluted, plated in triplicate on YEPD plates and then treated with different doses of X-ray. Final colony counts were taken after one week at 30 °C.

Strains. The transformable yeast strain XS95-6C (MAT~ rad52-1 ura3.52 leu2-3 leu2-112 trpl and his3.Lxl) was used in most of the experiments. It was constructed by first crossing g160/2d (MATa rad52-1 ade2-1 arg4 arg9 trpl his5 leul-1 ilv3 and leu2) from the Yeast Genetic Stock Center (Berkeley, Cal.) to RH218 (MA Ta trp1-289) and then back crossinga resulting MA Ta tad52-1 trpl derivative twiceto DBY746 (MA Ta trp1-289 ura3-52 his3-/,1 leu2-3 Ieu2-112) (similar to SHY strains in Botstein et al. 1980), kindly supplied by D. Botstein. The bacterial strains used were HB101 (CA600 leu- B 1 - thr- pro- lacz- S m - recA-, rBroB- SUI1) supplied by A. Clarke, JA300 (thr- leuB6- thithyA- trpCl l 7- hsrK- hsmK- strR) (Tschumper and Carbon 1980) supplied by John Carbon, and DB6507 (pyrF- in a HB101 background) supplied by D. Botstein. Plasmids. A bank of near random (Sau3A) yeast fragments cloned into the BamH1 site of the vector YEpI3 was kindly supplied by K. Nasmyth (Nasmyth and Tatchell 1980). YRp7 and Ylp5 were obtained from D. Botstein. Yeast Transformation. Yeast transformation was performed using a modified version of the method of Hinnen et al. (1978), as suggested by V. MacKay and R. Hitzeman. 100 ml of YEPD culture, grown to 2 x 107 cells/ml, was collected and washed in water. The resulting pellet was resuspended in 5 mlof S.E.D. (1 M sorbitol, 25 mM EDTA pH 8.0 and 50 mM dithiothreitol) and incubated 10 rain at 30 °C. After a wash in 10 ml 1 M sorbitol, cells were resuspended in S.C.E. (1 M sorbitol, 100 mM Na citrate

E. coli Transformation and Plasmid Preparation. E. coli transformation was performed as described by R. Davis et al. (1980). Small scale plasmid preparations were performed by the method of Holmes and Quigley (1981) and large scale isolations of plasmids were carried out using a protocol of Birnboim and Doly (1979) scaled up to 1 I by S. Conrad. Restriction, Ligation, $1 Nuclease Digestion andBAL31Digestion. All of the enzymes except BAL31 were obtained from Bethesda Research Laboratories and the B.R.L. suggested procedures were followed. BAL31 was obtained from New England Biolabs and their procedure was followed. X-ray Source. A Machlett OEG-60 X-ray source operated at 50 kV and 25 mA was used. The exposure rate was 240 R/s. The f factor for the exposure conditions used was 0.90 rad/R.

Ploidy Testing. The canavanine ploidy test (Schild et al. 1981) was used as a preliminary screen of transformants to differentiate haploids from cells of 2 N or greater ploidy. Viability and segregation of genetic markers after crosses to strains of known ploidy were used to confirm the ploidy of the haploid transformants and to differentiate diploid transformants from transformants of higher ploidy.

Results Cloning o f R A D 5 2 A leu2 rad52-1 yeast strain (XS95-6C) was transformed with YEp 13 (Broach et al. 1979) containing near random yeast DNA inserts (Nasmyth and Thatchell 1980) and Leu + transformants were selected. Approximately 2,000 Leu + transformant colonies were picked to master plates (50 per - l e u plate) and grown up for 2 days at 30 °C. They were then replica plated to YEPD plates and the replicas were treated with 50 krads o f X-ray. Two transformants showed significant X-ray resistance and these

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Fig. 1. Restriction map of YEpI3-[RAD52]. This plasmid consists of a 5.9 kb yeast Sau 3A fragment, containing the RAD52 gene, cloned into the BamH1 site of the YEp13 vector (pBR322, the yeast LEU2 gene and the part of the yeast 2 # plasmid which includes the origin of replication) (3). The (BamH1/Sau3A) sites can be cut by Sau3A but not by BamH1. The underlined BarnH1 sites and SalI sites were used for subcloning (see Fig. 3). The area in dashed lines represents the region presumed to contain RAD52 (see Discussion)

were tested further. Survival curves on these transformants demonstrated that they had near wild type survival after X-ray treatment (data not shown). Cells derived from these transformants which had lost their plasmid became simultaneously leucine auxotrophs and radiation sensitive. It was also evident that the colonies in which most of the cells retained the plasmid, being both Leu + and Rad +, were much larger ( 2 - 4 times as large after about 5 days at 30 °C) than colonies in which all the cells had lost the plasmid. This larger colony size was not noted when the tad52-1 mutant strain contained the YEpl3 vector alone or YEpl3 with other inserts. Diploids homozygous for rad52-1 with and without the RAD52 plasmid showed larger differences in colony size than similar haploids. Our observations indicates that rad52-1 mutant cells grow more slowly than identical cells containing the RAD52 gene on a plasmid. This is consistant with earlier observations in many laboratories that the rad52-i mutation is deleterious to normal growth of yeast cells and more deleterious to diploids than haploids. Plasmid DNA was isolated from the two Rad + transformants by transforming E. coli with a crude yeast DNA preparation and selecting for ampicillin-resistant E. coli colonies. From these E. coli transformants, plasmid DNA was isolated and restriction analyses carried out. One plasmid contained a 5.9 kb insert and the other one an insert slightly over 20 kb. Preliminary restriction

analysis of the two inserts was consistent with the smaller insert being part of the larger insert. Because of complications associated with obtaining large amounts of plasmid DNA and a detailed restriction map from the plasmid with the 20 kb insert, we concentrated on the plasmid containing the smaller insert. A restriction map of the smaller plasmid is shown in Fig. 1. Reintroduction of either of the "RAD52" plasmids into XS95-6C (rad52-1 leu2) resulted in a Leu + Rad + phenotype for all of the transformant colonies tested (100 for each plasmid). These results are consistent with the plasmids containing either the RAD52 gene or a suppressor of the rad52-1 mutation; experiments discussed below demonstrate that the smaller plasmid contains the RAD52 gene and not some type of suppressor. Survival Curves

Transformants were normally tested qualitatively for X-ray sensitivity by replica plating colonies or patches of cells to YEPD plates which were then treated with 50 krads and incubated at 30 °C. Wild type cells and rad52-1 cells containing the YEp 13-[RAD52] plasmid always had high enough survival to give near confluent growth, while replicas of tad52-1 cells lacking the plasmid were completely killed. This test has been shown to work equally well with haploids, diploids and triploids. More quantitative survival curves were mn on some of the transformants. In order to interpret these curves and use suitable control strains, it was necessary to determine the ploidy of the transformants. A genetic cross of the original transformant to a standard haploid strain yielded a hybrid strain which upon sporulation exhibited high spore viability and segregation ratios indicating that it was a tetraploid, not a diploid as had been expected. The segregation ratios also demonstrated that the original transformant was a triploid, presumably due to spheroplast fusion during the transformation procedure. Genetic analysis of several of our retransformed XS95-6C clones yielded some haploid and some diploid transformants. Survival curves on both haploid and diploid transformants, both with and without the plasmid, were performed. Figure 2 shows such a curve for a diploid transformant and shows that near wild type survival is observed. Even though the cells prior to X-ray treatment were grown in media lacking leucine, between 20% and 30% of the cells at the time of treatment gave rise to totally leucine minus colonies. These cells which have lost the plasmid can account for much of the difference between the top two curves in Fig. 2 since the bottom curve (rad52/rad52) shows that cells without the plasmid are extremely X-ray sensitive. X-ray resistance has also been observed in rad52-1 haploid cells with the plasmid. These results indicate that the plasmid is restoring near complete radiation resistance to rad52-1 mutant cells.

88

D. Schild et al. : Cloning of the Yeast RAD52 Gene

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In order to test whether the yeast RAD52 plasmid could complement the E. coli recA mutation, the YEp13[RAD52] plasmid was transformed into the recA mutant E. coli strain HB101, kindly supplied by A. Clarke. After 30 J/m 2 o f U.V. light, survival dropped to about 1 0 - 6 for both HB101 and HB101 with the RAD52 plasmid, indicating no effect of the RAD52plasmid on recA sensitivity to UV. The wild type RECA control (strain AB1157) showed only about a 10 -2 drop in survival following the same treatment. The RAD52 plasmid was also shown not to complement the recombination defect associated with the recA mutation (G. Warren, personal communication).

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Fig. 2. X-ray survival curves of a wild type diploid ($395D-1), a rad52-1 homozygous diploid containing the YEp13-{RAD521 plasmid (rad52/rad52 [RAD52]) and the same tad52-1 homozygous diploid, which had lost the plasmid (rad52/rad52)

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D. Schitd et al.: Cloning of the Yeast R A D 5 2

89

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FD, First-division segregation; SD, Second-division segregation; PD, Parental ditype; NPD, Nonparental ditype; T, Tetratype YRp7-INT1 and -INT2 are independent integration events of the plasmid Y R P 7 - A 4 - S a l l - [ R A D 5 2 ] and YIp5-INT3 and -INT4 are independent integration events of Y I p 5 - [ R A D 5 2 ] . The centromere marker used in the crosses with gRp7-INT and L Y S 7 was U R A 3 , and in the crosses with YIp5-INT and T S M 0 1 1 1 was T R P 1 Map distance and standard error analyses were calculated according to the method of Snow (1979) Published map distances (Mortimer and Schild 1980) F. Boutelet and F. Hilger, personal communcation

BamH1 fragment in YRp7-C9 had been mutated or modified either by growth in E. coli or by the various biochemical manipulations during the subcloning procedures. We can rule this out because we have subcloned the 1.97 kb fragment from YRp7-C9 back into YRp7 in the same orientation as YRp-C2.[RAD52] and this subclone now has RAD52 activity. The BamH1-BgllI subclones all lacked activity, indicating that the BgllI site is probably either in the structural gene or between the gene and its regulatory region.

Integration Analysis of RAD Plasmids In order to prove that we had cloned the RAD52 gene, rather than a suppressor of rad52-1, we examined integration events of two of the subclones. It has been demonstrated that integration of plasmids in yeast occurs via homologous recombination (Hicks et al. 1978; Scherer and Davis 1979; Orr-Weaver et al. 1981). Therefore, integration events at the chromosomal location of RAD52 would indicate that the plasmid contains the RAD52 region. Four independent spontaneous integra-

tion events were found in which the YRp7-A4-Sa/I[RAD52] plasmid had integrated into the genome of XS95-6C (~x tad52-1 trpl leu2 his3 ura3). Two of these integration events were at the chromosomal location of TRP1 (data not shown) and two integrated at the chromosomal site of RAD52. The chromosomal location of these integrants was established by two types of crosses. 1) These integrants were crossed to an a rad52-1 lys7 trpl strain with the ura3 centromere linked marker and asci from these crosses were dissected. The Rad ÷ Trp + phenotypes usually segregated together, as expected for an integrated plasmid containing both TRP1 andRAD52. Out of 196 individual segregants from these crosses however, there were three that were tad52 TRP1 and two that were RAD52 trpl; these presumably represent gene conversion events of some sort. The genetic analysis of these crosses (Table 1) indicates that both of these integrated plasmids are linked to L YS7 at a distance similar to the published distance betweenRAD52 andL YS7. The integrated plasmids were also both centromere linked, at a distance similar to the published distance between RAD52 and the centromere of chromosome XIII (Resnick 1968; Mortimer and Schild 1980). Examination of data

90 from individual asci clearly demonstrated that the plasmids had integrated on the opposite arm of chromosome XIII from LYS7. All of the data from these two crosses are consistent with the integration events having occurred at the chromosomal site of RAD52. 2) One of the integrants was also crossed to a Rad ÷ haploid of the opposite mating type; 10 asci from the resulting diploid (a/a rad52-1[YEp13.RAD52]/RAD52) were dissected. All eight asci with four viable spore colonies yielded 4 Rad÷: 0 rad- segregants and the two asci with three viable spore colonies yielded 3 Rad+:0 rad- segregants. This segregation pattern establishes that the integration event occurred at or very close to the RAD52 chromosomal site on chromosome XIII. If the YRp7-A4-Sall[RAD52] plasmid had not integrated at or very near the site of rad52-1 one would have expected some asci with 3 Rad+: 1 rad52-1 or 2 Rad+:2 rad52-1 segregations. The 1.97 kb BamH1 fragment has also been subcloned into YIp5, the URA3 integrating vector (Struhi et al. 1979; Botstein et al. 1980), and our results indicate that this plasmid also integrates at the chromosomal location of RAD52 on the left arm of chromosome XIII. When the strain XS95-6C (rad52-1 ura3 trpl, etc.) was transformed with the YIp5.[RAD52] plasmid, Ura ÷ transformants were selected. Two transformants were crossed to a strain containing ura3 and tsm0111, a new temperature sensitive mutation tightly linked to the centromere and on the right arm of chromosome XIII (F. Boutelet and F. Hilger, personal communication). The URA3 on the integrated plasmid now maps near tsm0111 (Table 1) at a distance consistant with the map positions of tsm0111 and RAD52. In the only ascus in which tsm0111 segregated second division the integrated plasmid segregated first division. Since tsm0111 is closer to the centromere, this one ascus indicates that the plasmid had probably integrated on the opposite arm from tsm0111, which is consisted with it integrating at the site of RAD52. Unexpectedly, the two original transformants and all of the URA3 segregants in these crosses were still mutant for rad52. This indicated that the 2 kb BamH1 fragment, which complements the rad52-1 mutation when on a replicating YRp7 plasmid, fails to complement when integrated with a YIp5 plasmid.

Deletion of BgllI Site The previously discussed subcloning experiments indicated that the BgllI site in the 1.97 kb BamH1 fragment was probably in the RAD52 gene. We decided to test this by constructing a four base pair deletion of the BgllI site using SI nuclease and determining if this deletion inactivated the gene. The 1.97 kb BamH1 fragment was first subcloned into pBR322 so that the BgllI site in the BamH1 fragment was the only BgllI site in the plasmid.

D. Schild et al. : Cloning of the Yeast RAD52 Gene This plasmid was restricted with BgllI, treated with S1 single-strand exonuclease, blunt-end ligated, recut with BgllI (to decrease transformation ability of plasmids still containing a BgllI site), and transformed into HB101. Mini plasmid preparations on the transformants showed that the BgllI site was missing from plasmids in most of the transformants. Following large scale preparations of two plasmids with independently derived deletions of BgllI, we subcloned these deletions into YRp7 in the proper orientation for RAD52 activity. When the deletions were introduced into yeast they failed to complement rad52-1 (Fig. 3). This demonstrates that the BgllI site is either in the structural gene or in a region essential for transcription or translation.

BAL31 Deletions In order to more precisely define where the RAD52 gene is located within the 1.97 kb BarnH1 fragment, we have isolated deletions into the insert from one end. Since the subcloning experiments (Fig. 3) and the BgllI deletion experiment discussed above indicated that the RAD52 region included the BgllI site, we isolated deletions into the insert from the side furthest away from the BgllI site (the left side in YRp7-C2-[RAD52], see Fig. 4a). Because the isolation of BAL31 deletions is simplified by starting from a restriction site that is unique in the plasmid, we first deleted the short SalI fragment of YRp7-C2[RAD52], using standard restriction and religation procedures. The resultant plasmid (YRp7.C2.2~al.[RAD52]) contains both unique Sall and BamH1 sites (Fig. 4a). This plasmid still complements the rad52-1 mutation, which is expected since the previously subcloned 3.3 kb Sall fragment, which lacks the short yeastBamH1 to SalI region, has RAD52 activity (Fig. 3). The YRp7-C2- ~SalI. [RAD52] plasmid was linearized by restriction with Sall and digested with BAL31 for various lengths of time from 15 min to 2 h. Most of the time points gave large deletions; all of the deletions we used were from the 15 and 30 min time points. Since BAL31 causes deletions in both directions from the Sail site, we restricted with PvulI before ligations so that we could later determine the length of the deletion within the RAD52 region. PvulI leaves blunt ends, but since BAL31 ends are frequently not blunt we increased the number of blunt ends by ftlling in some of the overhangs using the Klenow fragment of DNA PolI, prior to blunt end ligation. Following ligation, we transformed E. coli and used restriction analysis (PstI and BamH1 double digest) on mini plasmid preparations to screen for plasmids with different sized deletions up to about 1.5 kb. Large scale plasmid preparations were done on 24 plasmids and these plasmids were reintroduced into the rad52-1 yeast strain XS95-6C in order to score for RAD52 activity. The dele-

D. Schild et al.: Cloning of the Yeast RAD52 Gene Discussion

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Fig. 4A and B. BAL31 deletions. A The short Sall fragment from YRp7-C2-[RAD52] was removed before BAL31 deletions isolated; B BAL31 deletions were isolated from the SalI site into the RAD52 region. Line represents the length ofthedeleted segment. The left hand colomn is the designation of each deletion and its approximate length

tions which were reintroduced into yeast are diagrammed in Fig. 4b. The size of the deletions are approximations since agarose gel electrophoresis does not give exact lengths, but each deletion diagrammed did appear to be slightly different in length. Some deletions appeared to be of the same size and these duplications are not listed in Fig. 3b although the RAD52 activity results for these apparent repeats were consistent. Some of the sizes of the deletions might be overestimates ifBAL31 had actually digested much further in the SalI to PvulI direction and had deleted past the PvulI site. This seems unlikely for most of the deletions since the distance from SalI to PvulI is 1.4 kb and most of our deletions were 1 kb or shorter. The results for two deletions (G1 and 02) are inconsistent with the results from the other deletions; although they appeared to be short deletions they lacked activity. These cases probably represent longer deletions which appear shorter because of the addition of random short DNA fragments during ligation. The rest of the deletions are consistent with deletions of about 900 bases retaining RAD52 activity, while longer deletions inactivate the gene. Therefore, the RAD52 gene is probably at most 1 kb in length.

Recombinant DNA and gene cloning techniques have simplified the examination of many genes at the molecular level. For example, these techniques have been of great value in elucidating the role of RECA and other genes involved in DNA repair and genetic recombination in E. coli (reviewed by Rupp 1982). We are currently adapting these techniques for studying DNA repair and recombination in yeast. Here we report on the cloning of the yeast RAD52 gene and in a subsequent paper on the cloning of RAD51, RAD54 and RAD55 (Calderon et al. 1983). The RAD50 - R A D 5 7 genes are of particular interest because many mutations in this series affect both DNA repair and genetic recombination. TheRAD52 gene is the best characterized of these genes and, as mentioned earlier, the tad52-1 mutation has been shown to affect many aspects of DNA metabolism. The cloned RAD52 gene was isolated by first transforming a tad52-1 mutant strain and then screening transformants for resistance to X-rays. Since this plasmid, which complemented the X-ray sensitive phenotype of the tad52-1 mutation, could have either contained the RAD52 gene or some type of suppressor of tad52-1, we examined chromosomal integration events of plasmids containing the tad52-1 complementing insert. Since these plasmids frequently integrated at the chromosomal site of RAD52, presumably by homologous recombination, the insert appears to contain the structural RAD52 gene. From the original RAD52 plasmid, which had a 5.9 kilobase (kb) insert, we have subcloned an 2 kb BamH1 DNA fragment which also complemented the tad52-1 mutation. This fragment is unusual in that in one orientation it complements tad52-1, but it fails to complement the mutation when the insert is oriented in the opposite direction in the BarnH1 site of the tetracycline gene. Kenji Adzuma (personal communication), in the laboratory of Dr. H. Ogawa (Osaka University, Osaka, Japan), has independently isolated a 2.0 kb BamH1 fragment which complements tad52-1; this fragment appears to be identical to our 1.97 kb BamH1 fragment, since both share a common restriction map and his BamH1 insert exhibited the same orientation effect as our insert. K. Adzuma has sequenced much of the BamH1 fragment and has determined that the fragment lacks a yeast translational terminator sequence for the RAD52 gene, which results in the production of a fusion protein with the C-terminus coded for by pBR322 DNA. He explains the orientation difference by the fact that the fusion proteins resulting from the two orientations are of different sizes, with only the smaller one having RAD52 activity. Using BAL31 exonuclease we have determined that about 1 kb of the 2 kb BamH1 fragment can be deleted without destroying the ability of the plasmid to complement rad52-1. This indicates that the RAD52 gene is

92 probably about 1 kb in length, which is similar to I(. Adzuma's sequencing results (personal communication). Since the exact size o f the RAD52 gene is n o t known, it is possible that the RAD52 gene is larger than 1 kb if the protein contains a large non-essential C-terminus region or if the 1 kb fragment complements the rad52-1 mutation b y a type o f intragenic complementation. It is also possible that the coding sequence o f the gene is less than 1 kb in size if it contains an intron. Because the yeast rad52-1 mutation and the E. coli recA mutation b o t h affect recombination and repair, we tested whether our RAD52 containing plasmid could complement the recA mutation. We found no complementation of either the repair or recombination defect o f recA. This lack o f complementation could indicate that the RAD52 gene product can not functionally replace the RECA gene product. An alternate possibility is that the RAD52 protein is n o t produced in E. coli, either because o f problems with transcription, m-RNA processing, translation or post translational modifications, if they occur. Previous examinations o f many yeast genes have revealed that some complement mutations in the analogous E. coli gene, while other yeast genes do not. Our results cannot therefore be used to indicate that the RAD52 and RECA genes code for non-analogous proteins. Plasmids containing the RAD52 gene are currently being used to determine whether RAD52 is transcriptionally regulated and whether it codes for an essential function. Some o f the BAL31 deletions have created DNA fragments which presumably will only hybridize to the m R N A from RAD52. These are being used to directly determine the level o f RAD52 m R N A under different conditions. We have demonstrated, and others have observed previously, that the rad52-1 mutation is deleterious to normal growth. Since rad52-1 is the most sensitive allele o f RAD52 (Game and Mortimer 1974),it is possible that is is a slightly leaky allele and that RAD52 actually codes for an essential function. The four base deletions in the middle o f the RAD52 gene is being used to determine i f R A D 5 2 codes for an essential function.

Acknowledgements. We especially wish to thank Isabel Calderon, Rebecca Contopoulou and Michael Botchan for useful discussions during this research and Noel Fong and Tommy McKey for excellent technical assistance. This work was supported by N.I.H. grant GM07988 to D. S. and by the Office of Health and Environmental Research of the U.S. Department of Energy under Contract NO. DE-AC0376SF00098. References Birnboim HC, Doly J (1979) Nucleic Acids Res 7:1513-1523 Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis R (1980) Gene 8:17-24 Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121-133 Calderon IL, Contopoulou CR, Mortimer RK (1983) Curr Genet 7:93-100 Davis RW, Botstein D, Roth JR (1980) Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

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