of mutations of saccharomyces cerevisiae acts by dna lesions … · 2002-07-08 · the rad6 rad6...

Copyright 0 1990 by the Genetics Society of America

The SRS2 Suppressor of rad6 Mutations of Saccharomyces cerevisiae Acts by Channeling DNA Lesions Into the RAD52 DNA Repair Pathway

Robert H. Schiestl,**’ Satya Prakash* and Louise Prakasht

*Department of Biology, University of Rochester, Rochester, New York 14627 and tDepartment o f Biophysics, University of Rochester School of Medicine, Rochester, New York 14642

Manuscript received October 24, 1989 Accepted for publication December 22, 1989

ABSTRACT rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage

induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6A) mutations and show that they also suppress the y-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of y-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6A is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6A SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

T HE RAD6 gene of Saccharomyces cerevisiae is required for a variety of cellular functions. rad6

mutants are highly sensitive to DNA damaging agents including UV, ionizing radiation, and alkylating agents (HAYNES and KUNZ 1981; GAME 1983). In rad6 mutants, DNA strand discontinuities, formed during DNA replication in the newly synthesized DNA strand across from non-coding lesions, are not repaired (PRAKASH 1981). rad6 mutants are defective in mutagenesis induced by a variety of DNA damaging agents (PRAKASH 1974; LAWRENCE and CHRISTENSEN 1976; LAWRENCE 1982) and are also defective in meiotic recombination and sporulation (GAME et al. 1980; MONTELONE, PRAKASH and PRAKASH 1981). In addition, rad6 mutants grow poorly and have reduced plating efficiency.

RAD6 encodes a protein of 172 amino acids of 19.7 kD, which contains a highly acidic carboxyl terminus in which 20 of the 23 amino acids are acidic (REY- NOLDS, WEBER and PRAKASH 1985). Deletion of the polyacidic domain of RAD6 causes a defect in sporulation but not in the DNA repair and mutagenesis functions (MORRISON, MILLER and PRAKASH 1988). RAD6 is a ubiquitin conjugating (E2) enzyme which has been shown to conjugate ubiquitin to histones H2A and H2B in vitro ( JENTSCH, MCGRATH and VAR- SHAVSKY 1987; SUNG, PRAKASH and PRAKASH 1988).

Chapel Hill, North Carolina 27599. I Present address: Department of Biology, University of North Carolina,

of page charges. This article nust therefore be hereby marked “aduertisement” T h e publication costs of this article were partly defrayed by the payment

in accordance with 18 U.S.C. $1734 solely to indicate this fact.

Genetics 1 2 4 817-831 (April, 1990)

T o identify DNA repair genes that can substitute for the absence of RAD6 function, we have isolated suppressors of the UV sensitivity of a rad6A strain in which the entire genomic RAD6 gene has been deleted. Such suppressor mutations could arise in genes which, when overexpressed, replace the missing RAD6 activity. For example, increased amounts of another ubiquitin conjugating enzyme, such as that encoded by the CDC34 gene (GOEBL et al. 1988), might substitute for the missing RAD6 function. One could also obtain bypass suppressor mutations which act by providing access to an alternate pathway that can substitute for the blocked pathway. Suppressor analysis has been immensely useful in the identifica- tion of new recombination genes in Escherichia coli. CLARK and his colleagues isolated the sbcA and sbcB suppressors of the recombination deficiency in recBC mutants (BARBOUR et al. 1970; KUSHNER et al. 197 1). A new ATP independent exonuclease activity (exo- VIII) is overproduced in sbcA mutants (KUSHNER, NAGAISHI and CLARK 1974), and loss of exonuclease I activity occurs in sbcB mutants (KUSHNER et al. 197 1). Isolation of mutations that cause recombination deficiency in the recBC sbcB strain revealed the existence of the RecF pathway of recombination that can substitute for the RecBC recombination pathway in the sbcA or sbcB mutant background (HORII and CLARK 1973; SMITH 1988). The RecE, RecF, RecJ, RecN, RecO, RecQ and R u v genes participate in the RecF recombination pathway (HORII and CLARK 1973; SMITH 1988).

All the suppressors of the rad6A mutation we isolated are alleles of one locus and they are allelic to the

818 R. H. Schiestl, S. Prakash and L. Prakash

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previously isolated SRS2 suppressor of rad6-1 (LAW- RENCE and CHRISTENSEN 1979), an amber mutation of the RAD6 gene (REYNOLDS, WEBER and PRAKASH 1985). We show that SRS2 suppresses both the UV and y-ray sensitivity of rad6A strains. Previously, SRS2 was reported to suppress only the UV sensitivity of rad6 mutants (LAWRENCE and CHRISTENSEN 1979). SRS2 also suppresses the growth defect in rad6A strains to a limited extent; however, the UV mutagenesis and sporulation defects of the rad6A mutant are not suppressed. From our observations, we conclude that SRS2 acts by channeling unrepaired DNA lesions remaining in rad6A mutants into the RAD52 DNA repair pathway. We suggest that SRS2 is involved in the activation of the RAD52 DNA repair pathway and discuss possible ways by which this could occur.

MATERIALS AND METHODS

Strains and media: The E. cola strain HBlOl was used for plasmid propagation. The yeast strains used are listed in Table 1. Growth, minimal, and sporulation media were prepared as described previously (SCHIESTL and WINTERS- BERCER 1982; SHERMAN, FINK and HICKS 1986).

Transformation and other procedures: Large scale plasmid isolation from E. coli, E. coli transformation and electro- phoresis of DNA were performed according to MANIATIS, FRITSCH and SAMBROOK ( 1 982). Isolation of DNA fragments from agarose gels was carried out with the Geneclean Kit obtained from BIO 101, La Jolla, Calif. and used as rec- ommended by the supplier. A modification of the boiling method was used for small scale plasmid preparations from E. coli (HATTORI and SAKAKI 1986). Transformation of yeast was carried out by treating intact cells with lithium acetate (ITO et al. 1983) to promote DNA uptake.

Isolation of SRSZ mutations: Strains SX46A/6A and LP2752-4B/6A were subcultivated several times to accu- mulate suppressors of the rad6A mutation. Thereafter, single colonies were screened for their UV sensitivity, and six independent isolates were obtained, two in strain SX46A/ 6A, which were designated SUSl (suppressor of UV sensitivity) and SUSP, and four isolates in the LP2752-4B/6A background, designated SUS3 through SUS6. After it was established that all suppressor mutations were allelic to one another, as well as to SRS2 (suppressor of radsix), which was originally isolated by LAWRENCE and CHRISTENSEN (1979), the alleles were renamed SRS2-9 through SRS2-14.

Irradiation of cells: Cells were grown in liquid YPAD for two to three days at 30° , sonicated to disperse clumps, washed in distilled water, collected by centrifugation, and appropriate dilutions plated onto solid YPAD medium. UV irradiation was as described in PRAKASH and PRAKASH (1977). After UV irradiation, the plates were incubated at 30" for three to four days in the dark to avoid photoreactivation. For y-irradiation, cells were suspended in sterile distilled water and irradiated with a cobalt-60 source at a dose rate of 9 kilorad (kr) per min. Cells were then plated onto YPAD medium, incubated for 3-4 days, and survivors counted.

Determination of UV induced mutation frequencies: Single colonies were incubated in liquid YPAD medium for 48 hr, cells were sonicated to disperse clumps, collected by centrifugation, and washed with distilled water. Appropriate dilutions were plated onto synthetic complete (SC) medium for determination of the fraction of surviving cells, SC-

820 R. H. Schiestl, S . Prakash and L. Prakash

ARG+CAN medium for determination of can2' forward mutation frequency, and SC-LYS medium, for determination of LYS2+ reversion frequency. Plates were irradiated with UV light ( s e ~ above), incubated in the dark to avoid photoreactivation, and colonies counted after incubation of plates for 3 to 5 days at 30".

Deletion of RAD genes. Genomic deletions of different RAD genes (radA) were made by deleting the entire or most of the open reading frame (ORF) of these genes using the one step gene disruption method (ROTHSTEIN 1983).

The genomic RAD2 gene from position +129 to +2911 in the ORF (MADURA and PRAKASH 1986) was replaced with the URA3 gene after digestion of plasmid pKM55 (constructed by Kiran Madura) with BamHI and BglII, and transformation of yeast with this DNA fragment to Ura+.

A 2.5-kbHindI1I-BamHI fragment from pSCW218 (MOR- RISON, MILLER and PRAKASH 1988) was used to delete the genomic RAD6 gene from position -44 to +566 (REYNOLDS, WEBER and PRAKASH 1985) and replace it with the URA3 gene. This deletes the entire RAD6 protein coding region.

A 7.1-kb SalI-EcoRI RAD9 containing fragment obtained from plasmid pRR330 was used for replacement of the genomic RAD9 sequence from positions +1166 to +3546 within the ORF (SCHIESTL et al. 1989). A 6.7-kb RAD28 containing fragment from plasmid pJJ240 was used to delete the genomic RAD28 gene from position +132 to +1484 within the ORF (JONES, WEBER and PRAKASH 1988) and replace it with the 3.8-kb BamHI-BglII fragment with the URA3 gene flanked by direct repeats of the Salmonella hisG DNA from plasmid pNKY51 (ALANI, CAO and KLECKNER 1987).

The genomic RAD51 gene was replaced with the LEU2 gene. This was carried out by transformation of yeast cells with an XbaI-PstI fragment from plasmid pAM50 (provided by MARI AKER and R. MORTIMER) in which a StuI-HpaI fragment of the RAD51 region was replaced by a HpaI-NruI fragment from YEpl3 containing the LEU2 gene.

The genomic RAD52 gene was replaced with TRP2 to produce rad52-8, henceforth termed rad52A in this paper. This was carried out by BamHI digestion of plasmid pSM21 (provided by DAVID SCHILD and R. MORTIMER), and transformation of yeast cells with the BamHI fragment in which the TRP1 gene had been inserted at the BglIl site in the RAD52 ORF.

The genomic RAD54 gene was replaced with the LEU2 gene. This was done by transformation of yeast cells with a Bgl II-StuI fragment of plasmid pSM31 (provided by DAVID SCHILD and R. MORTIMER), in which the BamHI fragment containing most ot the open reading frame of RAD54 was replaced by a BglII fragment containing the LEU2 gene.

The genomic RAD57 gene was replaced with the LEV2 gene. Plasmid pSM51 (provided by DAVID SCHILD and R. MORTIMER), was digested with SacI and yeast cells trans- formed with the 4.5-kb SacI fragment in which a 0.5-kb PvuII-Sal1 fragment of the RAD57 region had been replaced by a 2-kb LEU2 fragment.

Deletion of each RAD gene was confirmed by complementation analysis with known rad mutations.

RESULTS

Suppression of the radiation sensitivity of the radfih mutation: Spontaneously arising mutations that suppress the UV sensitivity of the rad6A mutation were isolated in two strains deleted for the entire protein coding region of RAD6. Single colonies which showed a higher degree of UV resistance than the

rad6A mutant were isolated from strains SX46A/6A and LP2752-4B/6A. The mutants were initially termed SUS (suppressor of the UV sensitivity). SUSl and SUS2 mutations were isolated in strain SX46A/ 6A and SUS3 to SUS6 were isolated in strain LP2752- 4B/6A.

Figure 1 shows the UV and 7-ray sensitivities of rad6A strains carrying the suppressor mutations (rud6A SUS) and of the isogenic RAD+ and rad6A strains. The rad6A SUS strains display a much higher degree of UV resistance than the rad6 mutant. How- ever, the UV resistance of rad6A SUS strains was still below that of the RAD+, and UV survival curves showed a resistant tail (Figure 1A) which is typical for y-ray but not of UV survival curves. The suppressor mutations restored RAD+ levels of y-ray resistance to the rad6A mutation (Figure 1 B).

Allelism of the suppressor mutations: Since all SUS mutations are semidominant or dominant (see below), their allelism could not be determined by complementation analyses; therefore, we examined this by tetrad analysis. Since the suppressor mutations did not suppress the sporulation deficiency of the rad6A strains (see below), the rad6A strain carrying a suppressor was crossed to a RAD+ strain carrying a different suppressor mutation. For obtaining RAD+ SUS strains, the rud6A SUS strains were first crossed to a RAD+ strain. Putative RAD+ SUS strains were then crossed to a rad6A mutant strain, and the presence of the SUS mutation verified by tetrad analysis. These crosses established that the SUS phenotype is caused by a single mutation which shows Mendelian inheritance. The results of crosses between the RAD+ SUS strains and the original rad6A SUS isolates in different combinations are shown in Table 2. If any two SUS mutations were unlinked, then '/s of all segregants or '/i of the rud6A segregants should have resulted in UV and y-ray sensitive spore colonies. In most crosses, all segregants were UV and y-ray resistant, indicating that all SUS mutations are allelic to one another (Table 2). Only two crosses gave one radiation sensitive spore colony each. These segregants may have arisen by meiotic gene conversion between the different SUS mutant alleles.

The previously isolated SRS2 suppressor of the rad6-1 point mutation was reported to suppress the UV but not the y-ray sensitivity of rad6-1 (LAWRENCE and CHRISTENSEN 1979). To examine the allelism of the SRSB-1 mutation with the SUS mutations, the SRS2-1 mutation was outcrossed from the original strain F629 into the RAD+ background, and the presence of the SRS2-1 allele verified by crossing to a rad6A strain followed by tetrad analysis. We observed that the SRSB-1 mutation not only suppressed the UV sensitivity of the rad6A strain as reported previously (LAWRENCE and CHRISTENSEN 19'79), but, in contrast

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FIGURE 1.-Suppression of the radiation sensitivity of the rad6A mutation. Survival after U V irradiation (A) and after y-irradiation (B) of RAD', rad6A, rad6A SUS1, rad6A SUS2, rad6A SUS3, rad6A SUS4, rad6A SUS5, rad6A SUS6 strains. Strains, LP2752-4B R,4D+ (O), LP2752- 4B rad6A (0), LP2752-4B rad6A SUS3 (. .O.. .), LP2752-4B rad6A SUS4 (X), LP2752-4B rad6A SUS5 (O), LP2752-4B rad6A SUS6 (U), were used and all of these strains are isogenic. Strain SX46A rad6A SUS1 (A), and strain SX46A rad6A SUSP (A) are also isogenic. The SX46A RAD+ and LP2752-4B RAD+ strains, and their rad6A derivatives showed the same sensitivity to UV and y-rays.

to that study, SRS2-1 also completely restored y-ray resistance to rad6A up to the RAD+ level, much like the SUS mutations (Figue 1B). The RAD+ SRS2-1 mutant, when crossed to the rad6A SUS4 mutant, produced only UV resistant spores, establishing that both suppressors are allelic to one another. Therefore, we conclude that all SUS mutations are alleles of SRS2 (Table 2), and have renamed the SUSl to SUS6 mutations as SRS2-9 through SRS2-14, respectively.

Suppression of the radiation sensitivity of the radl8A mutation by SRS2: Like rad6 mutants, rad18 mutants are highly sensitive to UV, y-rays and to other DNA damaging agents (HAYNES and KUNZ 198 1; LAWRENCE 1982), and are defective in post- replication repair of UV damaged DNA (PRAKASH 1981). The rad6 and rad18 mutations show epistasis, since the radiation sensitivity of the r a d 6 A r a d l 8 A double mutant is the same as that of the rad6A or the r a d l 8 A single mutants (JEFF JONES, unpublished observations). To examine whether the SRS2 mutation also suppressed the radiation sensitivity of the r a d l 8 A

mutation, a r a d l 8 A mutant strain was crossed to a RAD+ SRS2 strain, and the UV and y-ray sensitivities of the resulting r a d l 8 A S R S 2 segregants determined. SRS2 suppressed the UV and y-ray sensitivities of the r a d l 8 A mutation (Figure 2), and the UV survival curve of the r a d l 8 A S R S 2 mutant also shows a resistant tail (Figure 2A).

Suppression of the slow growth phenotype of rad6A by SRS2: rad6A strains grow at a much slower rate than RAD+ strains. Competition experiments with the isogenic RAD+ us. rad6A and rad6A us. rad6A SRS2 strains were carried out to determine whether SRS2 suppresses the slow growth phenotype of the rad6A mutant (Figure 3). Cells were grown for over 30 generations, samples taken periodically, and the frequency of rad6A cells us. RAD+ or rad6A SRS2 cells determined. Figure 3A shows that the initial frequency distribution of 5% RAD+ and 95% rad6A cells was reversed after 35 generations, indicating a signif- icant growth advantage of the RAD+ strain over the rad6A strain. Figure 3B shows that the initial fre-

a22 R. H. Schiestl, S. Prakash and L. Prakash

TABLE 2

Allelism of SUS and SRS2 suppressors of rad6A mutation

Cross

No. of tetrads showing No. of spore colonies showing UV and y-ray

4 surviving 3 surviving spores spores Resistance Sensitivity

rad6A SUSl X RAD+ SUS3 rad6A SUSl X RAD+ SUS4 rad6A SUSl X RAD' SUS5 rad6A SUSP X RAD' SUSl rad6A SUSP X RAD' SUS4 rad6A SUS3 X RAD' SUS4 rad6A SUS3 X RAD+ SUS6 rad6A SUS4 X RAD+ SUS6 rad6A SUSS X RAD+ SUS4 rad6A SUS4 X RAD+ SRS2-I

2 8 7 9 3 13 10 13 9

13

7 6 8 2

10 2 4 2 5 1

29 50 51 42 42 58 52 58 51 55

0 1 0 0 0 0 0 1 0 0

~

All original rad6A SUS mutant isolates were crossed to RAD+ strains. RAD+ spores from tetrads showing two radiation sensitive mutants were crossed to a rad6A mutant to verify the RAD+ SUS phenotype. The RAD* SUS strains were then crossed to the original rad6A SUS isolates and in most cases, the rad6A gene could also be followed with the URA3 gene that was used for deleting the RAD6 gene. Strain F629, the original rad6-1 rad18-2 SRS2-1 isolate (LAWRENCE and CHRISTENSEN 1979) was crossed to a RAD+ strain, the RAD+ SRSP-1 phenotype verified through backcrossing to a rad6A strain, and the RAD+ SRSP-I strain crossed to the rad6A SUS4 strain. If the suppressors were unlinked, Ya of the spore colonies should show a radiation sensitive phenotype.

quency distribution of approximately 1 % rud6A SRS2- 14 and 99% rad6A srs2+ cells gradually changed to -30% rad6A SRS2-14 and 70% rad6A srs2+ cells. Similar results were obtained with the SRS2-12 and the SRS2-13 alleles. These observations indicate that rad6A SRSB strains have a growth advantage over the rad6A srs2+ strain; however, the growth rate of the rad6A SRS2 strain is still much lower than that of the RAD' strain. UV mutagenesis and sporulation defects of the

rad66 mutation are not suppressed by SRS2: UV induced forward mutations from canavanine sensitivity (CANl') to resistance (canl') and reversion of Zysl- 1 to LYSl+ were examined in the RAD+, RAD+ SRS2, and rad6A SRS2 strains (Table 3). canl' forward mutations and LYSI' revertants were induced by UV light in the RAD+ SRS2 strain as in the RAD+ strain. The rad6A SRS2 mutant strains, however, were completely defective in UV induced mutagenesis (Table 3). Thus, the SRS2 mutation does not suppress the lack of UV mutagenesis in rad6 mutant strains. The extreme UV sensitivity of rad6 mutants makes it quite difficult to unambiguously establish their non UV- mutable phenotype, and this has resulted in several reports that rad6 mutants are UV revertible [see LAWRENCE (1982) for a review]. The fact that UV mutagenesis is not decreased in the RAD+ SRS2 mutants and that rad6A SRS2 strains are much more UV resistant than the rad6A strains has made it possible to clearly establish the requirement of RAD6 in UV mutagenesis.

T o determine whether the sporulation defect of rad6AlradbA homozygotes is overcome by the SRS2 mutation, isogenic diploids homozygous or heterozy-

gous for RAD+ and radbA, as well as for SRS2 and srs2+, were constructed by crossing the appropriate LP2752-4B and SX46A strains. The diploids with at least one RAD+ copy sporulated with an efficiency of about 40%, whereas no sporulation occurred in any of the radbAlrad6A strains, regardless of the presence of the SRSB mutation. The SRSB mutation also had no effect on sporulation and spore viability in RAD+/ RAD+ strains.

Dominance of the SRS2 mutation: To determine whether the SRSB mutation acts in a recessive or dominant manner, a set of isogenic diploid strains all homozygous for rad6A were constructed. Strain RS53, homozygous for srs2+, strains RS54 and RS55, heterozygous for SRS2-9/srs2+ and SRS2-12/srs2+, respectively, and strain RS56, homozygous for SRS2, were constructed by intercrossing the rad6A parent strains and the original rad6A SRS2 isolates with each other. The UV and y-ray sensitivities of these diploids were determined (Figure 4). The SRSB mutation shows semidominance for the suppression of UV sensitivity of the rad6A mutation (Figure 4A) and dominance for the suppression of y-ray sensitivity (Figure 4B).

MATaIMATa diploid cells are more resistant to radiation, particularly to y-rays, than the haploid cells. The high degree of resistance of diploid cells is due to the presence of homologous chromosomes and the MATaIMATa mating type (GAME 1983). The survival curves in Figure 4 show that even though the rad6Al rad6A diploids are more radiation resistant than the rad6A haploids (Figure I) , the rad6Alrad6A SRS2I SRS2 diploids are no more radiation resistant than the rad6A SRS2 haploid strains (compare Figures 1 and

A

Yeast DNA Repair

B

823

0 1 0 20 30 4 0

UV, J/m2

L

.001 1 . 1 . 1 . 1

0 1 0 20 30 4 0

Gamma Ray, Kr FIGURE 2.--Suppression of the radiation sensitivity of the radl8A mutation. Survival after UV irradiation (A) and after y-irradiation (B)

of RAD+, radl8A and r a d l 8 A SRS2-9 strains. Strains LP2752-4B RAD+ (O), LP2752-4B r a d l 8 A (0), and RS171-IA radl8A SRS2-9 (0) were used. Similar levels of suppression of radiation sensitivity of the radl8A mutation have been observed in other r a d l 8 A SRS2-9 strains.

4). The SRS2 mutation confers a low level of sensitivity to UV and ionizing radiation in RAD+/RAD+ diploids (Figure 4).

Suppression of the rud6A mutation by SRS2 is dependent on the RAD52 DNA repair pathway: One possible mechanism for the suppression of the rad6A mutation by SRS2 is channeling of the DNA damage into a different DNA repair pathway(s). T o examine this possibility, we determined the radiation sensitivity of strains carrying rad6A SRS2 mutations in combi- nation with other radiation sensitive mutations. Mu- tations of genes in the RAD52 epistasis group render cells slightly UV sensitive and highly 7-ray sensitive, and many of these mutants are defective in double strand break repair and in genetic recombination (GAME 1983; ORR-WEAVER and SZOSTAK 1985). To determine the role of genes in the RAD52 epistasis group in suppression, we constructed deletion mutations of different RAD52 group genes in the rad6A SRS2 strain, and examined the radiation sensitivity of these strains. Deletion of the RAD52 gene in a RAD+ strain renders the strain only very slightly UV sensitive

(Figure 5A). Deletion of the RAD52 gene in a rad4A SRS2 strain, however, caused a complete loss of suppression of the UV sensitivity of the rad6A mutation by SRS2, as indicated by the similar UV sensitivity of the rad6A SRS2 rad52A strain to that of the rad6A or the rad6A rad52A strains (Figure 5A). An apparent loss of suppression could have occurred by synergistic enhancement of the UV sensitivity in the rad52A SRS2 double mutant, since both the rad52 and SRS2 mutants are slightly UV sensitive. However, the rad52A SRS2 strain was somewhat more UV resistant than the SRS2 strain. Therefore, the loss of suppression in the rad6A SRS2 rad52A strain is not due to increased UV sensitivity of the rad52A SRS2 mutant (Figure 5A). The SRS2 mutation made the cells slightly 7-ray sensitive (Figure 5B), whereas both the rad52A and rad6A mutants are highly sensitive to y-ray irradiation. In contrast to the high degree of suppression of y-ray sensitivity of the rad6A mutation, SRS2 suppressed the y-ray sensitivity of the rad52A mutation only slightly. Deletion of the RAD52 gene in the rad6A SRS2 strain caused a complete loss of suppression as


A B

0 1 0 20 30 4 0

Generations

2o t 0 0 1 0 2 0 3 0

Generations FIGURE 3.-Competition experiment between RAD+ and rad6A (A) and rad6A and rad6A SRS2-14 (B) strains. Isogenic strains LP2752-

4B RADf (O), LP2752-4B rad6A (0), and LP2752-4B rad6A SRS2-14 (W) were used. In (A), liquid YPAD medium was inoculated with a logarithmic culture of RAD+ and rad6A cells at a density of approximately 1 X lo4 and 1 X lo6 per r n l , respectively. In (B), liquid YPAD medium was inoculated with a logarithmic culture of about 1 X l o4 cells of the rad6A SRS2-14 strain and 1 X lo6 cells of the rad6A strain per 1111. Every 12 hr, the cells were counted, again diluted to IO6 cells per r n l in fresh YPAD medium, and incubated for another 12 hr. This regimen of diluting the cells i n fresh medium every 12 hr was repeated for up to 3 days. At each dilution step, cells were plated onto three plates of' solid YPAD. To determine the genotype of the colonies, after 3 days of growth at 30", the colonies were replica plated to solid YPAD and irradiated with 30 kr of y-irradiation. After another three days of incubation at 30", y-ray resistant (RAD+ or rad6A SRS2-14 colonies) as well as y-ray sensitive (rad6A) colonies were counted. The frequencies of different genotypes were plotted against the number of generations, which was calculated as log x / l o g 2 where x represents the fold increase in cell number within a given period.

TABLE 3

UV induced CANIS to c a d ' forward mutations (A) and l y s l -1 to LYSl+ reversion (B) in RAD+, RAD+ SRS2 and rad6A SRS2 strains

CAN/, ' to can/' mutations ( er 10' viable cells) UV dose (Jfm')

Stwin Genotype 0 5 10 15 20 30

I\. LP2752-4B RAD+ 0.66 (100)" 0.85 (90) 9.5 (93) 28 (70) 68 (39) KS2OI-lC RAD+SRS2-9 2.4 (100) 11.7 (62) 52.1 (32) 132 (19) 176 (18.7) 275 (11.7) KS22- 1 A rad6A SRS2-9 1.6 (100) <2.7 (3.1) 6.3 ( 2 . 7 ) 5.1 (1.7) <13 (0.66) L.P2752-4B/GASRS rad6ASRS2-12 0.6 (100) 0.47 (35) 0.67 (12.4) 0.43 (5.3) 1.53 (1.3)

l y s l - / to I . Y S / + revertants (per 10' viable cells) U V dose ( J / d )

0 5 10 15 20 30

B. 1.P2752-4B RAD+ 0.8 (100)" 18 (90) 78 (93) 196 (70) 359 (39) KS20I-lC RADf SRS2-9 <1 (100) 10.6 (62) 31 (32) 85 (19) 90 (18.7) 108 (1 1.7) LP2906-8D R.4D+ SRS2-14 0.7 (100) 28 (100) 73 (93) 153 (77) 310 (42) KS22- 1 A rad6A SRS2-9 <0.1 (100) -3.8 (5.2) t 4 . 8 (2.8) <5.5 (2.5) C7.5 (1.9) (16.6 (0.86) lal'27.52-4B/6ASRS radbASRS2-12 0.4 (100) <0.2 (25.5) - 4 . 2 (20.3) <0.5 (8.7) <0.6 (6.6)

Str;tins 1.1'2752-48 and 12P2752-4B/6ASRS are isogenic. Frequencies of forward mutations in the C A N l " gene to canl' and reversion of 1y1-1 LO LYSI' were determined. The LP2752-4B/6A strain is too UV sensitive to be tested at the same doses. However, extensive experiments were carried out at 0-3 J/m' of U V light which did not result in induced mutations. N o more than 10' cells were spread onto plates for selection of mutants. This was done to avoid shielding from U V irradiation, which would result in an artifactual increase i n the frequency o f mutants. In one experiment with the rad6A strain, we observed a spontaneous frequency of 0.3 LYS'I' revertants per lo' cells. \Iherc;th a t 3 J / m 2 no LYSI' revertants were found among a total of 1.3 X 10' survivors, which were spread on 40 Petri dishes. Thus, the frrcluency of LYS'I+ reversion in the rad6A strain is less than 0.78 per 10' cells. I n the RADf strain, the frequency of spontaneous LYSI+ rcvertm~ts \vas 1.2 per 10' cells and at 3 J/nI', it was 9 per 10' cells.

" Pet-cenr survival is given in parentheses.

indicated by similar y-ray sensitivities of rad6A Because of the requirement of the RAD52 gene for rad52A and rad6A rad52A SRS2 strains (Figure 5B). suppression, we next examined whether any other

Yeast DNA Repair 825

A B

.0001 0 1 0 20 30 4 0

UV, J/m2

100

10

1

s 9 .1 - .- >

cn x L

.01

.0001 0 1 0 20 30 4 0

Gamma Ray, kr FIGURE 4,”Dominance of the SRSZ mutation. Survival after UV (A) and 7-irradiation (B) of isogenic diploid strains. Strain RS53 rad6Al

rad6A srs2’ (0). strain RS56 rad6Alrad6A SRSZ-Y/SRSZ-IZ (O), as well as an average of two experiments with the strains RS54 radbAlrad6A SRSZ-Y/srsZ+, and strain RS55 rad6A/rad6Asrs2+/SRS2-12 (0). Survival of the RAD+/RAD+ strain LP-3053 (A) and of the RAD+/RAD+ SRS2- 12/SRS2-9 strain LP-3054 (U) is also shown. Strains RS53, RS54, RS55, RS56 and LP-3053 are isogenic (see Table l ) , and strain LP-3054 is closely related.

gene(s) in the RAD52 DNA repair pathway are also needed. Even though the rad5lA and rad57A mutants are barely UV sensitive, rad6A SRS2 strains carrying the rad5IA or the rad57A mutation were as UV sensitive as the rad6A strain (Figure 6A). The y-ray survival curves in Figure 6B show that the rad5lA and rad57A mutants are very y-ray sensitive and that introduction of the rad5lA or the rad57A mutation in the rad6A SRS2 strain caused a complete loss of suppression. Thus, the RAD51 and RAD57 genes are also required for suppression of radiation sensitivity of the rad6A mutation.

The RAD54 gene was deleted in a RAD+ strain and the resulting rad54A strain was crossed to a rad6A SRS2 strain. The diploid strain RS151 (Table 1) was sporulated and tetrads analyzed. The UV and y-ray sensitivities of the segregants showed that RAD54 was also required for suppression (data not shown). A rad55-2 strain was crossed to a rad6A SRS2 strain, the diploid RS 1 16 (Table 1) sporulated, and the radiation

sensitivity of the segregants examined. The results show that the RAD55 gene greatly affects the suppression of UV sensitivity (Figure 7A) and is required for suppression of y-ray sensitivity of the rad6A mutation (Figure 7B). We could not determine the role of the RAD50 gene in suppression since we found that rad6A rad50A double mutants were inviable, regardless of the presence of the SRS2 mutation. From these studies, we conclude that the RAD51, RAD52, RAD54, RAD55 and RAD57 genes are required for suppression of radiation sensitivity of the rad6A mutation by SRS2.

Excision repair pathway is not required for suppression of the rad6A mutation by SRS2: T o determine the role of excision repair genes in suppression of radiation sensitivity conferred by the rad6A mutation, we examined the effect of the excision repair defective rad2A mutation in suppression. The rad2A mutants are highly UV sensitive. The SRS2 mutation suppressed the UV sensitivity of the rad2A mutation only slightly. The rad2A rad6A double mu-

826

A

R. H. Schiestl, S. Prakash and L. Prakash

B

.0001 0 1 0 20 30 4 0

UV, J/m2 Gamma Ray, Kr

tants are extremely UV sensitive due to the blocking of excision repair (RESNICK and SETLOW 1972) and post-replication repair pathways (PRAKASH 198 1). The rad2A rad6A SRS2 strain was also very UV sensitive, but at all UV doses, this strain was less UV sensitive than the rad2A rad6A strain (Figure 8A). Neverthe- less, the high level of UV sensitivity imparted by the rad2A mutation made it difficult to assess the role of the RAD2 gene in suppression of the UV sensitivity of the rad6A mutation by SRS2. Since the rad2A strain is not y-ray sensitive, we could unambiguously determine the role of RAD2 in suppression of y-ray sensitivity of the rad6A strain by SRS2. As shown in Figure 8B, the rad2A mutation imparts some additional y- ray sensitivity to the rad6A mutant strain, whereas the rad2A rad6A SRS2 strain is as y-ray resistant as the RAD+ SRS2 and the rad2A SRS2 strains. These results clearly show that the RAD2 gene is not required for suppression of y-ray sensitivity of the rad6A mutation.

SRS2 is not allelic to RAD52, RAD51, RAD55, RAD57 and CDC34: Since deletion mutations of RAD52 and of other genes in this pathway result in loss of suppression of radiation sensitivity of rad6A by SRS2 and since SRS2 shows dominance, we examined the possibility that SRS2 is an overexpression mutation of RAD52, or of another gene in this pathway. A

FIGURE 5.-Requirement of the RAD52 gene in suppression. Survival after UV irradiation (A) and after y- irradiation (B) of RAD+, rad52A, radbA, rad6A SRS2-9, rad6A SRS2-9 rad52A, rad6A rad52A, RAD+ SRS2-9, and rad52A SRS2-9 strains. Strains, RS22-8B RADf (O), RS22-8B/52A rad52A (A), RS22-14B rad6A (0), RS22-14BI52A rad6A rad52A (O), RS22-2A radhASRS2-9 (A), RS22-2Al 52A rad6A SRS2-9 rad52A (W), LP2873-12B RAD' SRS2-9 (- -0- -), and strain LP2873-12B152A RAD+ SRS2-9 rad52A (- -0- -) were used. Genotypes were confirmed by complementation analysis.

strain, which was obtained by rad6A SRS2 rad52A deleting the RAD52 gene in a rad6A SRS2 strain, was crossed to a RAD+ strain, the diploid was sporulated, and tetrads analyzed. We found that about 50% of the rad6A spore colonies were suppressed, indicating that SRS2 and rad52 are not allelic. No suppressed rad6A spore colonies are expected if SRS2 and rad52 were allelic, since incorporation of the rad52A mutation in the rad6A SRS2 strain would inactivate the SRS2 gene.

T o test the possibility that SRS2 was an overexpression mutation of another gene in the RAD52 group, a rad6A SRS2 strain was crossed to the rad54A, rad55- 2 and rad57A strains, and segregants were back- crossed to rad6A, rad54, rad55 and rad57 mutants to examine allelism and suppression. Results of these crosses indicated that SRS2 is not an allele of the rad54, rad55, or rad57 genes.

The CDC34 gene shows homology to RAD6 (GOEBL et al. 1988). Both genes encode ubiquitin conjugating (E2) enzymes, and these enzymes have been shown to ubiquitinate histones H2A and H2B in vitro ( JENTSCH, MCGRATH and VARSHAVSKY 1987; SUNG, PRAKASH and PRAKASH 1988; GOEBL et al. 1988). We checked whether overexpression of CDC34 might have caused the suppression of rad6A. T o determine whether

A

Yeast DNA Repair

B

827

UV, J/m2

1001

10

1

s 9 .I - .- >

rn 3 L-

.o 1

.001

.0001 0

CDC34 is allelic to SRS2, the diploid RS 169 (Table 1) was sporulated and tetrads analyzed. Sporulation of strain RS169 was achieved by introducing the RAD6 plasmid pTB227 (MORRISON, MILLER and PRAKASH 1988) into RS169 by transformation and subsequently selecting for its loss on medium containing 5-fluo- roorotic acid. The results showed that SRS2 is not allelic or linked to cdc34 and that CDC34 does not affect suppression of rad6A.

DISCUSSION

SRS2 suppresses the DNA repair but not the UV mutagenesis and sporulation defects of the rud6A mutation: rad6 mutants are defective in DNA repair, induced mutagenesis and sporulation. We isolated six independent spontaneously arising suppressors of a rad6A mutation that is missing the entire protein coding region of RAD6. Genetic analyses have shown that all of the suppressors are alleles of a single gene. We also examined whether these suppressor mutations were allelic to a previously identified SRS2 suppressor of a rad6 point mutant (LAWRENCE and CHRIS- TENSEN 1979). Our observations clearly indicate that the newly isolated suppressor mutations are allelic to SRS2 and thus have designated the alleles identified by us as SRS2-9 to SRS2-14. Unlike the previous

t I I I

1 0 20 30 4 0

Gamma Ray, Kr

FIGURE 6.-Requirement of the RAD51 and RAD57 genes in suppression. Survival after U V irradiation (A) and after y-irradiation (B) of RAD', rad516, rad57A, rad6A, rad66 SRS2- 12, rad6A SRS2-12 rad516, rad66 SRS2-12 rad576 strains. Strains, RS22- 8 B RAD+ (O), XS803-3A/51A rad516 (A), XS803-3A/57A rad576 (X), RS22-14B rad66 (O), RS151"LC rad6A SRS2-12 (A), RS151-2C/516 rad66 SRS2-12 rad513 (M), and strain RS152-5C/576 rad6A SRS2-12 rad57A (0) were used. Genotypes were confirmed by corllplemet1t;rtioll m a l y - sis.

observations which showed that SRS2 suppressed the UV but not the y-ray sensitivity of rad6 mutants, our results show a suppression of both UV and y-ray sensitivity of rad6A mutants. SRSB also suppressed the UV and y-ray sensitivity of r a d l 8 A mutants. The rad6A mutants display slow growth and the SRS2 mutation suppressed this defect to a limited extent. T o determine the role of SRS2 in suppression of the UV mutagenesis defect of rad6 mutants, we examined the frequency of UV induced C A N I S to canl' forward mutations and lysl-1 to LYSl+ revertants in the RAD+, SRS2, and rad6A SRS2 strains. UV mutagenesis occurred normally in the SRS2 strain, whereas we observed no UV mutagenesis in rad6A SRS2 strains. Previously, the rad6-1 SRSB strain was found to be defective in UV mutagenesis of the cyc l -9 allele (LAW- RENCE and CHRISTENSEN 1979). All of these observations indicate that SRS2 does not suppress the UV mutagenesis defect of rad6 mutants. SRS2 also did not suppress the sporulation defect of rad6Alrad6A mutants. In the RAD+ background, SRS2 confers a slight degree of radiation sensitivity, and the sensitivity of RAD+ SRS2 and RAD+/RAD+ SRS2ISRS2 strains is about the same. The SRS2 mutation has no effect on sporulation or spore viability in the RAD+ background.

828

A

R. H. Schiestl, S. Prakash and L. Prakash

B

0 0 1 0 20 30 4 0

UV, Jm2

.01 ' I I I I

0 1 0 2 0 3 0 4 0

Gamma ray, Kr FIGURE 7.-lnvolvement of the RAD55 gene in suppression. Survival after UV irradiation (A) and after ?-irradiation (B) of RAD+, rad6A,

rad55-2, rad6A SRS2-9, rad6A rad55-2, rad6A SRS2-9 rad55-2 strains. Strains, RSl16-3A RAD' (O), RSl16-1A rad55-2 (A), RSl16-3C rad6A (0), RSl16-1C rad6A SRS2-9 (A), RS116-2D rad6A rad55-2 (O), and strain RSl16-3D rad6A SRS2-9 rad55-2 (W) were used. The genotypes of segregants were established by backcrosses to the rad6A and rad55 mutant strains. The radiation sensitivity in this experiment was examined at 23"C, since rad55-2 mutants show sensitivity at this temperature (LOVETT and MORTIMER 1987).

SRS2 channels DNA lesions into the RAD52 DNA repair pathway: Since SRS2 suppressed the DNA repair defect but not the mutagenesis and sporulation defects of the rad6A mutant, we examined the possibility that SRSZ acts by channeling unrepaired DNA lesions in the rad6 mutant into other DNA repair pathways. Genes in the RAD52 epistasis group are required for DNA double strand break repair and for genetic recombination (RESNICK and MARTIN 1976; GAME et al. 1980; PRAKASH et al. 1980; GAME 1983; ORR-WEAVER and SZOSTAK 1985). To examine the role of these genes in suppression, we coupled the rad6A SRS2 mutations with a mutation in one of the RAD52 group genes. Mutations in the RAD52 gene or in other genes of this group affect UV sensitivity only marginally. However, coupling of a rad52A, rad5lA, rad54A, rad55-2 or rad57A mutation into a rad6A SRS2 strain increased the UV sensitivity to a level similar to that of the strain carrying the rad6A mutation and a mutation in one of the RAD52 group genes. Thus, suppression of UV sensitivity of the rad6 mutant by SRS2 is mediated by genes in the RAD52

epistasis group. A similar genetic analysis for 7-ray resistance showed the requirement of the RAD52 epistasis group genes. Since SRS2 suppresses both the UV and 7-ray sensitivity of rad6A mutants, we did not expect the excision repair pathway to affect suppression. The genes in the RAD3 epistasis group are involved in excision repair of DNA damaged by UV light and by other agents that distort the DNA helix (REYNOLDS and FRIEDBERG 1981; WILCOX and PRAKASH 198 1 ; MILLER, PRAKASH and PRAKASH 1982a, b). Mutations in these genes render cells highly sensitive to UV light but not to 7-rays. T o determine whether the excision repair pathway contributes to suppression, we examined the UV and y-ray sensitivities of a rad6A SRS2 strain carrying the excision defective rad2A mutation. Our observations indicate that RAD2, and by inference the excision repair pathway, is not involved in suppression.

Dominance of SRS2: The SRSZ suppressor displays semidominance for the suppression of UV sensitivity and dominance for the suppression of y-ray sensitivity of rad6A mutants. Since suppression requires the

Yeast DNA Repair

A

829

.0001 1 ;

0 0 2 4 6 8 1 0

UV, J/m2

B

*0°01 0 0 L 1 0 20 30 4 0

Gamma Ray, Kr FIGURE 8.-RAD2 is not required for suppression. Survival after UV irradiation (A) and after 7-irradiation (B) of RAD’, rad2A, rad6A,

rad2A, RAD+ SRS2-9, rad6A SRS2-9, rad2A SRS2-9, rad6A SRS2-9 rad2A strains. Strains, RS22-8B RAD+ (O), LP2752-4B/2A rad2A (m), LP2752-4B/6A rad6A (0), LP2873-12B RAD+SRS2-9 (A), LP2873-12B12A rad2A SRS2-9 (X), RS22-2A rad6A SRS2-9 (A), RS47-5A rad2A rad6A (O), and strain RS84-1C rad2A rad6A SRS2-9 (- -0- -) were used. The genotypes were established by backcrosses to the rad2A and rad6A mutant strains.

genes in the RAD52 epistasis group, SRS2 may be an overproduction mutation of one of the RAD52 group genes. However, results of genetic analyses indicate that SRS2 is not an allele of the RAD51, RAD52, RAD55 and RAD57 genes. We also examined the possibility that SRS2 was an overexpression mutation of the CDC34 gene which, like the RAD6 gene, encodes a ubiquitin conjugating (E2) enzyme. Both RAD6 and CDC34 enzymes have been shown to ubiquitinate histones H2A and H2B in vitro (JENTSCH,

MCGRATH and VARSHAVSKY 1987; SUNG, PRAKASH and PRAKASH 1988; GOEBL et al. 1988). Our results indicate that SRS2 is not an allele of CDC34.

Eukaryotic cells, including S. cerevisiae, treated with DNA damaging agents arrest cell division in the G2 stage of the cell cycle to ensure repair of damaged DNA before entry of cells into mitosis [see WEINERT and HARTWELL (1 988) and SCHIESTL et al. (1 989) for references]. The RAD9 gene of S. cerevisiae is required for this GP arrest phenomenon (WEINERT and HART- WELL 1988; SCHIESTL et al. 1989). SRS2 could act by

extension of the G2 stage of the cell cycle. This could be achieved by overproduction of the RAD9 protein or of another protein(s) involved in G2 inhibition. We have observed no obvious lengthening of the G2 phase in the rad6A SRS2 mutants and also have observed no effect of the rad9A mutation on suppression of the radiation sensitivity of the rad6A mutation by SRS2 (results not shown); therefore, we consider this possibility unlikely.

Suppression in haploids and diploids: The resistant tail observed in UV survival curves of rad6A SRS2 and r a d l 8 A SRS2 haploid strains suggests that suppression of UV sensitivity of rad6 and rad18 mutants is dependent on the presence of G2 cells in the culture. Both rad6 and rad18 mutants are defective in post-replication repair of gaps that are formed across from the non-coding DNA lesions during DNA replication (PRAKASH 1981). Since rad6 and rad18 mutants are proficient in spontaneous and radiation induced mitotic recombination (BORAM and ROMAN 1976; MONTELONE, PRAKASH and PRAKASH 1981;


HAYNES and KUNZ 1981), it has been suggested that the RAD6 and RAD18 genes carry out post-replication repair in a nonrecombinational manner (PRAKASH 1981). The increased UV resistance of G2 cells in rad6 SRS2 and rad18 SRSB mutants may indicate that SRS2 acts by channeling DNA lesions into a recombination pathway involving sister chromatids.

In MATaIMATa diploid cells, the level of suppression of the UV and y-ray sensitivities of the rad6A strain by SRSB is less than in haploids (compare Fig- ures 1 and 4). This is interesting since particularly for y-rays, MATaIMATa diploid cells show a much higher degree of resistance than haploid cells. The lower level of suppression in radbAlrad6A SRS2ISRS2 diploids than in rad6A SRS2 haploids could arise from a/ a repression of SRS2 transcription.

Possible mechanism of action of SRSZ: The dominance of suppression by SRS2 suggests that SRS2 could be an overexpression mutation. In SRS2, the unrepaired gaps and breaks left in the newly synthesized DNA strand in the rad4 and rad18 mutants might be channeled more efficiently into a recombination pathway involving sister chromatids. SRS2 may either regulate the synthesis of a nuclease(s), or it may itself encode a nuclease that converts single strand gaps and breaks into double strand breaks which can then be processed by recombination proteins encoded by the RAD52 group genes. SRS2 could thus be re- sponsible for increasing the amount of a rate-limiting enzyme in the recombination process. The low efficiency of the RAD52 recombinational repair pathway in srs2+ cells can then be ascribed to limiting levels of this nuclease. In E. coli, the recombination deficiency of recB recC mutants can be suppressed by the sbcA and sbcB mutations. In sbcA mutants, increased levels of exonuclease VI11 channel recombination interme- diates into an alternate recombination pathway con- trolled by genes in the RecF pathway (BARBOUR et al. 1970; KUSHNER, NACAISHI and CLARK 1974; HORII and CLARK 1973).

If the SRS2 and RAD52 gene products acted sepa- rately and sequentially, then one might expect the SRS2 rad52 double mutant to exhibit a higher level of radiation sensitivity than the SRS2 or rad52 single mutants, because double strand breaks generated by SRS2 would go unrepaired in the SRS2 rad52 mutant. The observation that the SRS2 rad52 double mutant is no more sensitive to radiation than the single mutants, however, can be explained if we assume that SRSZ nuclease is a component of a multi protein complex that includes the products of the RAD52 and of other genes in this epistasis group, and conversion of single strand breaks into double strand breaks occurs only when the entire functional complex is present.

Rather than affecting the synthesis of a nuclease,

SRS2 could increase the amount or activity of another rate limiting enzyme, as for example, an activity required for the displacement of DNA strands and annealing of complementary DNA strands. The RecA protein of E. coli promotes a variety of ATP dependent reactions, including homologous pairing of complementary single strands of DNA and formation of long heteroduplex DNA regions (SMITH 1988). SRS2 could regulate the synthesis of or encode such a protein.

Another possibility is that SRSB affects the level, stability, or activity of proteins encoded by the RAD52 groups genes. We have not observed any effect of SRS2 on RAD52 or RAD54 transcript levels (M. BANK- MANN and L. PRAKASH, unpublished observations). Increased activity of a protein required for post trans- lational modification of recombination proteins could also account for suppression by SRS2; for example, SRS2 may encode or control the levels or activity of a protein kinase. The CDC7 gene of S. cerevisiae is required for the initiation of mitotic S phase (HART- WELL 1973) and encodes a protein kinase (PATTERSON e t al. 1986). The CDC7 gene is also involved in UV mutagenesis, since cdc7 mutants exhibit a reduction in mutagenesis induced by DNA damaging agents (NJAGI and KILBEY 1982), and increased levels of CDC7 lead to an increase in UV mutagenesis (SCLA- FANI et al. 1988). Elevated levels of a protein kinase in SRS2 mutants may cause further activation of the RAD52 recombination pathway. Cloning and analysis of the structure of the SRSB gene and characterization of the biochemical properties of its encoded protein should help elucidate the mechanism of suppression.

This work was supported by U. S. Public Health Service grants GM19261 and CA41261 from the National Institutes of Health.

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Communicating editor: G. S. ROEDER

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