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The Genetic Control of Direct-Repeat Recombination In Copyright 0 1989 by the Genetics Societyof America The Genetic Control of Direct-Repeat Recombinationin Saccharomyces: The Effect of rad52 and radl on Mitotic Recombination atGALlO, a Transcriptionally Regulated Gene Barbara J. Thomas’ and Rodney Rothstein Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York 10032 Manuscript received May 17, 1989 Accepted for publication September 8, 1989 ABSTRACT We have previously showndirect-repeat recombination events leadingto loss of a plasmid integrated at the GALlO locus in Saccharomyces cerevisiae are stimulated by transcription of the region. We have examined the role of two recombination- and repair-defective mutations, rad1 and rad52, on direct repeat recombination in transcriptionally active and inactive sequences. We show that the RAD52 gene is required for transcription-stimulated recombination events leading to loss of the integrated plasmid. Similarly, Gal+ events between the duplicated repeats that retain the integrated plasmid DNA (Gal+ Ura+ replacement events) are reduced 20-fold in the rad52 mutant in sequences that are constitutively expressed. Incontrast, in sequences that are not expressed, the rad52 mutation reduces plasmid loss events by only twofold and Gal+ Ura+ replacements by fourfold. We also observe an increase in disome-associated plasmidloss events in the rad52 mutant, indicative of chromosome gain. This event is not affected by expression of the region. Plasmid loss events in rad1 mutant strains are reduced only twofold in transcriptionally active sequencesand are not affected in sequences that are repressed. However, the radl and rad52 double mutant shows a decrease inplasmid loss events greater than the sum of the decreases in the rates of this event displayed by either single mutant in both constitutive and repressed DNA, indicating a synergistic interaction between these two genes. The synergism is limited to recombination since the radl rad52 double mutant is no more sensitive when compared with either single mutant in its ability to survive radiation damage. Finally, the recombination pathway that remains in the double mutant is positively affected by transcription of the region. ENETIC recombination in most organisms oc- orderingthese genes into genetic pathways(GAME G curs by multiple pathways. In prokaryotic sys- and MORTIMER1974; HAYNES and KUNZ198 1; KUNZ tems such as Escherichia coli or the bacteriophage X, and HAYNES198 I ; MALONE1983). the isolation and characterizationof mutations defec- The RAD52 gene was originallyidentified by a tive in various steps of the recombination process has mutation that confers sensitivity to x-rays (RESNICK contributed greatly tothe understanding of the mech- 1969). Based on physical analysis of irradiated cells, it anism of theseevents in vivo (reviewedin SMITH was concluded that the RAD52 gene product is nec- 1987). In the yeast Saccharomyces cerevisiae, mutants essary for the repair of double-strand breaks in the defective in meiotic or mitotic recombination or both DNA(Ho 1975; RESNICKand MARTIN 1976).In have been isolated by their sensitivity to various types addition,the RAD52 geneproduct is requiredto of radiation or by directrecombinational screens repair the double-strand lesion induced in HO-cata- (GAMEand MORTIMER1974; HAYNES and KUNZ1 ; 198 lyzed mating-type interconversion (MALONEand Es- KUNZand HAYNES 1981 ; ESPOSITOet al. 1984; HOL- POSITO 1980; WEIFFENBACHand HABER1981). De- LINGSWORTH and BYERS1989). Meioticrecombina- tailed analyses of specific mitoticrecombination events tion mutants have also been recovered in screensfor have shown differing effectsof the rad52 mutation in sporulation-defective cells (ESPOSITOand KLAPHOLZ different assay systems. In one case, direct-repeat re- 1981) or in screens for meiotic lethals (ENGEBRECHT ciprocal exchange events leading to loss of a plasmid and ROEDER1989). However, although some of these integrated at the HIS4 locus were found to be only mutations have been studied quite extensively in spe- slightly affected by a mutation in rad52 (JACKSON and cific assay systems, there has been little progress in FINK 1981). However, events described as gene con- versions weredramatically reduced (JACKSON and ’ Currentaddress: Department of Biological Chemistry,University of California, Los Angeles, California 90024. FINK 1981). Recombination events leading to loss of The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby“advertisement” marked a SUP4-o allele located within a clusterof the repeated in accordance with 18 U.S.C. 51734 solely to indicate this fact. element, delta, on chromosomeX were reduced 100- Genetics 123 725-738 (December, 1989) 726 B. J. Thomas and R. Rothstein fold by a mutation in the RAD52 gene (ROTHSTEIN, below) are recovered at nearly wild-type levelsin HELMSand ROSENBERG1987). Similarly, recombina- transcriptionally inactive rad52 cells, although this tion between small inverted repeats displays a 100- class of recombinant was previously shown to be ex- fold decrease in the rad52 mutant(WILLIS and KLEIN tremely sensitive to a mutation in rad52 (JACKSON and 1987). Recombination occurring by sister chromatid FINK1981). On the other hand, the rateof this event exchange is dependenton RAD52 (FASULLOand is reduced 20-fold in transcriptionally active cells in DAVIS1987). Integration of a gappedplasmid into the the rad52mutant, although transcriptiondoes not genome is blocked in therad52 mutant (ORR- affect the rate of this event in RAD wild-type strains. WEAVER,SZOSTAK and ROTHSTEIN198 1). Finally, In contrast to other studies (R. KEIL, personal com- directrepeat recombination events initiated by an munication), a mutation in RADl results in only a HO-induced double-strand-break also require a func- twofold reduction in plasmid loss events in expressed tional RAD52 gene (RUDINand HABER1988; NICKO- sequences, and shows no effect on plasmid loss events LOFF et d. 1989). Ingeneral, the rad52 mutation in repressed DNA. The radl rad52 double mutant results in a striking reduction in what have classically displays a dramatic decreasein the rateof plasmid loss been termedgene conversion events. Incontrast, events. These experiments show that a wild-type copy reciprocal exchange events are reduced by varying of RAD52 can compensate for a defect in radl, but a amounts in rad52 cells. It is clear that the RAD52 functional RAD1 gene only partially rescues the rad52 gene plays a major role in recombination in mitotic mutantphenotype, which suggests that RADl and cells; however, since recombination is not completely RAD52 are components of two overlapping pathways eliminated in rad52 mutant cells, it suggests that an- for mitotic recombination in yeast. Furthermore, other, RAD52-independent recombination pathway based on the different sensitivities of these events to must also beoperating (HABERand HEARN1985; a mutation in rad52 in transcribed versus repressed HOEKSTRA, NAUGHTONand MALONE 1986). DNA, we proposethat transcription-stimulated re- The RADl gene is a component of the excision- combination probably proceeds via a different path- repair pathway for UV damage in yeast (REYNOLDS way fromrecombination in transcriptionally silent and FRIEDBERG198 1 ; WILCOXand PRAKASH198 1). sequences. Finally, since the radl rad52 double mu- Recently, studies of mitotic recombinationevents tantdoes not exhibit a concomitant synergistic in- stimulated by the Pol1 rDNA promoter, HOT1 (KEIL crease in sensitivity to damage by radiation when and ROEDER 1984; ROEDER,KEIL and VOELKEL-MEI- compared with either single mutant, the recombina- MAN 1986), show that a mutation in RADl reduces tion function of these gene products is independent HOT1-stimulated recombination events (R. KEIL, per- of their role in survival of radiation damage. sonal communication). Similarly, the RADl gene has MATERIALS AND METHODS been shown to be required for recombinationbetween direct-repeats, as well as for homologous integration Strains: The strains used in these experiments are de- of linear plasmids (SCHIESTLand PRAKASH1988). scribed in Table l. A TRPl disruption of rad52 (SCHILDet al. 1983a, b) was introduced into the genome by transform- Based on analysis of recombination events between ing linearized DNA (ROTHSTEIN 1983) into yeastmade heteroallelic duplications, it was suggested that RADl competent by the LiCl method (ITO et al. 1983). The pres- may be involved in the processing of recombination ence of the disruption was confirmed byanalysis of the intermediates containing short stretchesof heterodu- genomic DNA of transformants by blots (SOUTHERN1975), genetic linkage analysis to known mutations, and sensitivity plex DNA (KLEIN 1988). Inaddition, studies of of themutant strain to ionizing radiation (50 krad, in a RAD5'Zindependent recombination demonstrate that cesium source emitting 7.8 krad/hr). The radl disruption exposure of rad52 cells to UV-irradiation results in a was previously constructed in this laboratory in an isogenic wild-type spectrum of recombination events (E. SUC- genetic background (RONNEand ROTHSTEIN1988), and is ARMAN and J. HABER, personal communication). marked with the LEU2 selectable marker. Segregants con- taining either a single rad mutation or both were made by These experiments suggest a role for excision-repair crossing the disrupted strainsto previously constructed iso- gene products in genetic recombination in yeast.
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