Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12711-12715, December 1994 Genetics Transcriptional induction of Ty recombination in yeast (repeated sequences/ectopic gene conversion/DNA repair/RAD genes) YAEL NEVO-CASPI AND MARTIN KuPIEC* Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel Communicated by Roger D. Kornberg, September 6, 1994 (receivedfor review February 18, 1994)

ABSTRACT Families of repeated sequences are present in of transcription (6). Evidence for induced recombination the genomes of all eukaryotes. Little is known about the upon transcription was also obtained in Schizosaccharomy- mechanism(s) that prevents recombination between repeated ces pombe (7) and in mammalian cells (8-11). sequences. In the yeast Saccharomyces cerevisiae, recombina- A close relationship between transcription and DNA repair tion between homologous sequences placed at nonhomologous has been found: Lesions in actively transcribed genes are locations in the genome (ectopic recombination) has been repaired faster than those in inactive ones and the template shown to occur at high frequencies for artificially created strand of actively transcribed genes is preferentially repaired repeats, but at relatively low frequencies for a natural family (12-15). In addition, it was also recently found that the DNA of repeated sequences, the Ty family. We have previously repair genes RAD3, RAD25, and SSLI encode proteins that shown that a high level ofTy cDNA in the cell causes an increase play a role also in transcription (16, 17). in the rate of nonreciprocal recombination (gene conversion) of In this paper we present evidence that elevated transcrip- a marked Ty element. In the present study, we show that it is tion of a Ty element causes an increase in its rate of also possible to elevate the rate of recombination of a marked recombination, and we investigate the role of different RAD Ty by increasing its transcription. This induction is different genes in this process. from, and acts synergistically to, the one seen upon increased levels of donor Ty cDNA. We show that the induction by transcription does not require the products of the RADSO, MATERIALS AND METHODS RADS5, and RAD57 genes. In contrast, cDNA-mediated re- Yeast strains. All the Saccharomyces cerevisiae strains combination is dependent on the product ofthe RADSI gene but used in the present study are isogenic and were derived from not on products of the genes RAD5O or RAD57. strain MK89 (4) (MATa ura3-Nco- his3-11,15 leu2-3,112 trpl-Xba- can.1-101) by transformation. They carry, at the Ty retrotransposons constitute the main family of naturally L YS2 locus, a Ty element marked by the insertion ofa URA3 occurring repeated sequences in the yeast genome, repre- gene (TyUra). Strains MK87 and MK104 have been de- senting 1-2% of the genome (for review, see ref. 1). Ty scribed (4); MK87 carries a Ty2Ura, and MK104 carries a elements are retrovirus-like transposons that code for an TylUra. MK95, MK116, and YN2 were created by trans- end-to-end transcript that is reverse-transcribed to cDNA by forming MK89 with plasmids pM106, pM128, and pYN10, Ty-encoded proteins. They transpose at very low frequencies respectively. In MK95, the first 237 nt of the Tyl have been (10-9 to 10-7 per locus per cell division). The two main replaced by 741 bp from the GAL] promoter (GalTylUra) classes of Ty elements, Tyl and Ty2, share extensive ho- (18); in MK116, a frameshift mutation was introduced in the mology. There are -30-40 copies ofTy elements per haploid GalTylUra by filling-in the Asp718 site of the Ty; in YN2, a genome (1). linker containing amber stop mutations in all three frames Recombination between homologous sequences located at was inserted at nt 475 ofthe Ty. YN3 carries a deletion ofthe nonhomologous positions in the genome (ectopic recombi- Ty promoter and was created by transformation with plasmid nation) occurs readily between artificially duplicated genes, pYN9. Strains MK172 (rad50:: TRPl), UB1, (rad51::LEU2), in mitotic and meiotic yeast cells. Both reciprocal and and UB2 (rad57: :LEU2) were created by a one-step replace- nonreciprocal recombination (gene conversion) have been ment transformation (19) using plasmids pME305 (20), observed. Ectopic reciprocal recombination causes chromo- pAM28 (21), and pSM51 [a Pvu II-Sal I fragment of the somal aberrations, such as translocations, deletions, and RAD57 gene replaced by the LEU2 marker (22)], respec- inversions (2). Since repetitive sequences are present in the tively. After transformation, all the relevant chromosomal genomes of all eukaryotes, the karyotypic stability might be configurations were confirmed by Southern blot analysis. maintained by (a) mechanism(s) that prevent(s) recombina- Plasmids. pM106, carrying the lys2::GalTyl Ura allele was tion between such repeats. We have been using the Ty family constructed in several steps: First, the 1.2-kb URA3 fragment of retrotransposons as a model to study recombination be- was cloned into the 3'Bgl II site ofa GalTy (18), thus creating tween naturally occurring repeated sequences. The rate at plasmid pRJ2. Then the BamHI fragment containing the which Ty elements engage in homologous ectopic recombi- whole GalTylUra was inserted in the unique Bgl II site ofthe nation is relatively low and is mainly nonreciprocal in nature LYS2 gene in plasmid pDP6 (23). Plasmid pM128 was con- (3). Recent experiments in our laboratory have shown that Ty structed by filling-in the Asp718 site in the Ty of pM106 with cDNA participates in gene conversion events involving a the Klenow fragment ofEscherichia coli DNA polymerase I, marked Ty element (4). followed by ligation; this process generates a SnaBI site in its Transcription by DNA-dependent RNA polymerase I has place. pYN10 was constructed in several steps: First, a been shown to cause an increase in recombination in yeast 6.2-kb BamHI-Cla I fragment of pM106 was replaced by a (5). Elevated levels of recombination between directly re- BamHI-Cla I 380-bp fragment of pBR322, thus creating peated GALIO genes have also been observed upon induction pYN6. Then, a Xho I-Asp718 fragment of the Ty was replaced by a similar one from pGTyl-H3His3 (Tya-475Am) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: LTR, long terminal repeat. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

12711 Downloaded by guest on September 27, 2021 12712 Genetics: Nevo-Caspi and Kupiec Proc. Natl. Acad. Sci. USA 91 (1994) (24) containing a triple amber linker insertion. Finally, the and calculated by the method of the median (29). Twelve to 6.2-kb BamHI-Cla I fragment deleted in the first step was 24 cultures grown in SD or SGal medium (4, 27) were used in restored (instead of the pBR322 fragment), recreating a each experiment. DNA was prepared from one Ura- Leu- full-length Ty element. This plasmid was called pYN10. colony (strains carrying pM97) or one Ura- His- colony pYN9 was created by first deleting a Nhe I-Xho I fragment (strains carrying pJefl678) from each culture and subjected to (containing the Ty promoter) from pYN6 and then recon- Southern blot analysis using LYS2, neomycin-resistance structing the whole Ty with the BamHI-Cla I fragment, as gene (neo), and URA3 sequences as probes (4). described above. Plasmids pM97, pM102, and pM109 have been described (4). pYN8, carrying the same triple-amber linker as pYN10 was constructed by replacing the Xho I-Cla RESULTS I fragment of pM97 with the corresponding fragment from Increased transcription of a marked Ty (GalTylneo), result- plasmid BJC28 (24). pJefl678 (a gift from Jef Boeke, Johns ing in high levels of Ty cDNA, causes elevated rates of Hopkins University, Baltimore) carries a GalTylneo on a recombination ofanother differently marked Ty (TyUra), due 2-,um plasmid-containing vector with a HIS3 selectable to gene conversion events in which cDNA copies of the marker. pM43 was used as a probe in Southern blot hybrid- GalTylneo act as donors of information (4). The level of ization experiments; it carries a Pvu II fragment of the LYS2 conversion of the marked TyUra can be estimated by select- gene (25). ing for Ura- cells on 5-fluoroorotic acid medium (28). Media, Growth Conditions, and General Procedures. Stan- To analyze the effect of transcription of the recipient dard molecular biology procedures such as cloning, restric- molecule on the level ofrecombination, we constructed yeast tion enzyme analysis, and Southern blot analysis were car- strain MK95 (Fig. 1). This strain carries a TylUra element on ried out as described in ref. 26. Yeast media and molecular chromosome II in which the natural promoter has b6en biology procedures (transformations, DNA preparations, replaced by the GAL] promoter (GalTylUra) (4). This Ty is etc.) were carried out as in ref. 27. Ura- colonies were not expressed in a glucose-containing medium, but high selected on SD complete medium with uracil (50 mg/liter) levels of transcription are seen in a medium containing and 5-fluoroorotic acid (0.85 mg/ml) (28). galactose (results from Northern blot analysis not shown). As Measurement of Ty Recombination. Recombination rates controls, isogenic strains with different TyUra elements were were measured by fluctuation test analysis as described (4) used: MK104, bearing a Tyl with its natural promoter, and A A URA3 B MK104 > . TyA neo TyB _PR N RT RH

A MK95 -1 Ll

A A YN3 pbs- pM109 - --{ 7

SnB MK116 SnB pMl1 02 - t _>~~~~~--

YN2 1am A am A pYN8 - _=

A B MK87 (Ty2)

FIG. 1. Schematic representation ofTy elements used in the present study. Ty elements are depicted as open boxes bounded by open triangles (LTRs). (A) Chromosomal copies of recipient Ty elements. The two open reading frames TyA and TyB and the proteins produced from the Ty mRNA are shown. PR, protease; IN, integrase; RT, reverse transcriptase; RH, RNase H. The hatched box at the 3' end of the Ty elements show the URA3 insert. MK104 carries a wild-type TylUra (4). In MK95, the first 237 nt of the Tyl have been replaced by 741 bp from the GAL] promoter (18). In YN3, this fragment was deleted. The Tyl in MK116 carries a frameshift mutation, created by filling-in the Asp718 site with the Klenow fragment of Escherichia coli DNA polymerase I; this process generates a SnaBI site in its place. In YN2, a 12-nt linker carrying amber stop codons (am) in all the three frames was inserted at nt 475 of the Ty (24). MK87 carries a Ty2 element (4). A, Asp718; B, BamHI; SnB, SnaBI. Stippled boxes indicate inactive regions in the Ty. (B) Plasmid-borne donor Ty elements. pM97 (4) is a high copy number plasmid that carries a GALl-promoted Tyl and a LEU2 marker. The solid box at the 3' of the Ty represents the bacterial neomycin resistance (neo) gene. Three other plasmids were constructed, based on pM97. pM109 (4) carries substitutions in the primer binding site (pbs) of the Ty. pM102 (4) carries the same Asp718 filled-in site as MK116. pYN8 carries the same linker insertion as the TylUra in strain YN2. Downloaded by guest on September 27, 2021 Genetics: Nevo-Caspi and Kupiec Proc. Natl. Acad. Sci. USA 91 (1994) 12713 MK87, carrying a Ty2 (4) (Fig. 1). Strain MK104 produces Table 2. Rate of appearance of Ura- colonies high levels of Ura- colonies by a mechanism that replaces the Rate whole Ty by a solo long terminal repeat (LTR) (4). There are several ways by which this result can be obtained: an Strain Glucose Galactose LTR-LTR reciprocal exchange (intrachromatidal recombi- MK87 (Ty2Ura)/pM97 3.66 ± 0.56 38.20 ± 5.29 nation), a conversion of the whole element by a solo LTR, an YN3 (Ty1UraALTR)/pM97 8.00 ± 1.33 83.10 ± 9.88 unequal sister chromatid exchange, or a single-strand anneal- MK95 (GalTylUra) 1.43 ± 0.33 33.18 ± 4.41 ing event between the direct repeats (30). To focus on ectopic MK95 (GalTylUra)/pM97 3.79 ± 0.65 269.10 ± 59.01 conversion between Ty elements, we constructed strain MK95/pM1O9 (pbs-) 3.69 ± 0.66 49.78 ± 5.85 YN3, in which part of the 5' LTR is deleted (Fig. 1). This MK95/pM102 (rt- rnh-) 3.22 ± 0.71 31.38 ± 2.79 strain shows a lower basal level of Ura- colonies (Table 1), MK95/pYN8 (TyA amber) 3.26 ± 0.61 23.00 ± 3.00 which are mainly the result ofconversion events in which the MK116 (GalTylUra rt- rnh-) 4.62 ± 0.89 18.58 ± 2.28 TyUra information is replaced by information from another MK116 (GalTylUra rt- Ty element (3, 4). The level of appearance of Ura- colonies rnh-)/pM97 3.22 ± 0.59 159.62 ± 1.0 of YN3 and that of the other control strains is not changed YN2 (GalTylUra TyA amber) 3.00 ± 0.59 35.90 ± 4.68 when the cells are grown on a galactose-containing medium YN2 (GalTylUra TyA (Table 1). In contrast, the rate ofappearance ofUra- colonies amber)/pM97 3.20 ± 0.62 334.03 ± 38.74 in MK95 cells grown on galactose is one order of magnitude Rate of appearance of Ura- colonies is expressed as number x higher than that ofcells grown under transcription-repressing 10-7 per cell per generation. conditions (Table 1). Independent Ura- colonies from induced and uninduced sustain a reverse-transcription reaction (4); pM102 has a cultures of MK95 were subjected to Southern blot analysis to frameshift mutation in the Ty, so that no reverse transcriptase determine the type of event that had created the Ura- or RNase H is produced (4); pYN8 carries amber mutations phenotype. LYS2 sequences were used as a probe, since the in all three frames, so that no protein is produced from this TylUra is located at the L YS2 locus. In glucose-grown cells, Ty (24). When these plasmids were introduced into strains 57% of the Ura- colonies (20 of 35 colonies) were the result MK104 (TylUra) or MK87 (Ty2Ura), no increase in the rate of gene conversion of the GalTylUra by other Ty elements, of appearance of Ura- colonies was seen in galactose, and in 31% (11 of35 colonies) the whole Ty had been replaced showing that high cDNA levels are essential for increased by a solo LTR. The remaining Ura- colonies (4 of 35 recombination between TyUra and GalTylneo (ref. 4 and colonies) still carried the URA3 insert at the Ty and were the data not shown). Introducing these plasmids into MK95 result of mutations in the URA3 gene of the Ty or ectopic resulted in a rate of Ura- colonies similar to the one obtained conversion by the ura3-Nco- allele on chromosome V. Even without any plasmid (Table 2). These results confirm that the though the rate of appearance of Ura- colonies was 20-fold additional increase in recombination seen in the presence of higher in galactose-grown cells, the ratio between the differ- pM97 in galactose is cDNA-mediated and imply that the ent types of Ty recombination remained similar: 60% (15 of induction of transcription of the GalTylUra cannot comple- 25 colonies) conversions and 36% (9 of25 colonies) solo LTR ment the defect in the GalTylneo on the plasmid. events. When the frameshift mutation or the triple amber mutation When MK95 cells carrying a GalTylneo on a high copy were introduced into the recipient Ty in MK95, creating number plasmid (pM97) were grown on galactose, thus strains MK116 and YN2, respectively, the high rate of producing increased levels of Tylneo cDNA, the rate of conversion in galactose was unaffected (Table 2). Thus, the appearance of Ura- colonies increased by another order of increase in recombination caused by transcription of the magnitude, to a rate of 2.7 x 10-5 per cell per generation recipient Ty, as opposed to that seen when the donor Ty, is (Table 2). Thirty-one of 36 colonies analyzed had the recip- highly transcribed, does not require reverse transcription, ient GalTylUra replaced by a GalTylneo. These events are and is probably due to transcription per se. Furthermore, due to recombination with the abundant Tylneo cDNA upon transformation of strains MK116 and YN2 with pM97, present in the cell (4). We note that while the presence of the level of Ura- colonies obtained was similar to the one increased levels of Tylneo cDNA causes a one order of seen in MK95/pM97 (Table 2), showing once more the magnitude increase in strains MK87 and YN3, MK95/pM97 synergistic nature of both types of induction. grown in galactose shows a two order of magnitude increase. To further characterize the genetic requirements of the This can be explained by a synergistic effect between the two cDNA-mediated and the transcription-induced recombina- causes for elevated rates of recombination-namely, in- tion, we created three isogenic strains, each carrying a creased donor cDNA levels and transcription ofthe recipient mutation in a different RAD gene. These repair genes have Ty. been shown to be required for homologous recombination in This hypothesis was tested by introducing changes in the yeast (for review, see ref. 2). radSl and radS7 mutants have donor and the recipient Ty elements. Three additional plas- been reported to be completely defective in meiotic and mids were used as donors: pM109 carries mutations in the mitotic allelic recombination (2, 31); in contrast, the RADSO primer-binding site (pbs) of the Ty, rendering it unable to gene product seems to be needed only for meiotic recombi- nation (2, 32). Table 3 shows the results obtained with the Table 1. Rate of appearance of Ura- colonies different rad strains that do or do not carry a GalTylneo Rate plasmid, in glucose- or galactose-containing medium. The radSO strain gave similar results to those obtained with the Strain Glucose Galactose Rad+ strain (MK95). Surprisingly, both radSl and radS7 MK87 (Ty2Ura) 3.03 ± 0.63* 3.31 ± 0.66* strains gave a very high rate of Ura- colonies. The basal rate MK104 (TylUra) 33.50 ± 7.41* 31.10 ± 2.79* of recombination in these mutants is two orders of magnitude YN3 (TylUra ALTR) 4.90 ± 0.86 2.36 ± 0.78 higher than that of MK95. This increase is due to a very high MK95 (GalTylUra) 1.43 ± 0.33 33.18 ± 4.41 rate of recombination between the LTRs of the recipient Ty: Rate of appearance of Ura- colonies is expressed as number x all the Ura- derivatives analyzed were the result of a LTR- 10-7 per cell per generation. LTR interaction. Table 3 also shows that both radSl and *Data are from ref. 4. radS7 strains still show an increase in the rate of appearance Downloaded by guest on September 27, 2021 12714 Genetics: Nevo-Caspi and Kupiec Proc. Natl. Acad. Sci. USA 91 (1994) Table 3. Rate of appearance of Ura- colonies in various rad strains and distribution of the different types of events obtained Events, no. Strain Rate Tyneo* Tyt LTRt TyUra§ Total MK95 (Rad+) Glu 1.43 ± 0.33 20 11 4 35 MK95 Gal 33.18 ± 4.41 - 15 9 1 25 MK95/pJefl678 Glu 2.47 ± 0.53 3 4 2 3 12 MK95/pJefl678 Gal 374.92 ± 53.43 11 0 2 0 13 MK172 (rad5O) Glu 2.02 ± 0.48 7 3 1 11 MK172 Gal 33.20 ± 5.45 5 3 4 12 MK172/pJefl678 Glu 2.52 ± 0.50 2 4 2 3 11 MK172/pJefl678 Gal 182.33 ± 32.97 9 7 1 1 18 UB1 (rad5l) Glu 238.37 ± 35.16 - 0 11 0 11 UB1 Gal 1040.00 ± 114.37 0 12 0 12 UB1/pJefl678 Glu 356.64 ± 56.24 0 0 18 0 18 UB1/pJefl678 Gal 1005.67 ± 140.09 0 0 12 0 12 UB2 (rad57) Glu 103.05 ± 13.27 0 12 0 12 UB2 Gal 379.65 ± 37.59 0 11 0 11 UB2/pJefl678 Glu 114.18 ± 22.23 0 0 16 0 16 UB2/pJefl678 Gal 507.93 ± 73.91 8 1 5 4 18 Rate of appearance of Ura- colonies is expressed as number x 10-7 per cell per generation. *Replacement of the TylUra by information from Tylneo. tReplacement of TylUra by information from an unmarked Ty element. tReplacement of the TylUra by a solo LTR. §Mutation at the URA3 gene in the TylUra or conversion by the ura3-Nco- allele on chromosome V. of Ura- colonies when grown in galactose, implying that the cells together with the transcriptional induction of the recip- induction due to transcription is still present. When they were ient Ty. These results imply that the induction of recombi- transformed with a plasmid carrying a GalTylneo, no change nation observed upon transcription of the recipient Ty is due in the rate of appearance of Ura- cells was seen in glucose- to a different mechanism than the induction caused by grown cells; on galactose-containing medium, however, overexpression ofTy cDNA. The synergistic effect observed these strains behaved differently from each other. Galactose- implies that two different factors or steps are rate-limiting for grown UB1 (rad5l) carrying the GalTylneo plasmid showed Ty recombination. One ofthem is the availability ofcDNA to the same level of recombination as the strain without the act as donor of information; the second one limits recombi- plasmid; in addition, no conversion events were detected. nation when the Ty is not expressed but is no longer limiting Thus, the RADSJ gene product is needed for Tylneo cDNA- upon transcription from the GAL promoter. mediated recombination. When the rad57 strain carrying the What is the reason for this increased recombination level plasmid was grown in galactose, however, a modest but upon transcription? We note that the distribution of recom- reproducible increase in the number of Ura- colonies was bination events in repressed or induced conditions remains seen. Analysis of independent Ura- colonies showed that 8 unchanged, implying that there is no induction of a specific of 16 carried a replacement of the TylUra by a Tylneo, type of event. More likely, a general mechanism increases all implying that cDNA-mediated recombination can take place types of recombination. Moreover, even in rad5l and rad57 in radS7 strains. strains, in which an unusually high level of LTR-LTR interactions is seen, transcription of the Ty still causes a DISCUSSION modest increase in the level ofrecombinants observed. Three nonexclusive models can be proposed to explain transcrip- Nonreciprocal recombination (gene conversion) between re- tion-induced recombination: (i) Recombination may result peated sequences constitutes an evolutionary mechanism from changes in chromatin caused by the passage ofthe RNA that promotes homogeneity between members of the family polymerase through the Ty. The transcription complex may [concerted evolution (33)] and can also generate new com- remove factors that normally prevent recombination or may binations with adaptive advantage. Reciprocal recombina- render the DNA more amenable to the recombination ma- tion between repeated sequences, on the other hand, can chinery by altering its topology or by allowing the interaction result in gross karyotypic changes, such as deletions, inver- ofunwound DNA with recombination proteins (8, 10, 11). (ii) sions, and translocations, which have immediate effects in Alternatively, the high levels of recombination could be due fitness and may also contribute to speciation mechanisms (2, to an increase in repair activity (34, 35). Such an activity can 33). Since all eukaryotic cells carry many repetitive se- result in secondary lesions in the DNA that may promote quences, the level of recombination between them is prob- recombination. (iii) Finally, the binding of transcription fac- ably regulated. The Ty elements of yeast constitute a good tors to the GAL] promoter under transcription conditions example of a naturally occurring family of repeated se- may be directly or indirectly responsible for the increase in quences. The level of recombination between Ty elements is recombination. A dual role for proteins that act in transcrip- relatively low and is almost exclusively nonreciprocal in tional silencing and DNA replication has been found lately nature (3). Furthermore, this low recombination frequency is (36-38). Similarly, it is possible that protein-protein interac- not increased by DNA damage (25). In the present paper, we tions exist between transcription and recombination factors, show that a higher rate of ectopic recombination can be as has been suggested for the immunoglobulin genes in obtained upon induction oftranscription of a marked Ty. This mammalian cells (10, 11, 39). elevated level of recombination is not dependent on gene This laboratory has shown (25) that UV irradiation causes products encoded by the Ty itself(proteins, RNA, or cDNA); an increase in Ty RNA levels without a concomitant increase moreover, a synergistic increase in the rate of recombination in recombination; this result seems to be in contradiction with can be observed when Tylneo cDNA is overproduced in the model ii. Support for model iii comes from the finding (40) Downloaded by guest on September 27, 2021 Genetics: Nevo-Caspi and Kupiec Proc. Natl. Acad. Sci. USA 91 (1994) 12715

that in the HIS4 gene of yeast, binding of the RAP1 tran- J. R. & Jones, E. W. (Cold Spring Harbor Lab. Press, Plainview, scription factor to the promoter increases meiotic recombi- NY), pp. 193-291. 2. Petes, T. D., Malone, R. E. & Symington, L. S. (1991) in Molecular nation in the region, showing that this protein can play a dual and Cellular Biology of the Yeast Saccharomyces, eds. Broach, role in transcription and recombination. It is possible that the J. R., Pringle, J. R. & Jones, E. W. (Cold Spring Harbor Lab. galactose-promoted transcription induction and the UV- Press, Plainview, NY), pp. 407-521. promoted transcription induction are mediated by different 3. Kupiec, M. & Petes, T. D. (1988) Mol. Cell Biol. 8, 2942-2954. sets of proteins and that in the latter case, no coupling to a 4. Melamed, C., Nevo, Y. & Kupiec, M. (1992) Mol. Cell Biol. 12, recombinational mechanism (or, alternatively, coupling to 1613-1620. 5. Voelkel-Meiman, K. & Roeder, G. S. 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Biol. 12, nism and are thus fully detected. The high level of LTR-LTR 3235-3246. interactions seen in these strains is still inducible by transcrip- 22. Kans, J. A. & Mortimer, R. K. (1991) Gene 105, 139-140. tion, implying that this induction is carried out by a general 23. Fleig, U. N., Pridmore, R. D. & Philipsen, P. (1986) Gene 46, mechanism, which is independent of the RADSJ and RADS7 237-245. gene products. 24. Curcio, M. J. & Garfinkel, D. J. (1992) Mol. Cell. Biol. 12, 2813- 2825. It is important to note that, although RADSJ and RADS7 25. Parket, A. & Kupiec, M. (1992) Mol. Cell. Biol. 12, 4441-4448. share DNA sequence homology (and are also homologous to 26. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular bacterial RecA proteins) (21, 43, 44), radS7 strains are Cloning: A Laboratory Manual (Cold Spring Harbor Lab. 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Genet. preclude us from detecting a normal level of conversion 18, 111-116. events. Alternatively, RAD57 may still be required for spon- 33. Edelman, G. M. & Gally, J. A. (1970) in The Neurosciences: taneous interchromosomal conversion events but not for Second Study Program, ed. Schmitt, F. 0. (Rockefeller Univ. cDNA-mediated events. Press, New York) pp. 962-972. We have shown that active transcription of a Ty element, 34. Selby, C. P. & Sancar, A. (1993) Science 260, 53-57. a member of a family of repeated sequences, affects its level 35. Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J. H., Chambon, P. & Eagly, J. M. (1993) Science of recombination. Further studies are required to elucidate 260, 58-63. the precise mechanism of this induction. These studies 36. Foss, M., McNally, F. J., Laurenson, P. & Rine, J. (1993) Science should contribute to the understanding of basic mechanisms 262, 1838-1844. by which eukaryotic cells control recombination between 37. Bell, S., Kobayashi, R. & Stillman, B. (1993) Science 262, 1844- repeated sequences. 1849. 38. Micklem, G., Rowly, A., Hardwood, J., Nasmyth, K. & Diffley, J. F. X. (1993) Nature (London) 366, 87-89. We thank Jef Boeke, Joan Curcio, David Garfinkel, Shirleen 39. Jenuwein, T., Forrester, W. C., Qiu, R. & Grosscheld, R. (1993) Roeder, and Lorraine Symington for plasmids; Cathy Melamed for Genes Dev. 7, 2016-2032. her contribution in the early steps of the project; Udi Barki for help 40. White, M. A., Wierdl, M., Detloff, P. & Petes, T. D. (1991) Proc. in strain constructions; and Rina Jaget and Rivka Steinlauf for Natl Acad. Sci. USA 88, 9755-9759. excellent technical assistance. These studies were supported by a 41. Steele, D. F., Morris, M. E. & Jinks-Robertson, S. 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