Genetics, Vol. 154, 543-556, February 2000, Copyright © 2000

The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition

Alison J. Rattraya, Brenda K. Shafera, and David J. Garfinkela
a Gene Regulation and Chromosome Biology Laboratory, ABL-Basic Research Program, NCI-FCRDC, Frederick, Maryland 21702

Corresponding author: Alison J. Rattray, Gene Regulation and Chromosome Biology Laboratory, ABL-Basic Research Program, NCI-FCRDC, P.O. Box B, Bldg. 539, Rm. 151, Frederick, MD 21702., rattray{at}mail.ncifcrf.gov (E-mail)

Communicating editor: L. S. SYMINGTON


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or {delta}) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

TY elements of yeast are members of a widely disseminated class of eukaryotic repetitive sequences functionally and structurally related to retroviruses (for reviews see TEMIN 1985 Down; BOEKE and SANDMEYER 1991 Down; FLAVELL 1995 Down). Ty1 elements and their solo long-terminal-repeat (LTR) derivatives ({delta} elements) are the most abundant dispersed repetitive sequence in Saccharomyces cerevisiae, representing ~4% of the genome (see GOFFEAU et al. 1996 Down). Ty elements can destabilize the genome in two ways: de novo transposition can result in insertional mutagenesis of cellular genes, and homologous recombination between the dispersed copies can lead to gross chromosomal rearrangements such as translocations, inversions, and large insertions or deletions (BOEKE and SANDMEYER 1991 Down). A majority of the full-length endogenous Ty1 elements appears to be competent for transposition (CURCIO et al. 1988 Down; CURCIO and GARFINKEL 1994 Down; JORDAN and MCDONALD 1998 Down; KIM et al. 1998 Down) and Ty1 RNA is very abundant, comprising 0.1–1% of the total RNA in haploid cells (ELDER et al. 1983 Down; CURCIO et al. 1990 Down). However, Ty1 proteins are not abundant (GARFINKEL et al. 1985 Down; CURCIO and GARFINKEL 1992 Down) and transposition occurs at a low rate (10-5 to 10-7 events/cell/generation; CURCIO and GARFINKEL 1991 Down). Ectopic expression of Ty1 from the inducible GAL1 promoter overcomes "transpositional dormancy" by a mechanism that is largely post-translational (CURCIO and GARFINKEL 1992 Down, CURCIO and GARFINKEL 1999 Down; CONTE et al. 1998 Down; LEE et al. 1998 Down). De novo Ty1 insertions occur preferentially upstream of genes transcribed by RNA polymerase III, and therefore they appear to target "nondeleterious" regions of the genome (JI et al. 1993 Down; DEVINE and BOEKE 1996 Down). Homologous recombination between genomic Ty1 elements occurs at rates much lower than at most artificially introduced ectopic sequences and are rarely associated with crossovers (KUPIEC and PETES 1988A Down, KUPIEC and PETES 1988B Down). Furthermore, recombination between the LTRs of a single element, leaving a solo {delta}, has been shown to remove the deleterious effects of some Ty insertions (ROEDER and FINK 1980 Down; FINK et al. 1986 Down; JORDAN and MCDONALD 1999 Down). Taken together, these data suggest that Ty1 elements and yeast cells have a commensal relationship in which new insertions are rare, and those that do occur are targeted to "safe" sites (BOEKE and DEVINE 1998 Down). Since most of the Ty1 elements in the genome appear to be functional, cDNA conversion of preexisting Ty1 elements should be of little consequence, and the unusually low level of crossovers between endogenous Ty1 elements should minimize rearrangements by ectopic recombination.

The relationship between Ty1 transpositional integration and RAD52-mediated recombination has been examined in two ways. First, the role of Ty1 integrase (IN) has been analyzed in spt3 cells that are defective in expression from Ty1 promoters (WINSTON et al. 1984 Down), but not defective for expression from GAL1-promoted Ty1 elements (BOEKE et al. 1986 Down). Transposition rates from GAL1-promoted Ty1 elements carrying the in-2600 mutation, which has no in vitro integrase activity (EICHENGER and BOEKE 1988), are not significantly different from wild type (SHARON et al. 1994 Down). However, a majority of Ty1 insertion events observed in an in-2600 mutant are RAD52 dependent. Interestingly, His+ events from an endogenous Ty1his3-AI element are markedly elevated in a rad52 mutant (CURCIO and GARFINKEL 1994 Down; CONTE et al. 1998 Down; LEE et al. 1998 Down), suggesting that the RAD52 gene product normally inhibits transpositional events. Here, we expand this observation by determining the consequences of defects in other DNA recombination and repair genes and by examining what stage of the transposition process they affect.

The RAD50–RAD57 members of the RAD52 recombinational repair pathway were initially identified by their sensitivity to ionizing radiation and were subsequently shown to be required for most homologous recombination events. A rad52 mutant exhibits the strongest defects (for reviews see PETES et al. 1991 Down; GAME 1993 Down; PAQUES and HABER 1999 Down), including a defect in cDNA-mediated homologous recombination (DERR and STRATHERN 1993 Down; SHARON et al. 1994 Down; LIEFSHITZ et al. 1995 Down). The Rad52p is known to bind to the ends of DNA molecules (DYCK et al. 1999 Down), to promote DNA strand annealing between single-stranded oligonucleotides (MORTENSEN et al. 1996 Down; SUGIYAMA et al. 1998 Down), and to potentiate the strand-exchange activity of Rad51p (SUNG 1997A Down; NEW et al. 1998 Down; SHINOHARA and OGAWA 1998). The Rad51 protein is homologous to the Escherichia coli RecA protein and, like RecA, it forms an extended helical filament on DNA (SHINOHARA et al. 1992 Down; OGAWA et al. 1993 Down) and can promote DNA strand exchange in vitro (SUNG 1994 Down; SUNG and ROBBERSON 1995 Down). The addition of one of several different factors including Rad52p, Rad54p, or the heterodimeric Rad55p/Rad57p complex stimulates strand exchange (SUNG 1997A Down; NEW et al. 1998 Down; PETUKHOVA et al. 1998 Down; SHINOHARA and OGAWA 1998). Rad55p and Rad57p are two additional yeast RecA homologs that have not been shown to possess strand-exchange activity by themselves or together (SUNG 1997B Down), but appear to function as a heterodimeric complex facilitating Rad51p function (JOHNSON and SYMINGTON 1995 Down; SUGAWARA et al. 1995 Down; SUNG 1997B Down). The Rad54p is a member of the SWI2/SNF2 family of DNA-dependent ATPases (EMERY et al. 1991 Down) and may function by opening up the DNA for repair and recombination. RAD50 was initially included in the RAD52 epistasis group on the basis of its severe sensitivity to ionizing radiation (GAME 1993 Down); however Rad50p is a member of a different protein complex that also includes Mre11p and Xrs2p (JOHZUKA and OGAWA 1995 Down; USUI et al. 1998 Down). Rad50p and Mre11p share homology with the E. coli SbcCD proteins, respectively (SHARPLES and LEACH 1995 Down), and have both single-stranded (ss) and double-stranded (ds) DNA exonuclease and ssDNA endonuclease activity in vitro (FURUSE et al. 1998 Down; MOREAU et al. 1999 Down). In addition to its role in DNA damage repair and meiotic double-strand break (DSB) repair (GAME 1993 Down; JOHZUKA and OGAWA 1995 Down), the Rad50p, Mre11p, and Xrs2p complex is also involved in telomere maintenance (NUGENT et al. 1998 Down) and nonhomologous end-joining (SCHIESTL et al. 1994 Down; NAIRZ and KLEIN 1997 Down; TSUKAMOTO et al. 1996 Down), but is not required for mitotic heteroallelic recombination (MALONE et al. 1990 Down).

Nucleotide-excision repair (NER) is the process by which distortions of the helical structure of DNA are repaired. This complex involves the products of ~30 genes including the RAD1 and RAD2 genes, which participate in the recognition and incision of the damaged DNA (GUDZER et al. 1995 Down). Mutations in RAD1 or RAD2 result in extreme sensitivity to ultraviolet light (UV) (FRIEDBERG et al. 1995 Down; BHATIA et al. 1996 Down). Rad1p forms a complex with Rad10p that specifies a ssDNA endonuclease (SUNG et al. 1993 Down; TOMKINSON et al. 1993 Down; DAVIES et al. 1995 Down). A rad1 mutant is also deficient in a subset of mitotic recombination events that require removal of nonhomologies from the ends of the recombining molecules (KLEIN 1988 Down; SCHIESTL and PRAKASH 1988 Down; THOMAS and ROTHSTEIN 1989 Down; FISHMAN-LOBELL and HABER 1992 Down; IVANOV and HABER 1995 Down; PAQUES and HABER 1997 Down). RAD2 encodes a flap-endonuclease (HABRAKEN et al. 1995 Down) and is a subunit of the TFIIH repairosome essential for NER (SVEJSTRUP et al. 1995 Down). However, rad2 mutants have only minor defects in mitotic recombination (IVANOV and HABER 1995 Down).

Mismatch-directed repair (MMR) is required for the correction of mismatched bases that are generated by replication errors or by heteroduplex formation during homologous recombination (MODRICH and LAHUE 1996 Down; JIRICNY 1998 Down; PROLLA 1998 Down). A msh2 null mutant has a strong spontaneous mutator phenotype and is defective in the removal of nonhomologies generated during recombination when these are between 30 bp and ~1 kb in length (SAPARBAEV et al. 1996 Down; PAQUES and HABER 1997 Down; SUGAWARA et al. 1997 Down). Although MSH2 does not appear to affect the level of recombination between identical sequences, it normally inhibits recombination between slightly diverged (homeologous) sequences (ALANI et al. 1994 Down; DATTA et al. 1996 Down, DATTA et al. 1997 Down; CHEN and JINKS-ROBERTSON 1998 Down). The major replicative DNA ligase is encoded by the essential CDC9 gene (TOMKINSON et al. 1993 Down). Mutants show increased mitotic recombination, even when grown at the permissive temperature (GAME et al. 1979 Down), suggesting that these conditions do not restore wild-type levels of activity.

Our results indicate that mutations in RAD50, RAD51, RAD52, RAD54, RAD57, and CDC9 genes lead to an increase in Ty1 transposition, whereas mutations in the RAD1, RAD2, or MSH2 genes do not. In all cases, the increased transposition rate is due to a post-translational mechanism because Ty1 RNA and protein levels remain at wild-type levels, but the amount of unincorporated Ty1 cDNA is significantly increased.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Media, growth conditions, and genetic methods:
Rich medium (YEPD) and synthetic complete (SC) medium lacking the appropriate amino acid or nucleic acid base were prepared as described (ROSE et al. 1990 Down). Cells were grown at 30° unless otherwise indicated. Transformations were performed by the lithium acetate method (ITO et al. 1983 Down), and tetrad dissection was performed as described previously (ROSE et al. 1990 Down). Methyl methane thiosulfonate (MMS; Sigma, St. Louis) was used to assay the sensitivity of strains by replica plating test strains to YEPD medium containing 0.05% MMS. The UV sensitivity of strains was scored by replica plating test strains to YEPD medium and irradiating with 150 J/m2 at 254 nm. Forward mutation rates were measured by plating on SC - Arg + canavanine media (ROSE et al. 1990 Down). Genomic DNAs were isolated by the glass bead disruption method (HOFFMAN and WINSTON 1987 Down), unless otherwise noted.

Plasmids:
The plasmids utilized in this study are described in Table 1. The disruption plasmids were digested with the appropriate restriction enzymes and were used for one-step transplacement (ROTHSTEIN 1983 Down).


 
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  		Table 1. Plasmids

Yeast strains:
The strains used in this study are listed in Table 2. Mutant phenotypes of the different disruptions were scored phenotypically by measuring sensitivity to 0.05% MMS for the rad50, rad51, rad52, rad54, and rad57 disruptions; sensitivity to UV (150 J/m2) for the rad1 and rad2 disruptions; and an increase in spontaneous CAN1 mutations for the msh2 disruption. Gene disruptions were also confirmed by Southern blot analysis. A congenic cdc9-1 strain, yAR314, was constructed by crossing strain JC364 with strain GRY645 and selecting spores that were temperature sensitive and papillated to His+. Strain yAR314 was then backcrossed twice with strain JC364 to yield the cdc9-1 strain yAR348, which was used in these studies.


 
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  		Table 2. Yeast strains

Transposition assays:
Qualitative assays were performed by spreading cells onto YEPD agar in 1.5 x 1-cm patches followed by incubation at 20° for 5 days. Cells were then replica plated to SC-histidine and incubated at 30° for 2–3 days prior to photographing. Due to the temperature sensitivity of cdc9 strains, plates were incubated at 26° instead of at 30°, along with a wild-type control. Transposition rates were determined as described previously (CURCIO and GARFINKEL 1991 Down). Briefly, 9 single colonies grown on solid YEPD media at 30° (or 26° for cdc9 strains and a wild-type control) were resuspended in distilled water and ~1000 cells were inoculated into 3 ml YEPD broth. At this time an aliquot was removed to determine colony-forming units (cfu/ml) inoculated. Cells were then grown for 3 days at 20° with aeration and aliquots were plated to determine cfu and the proportion of His+ cells. Rates were calculated from the median frequency by the Drake equation (DRAKE 1970 Down). To examine whether transposition events were producting multimeric arrays, or whether there were any obvious gross changes in the distribution of transposition events, 104 independent His+ colonies were isolated for each strain, purified, and DNA isolated in pools of eight. DNA was digested with PvuII and separated by agarose gel electrophoresis and Southern blots were probed with a 32P-labeled his3-AI probe, which hybridizes to the genomic junction fragment containing his3-AI or HIS3 sequences.

Northern blot analysis:
Total RNA was extracted by resuspending pelleted cells in 0.2 ml 0.1 M NaCl, 10 mM EDTA, 5% SDS, 50 mM Tris-HCl, pH 7.5, 0.2 ml phenol:chloroform:isoamyl alcohol 24:24:1 (PCI), and 0.3 ml glass beads. Samples were vortexed for 5 min and centrifuged. The supernatant was reextracted with PCI and precipitated with absolute ethanol. Samples were resuspended and treated with RNase-free DNase (Promega, Madison, WI), reprecipitated, and stored at -20°. Approximately 10 µg of RNA was separated electrophoretically on formaldehyde gels (AUSUBEL et al. 1994 Down) and was blotted to Hybond N (Amersham, Piscataway, NJ) nylon membranes. Strand-specific probes were synthesized from plasmids pBDG689-ACT1, pBDG456-Ty1, and pBJC-his3-AI by in vitro transcription according to manufacturer's instructions (Promega). Filters were hybridized with either the Ty1 and actin probes or the his-AI and actin probes in formamide at 42° (AUSUBEL et al. 1994 Down) and washed to a final stringency of 0.1x SSPE at 65°. Quantitation was performed by analysis on a STORM 8600 phosphorimager and analyzed with ImageQuant software. The data were normalized by dividing the total counts per minute for the Ty1 or his3-AI probe by the total counts per minute for the actin probe from the same blot and were divided by the corresponding ratio for the wild-type strain.

Protein analysis:
Total protein extracts were prepared from 108 mid-to-late log phase cells by disrupting washed cells in 0.4 ml buffer (0.15 mM KCl, 10 mM HEPES-KOH, pH 7.8, 5 mM EDTA, 30 mM DTT, 2 mM PMSF, 1x complete protease inhibitors (Boehringer Mannheim, Mannheim, Germany), and 0.2 ml acid-washed glass beads, followed by vortexing for 15 min at 4°. Supernatants were centrifuged for 5 min, and protein concentration was determined with the Bio-Rad colorimeter assay reagent (Bio-Rad, Richmond, CA). Protein (12 µg) was separated by SDS-PAGE and transferred to Immobilon P (Millipore, Bedford, MA) by a semi-dry Millipore apparatus. Immunodetection was performed with polyclonal antiserum to Ty1 virus-like particles (VLPs; YOUNGREN et al. 1988 Down) and the ECF immunodetection kit (Amersham). The blots were stripped according to manufacturer's instructions (Amersham), reprobed with antiserum to the heat-shock protein histidyl tRNA synthetase (a gift from T. Mason), and analyzed by ECF immunofluorescence. ECF immunofluorescence was visualized using a STORM 8600 phosphorimager and quantitated with ImageQuant software. Endogenous VLPs were partially purified from 1 liter of mid-to-late log phase cells grown in YEPD at 20° by sedimentation in sucrose step gradients as described previously (EICHINGER and BOEKE 1988 Down). Protein (25 µg) was separated on 8% SDS-PAGE gels and transferred to Immobilon P. Western blot analysis was performed as described above by successively probing with antiserum against Ty1-IN (b2), Ty1-reverse transcriptase (RT) (b8), and Ty1-VLPs (GARFINKEL et al. 1991 Down), except that ECL (Amersham) and autoradiography were used for immunodetection. ß-Galactosidase assays were performed on total protein glass bead extracts from 1 ml of the appropriate cells grown at 20° to mid-log phase in YEPD as described (ROSE et al. 1990 Down).

cDNA analysis:
Approximately 500 cells from single colonies were resuspended in 10 ml YEPD broth and grown for 2 days at 20°. About 108 washed cells were embedded in 0.5 ml 1% LMP agarose (SeaKem) in 150 mM EDTA and digested for 10 hr with zymolyase (Seikagaku America). Cells were lysed by incubating agarose plugs in a solution containing 2 mg/ml proteinase K (Sigma) and 1% Sarkosyl (Sigma) overnight at 50° (GERRING et al. 1991 Down). Approximately 50 µl of the plugs was subjected to 0.6% agarose gel electrophoresis and blotted to Hybond N+. The resulting filters were first hybridized with randomly primed 32P-labeled fragments from pBJC42-his3-AI or pBDG456-Ty1. After autoradiography and phosphorimage analysis, the blots were stripped and hybridized with a randomly primed 32P-labeled 1.9-kb SpeI fragment from YEp24, containing sequences from the yeast 2-µm plasmid. Quantitation was performed by phosphorimage analysis using ImageQuant software. The relative increase in Ty1 cDNA is defined as the ratio of the mutant cDNA to 2-µm hybridization signal, divided by the ratio of the wild-type cDNA to 2-µm hybridization signal.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Recombinational-repair-deficient mutants lead to increased levels of Ty1 transposition:
We used the his3-AI indicator gene (CURCIO and GARFINKEL 1991 Down) to follow the transposition of a single genomic Ty1 element under control of its native promoter. Reverse transcription of a spliced transcript and insertion into the genome result in the formation of His+ prototrophs, providing a phenotypic selection for reverse-transcription-mediated events. Previous results indicate that His+ events are elevated in rad52 mutant strains (CURCIO and GARFINKEL 1994 Down; CONTE et al. 1998 Down; LEE et al. 1998 Down), suggesting that Rad52p normally inhibits transposition. Although rad52 mutants show the most severe defects in homologous recombination, certain recombination events are only dependent upon a subset in the RAD52 epistasis group. Therefore, we examined the effect of mutations in other members of this repair pathway on Ty1 transposition. Isogenic strains bearing disruptions in the various rad genes were constructed by transplacement of the disrupted gene into the wild-type strain JC364, which has a chromosomally marked Ty1-270his3-AI element (Table 2). The presence of each mutation was confirmed by both phenotypic and physical analyses (see MATERIALS AND METHODS).

Qualitative transposition assays with strains bearing the different disruptions are shown in Figure 1, and quantitative analyses are shown in Table 3. The rate of His+ formation was 7.8 x 10-7 (events/cell/generation) in the wild-type strain JC364. We found a 24-fold increase in His+ events in a rad52 mutant, to a rate of 1.9 x 10-5. Although the actual level of stimulation varied when different members of the RAD52 epistasis group were analyzed, all mutants had increased levels of transposition.



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  		Figure 1. Qualitative measurement of His+ transposition events in wild-type and DNA-repair-deficient strains. Four individual colonies were patched to YEPD and were grown at 20° for 5 days, after which they were replica plated to SC-His and incubated at the indicated temperature for an additional 3 days prior to photographing. (A) Strains JC364, DG1644, yAR291, yAR306, DG1520, and yAR294. Only genotype of specific repair function disrupted in each strain is indicated. SC-His plates were incubated at 30°. (B) Strains JC364 and yAR348. SC-His plates were incubated at 26°.


 
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  		Table 3. Effect of recombination repair mutants on Ty1 transposition

Both RAD51 and RAD57 encode RecA homologs that are not functionally redundant (GAME 1993 Down; RATTRAY and SYMINGTON 1995 Down; BENSON et al. 1998 Down). Rad51p is necessary for cDNA conversion of a chromosomal Ty1 element, whereas Rad57p is not (NEVO-CASPI and KUPIEC 1994 Down; LIEFSHITZ et al. 1998 Down). We observed an 11-fold stimulation of transposition events in rad51 mutants (Table 3). rad57 mutants were similar to rad52 mutants, with a 21-fold stimulation in transposition (Table 3). The recombination defect of rad54 null mutants is most similar to that of rad51 mutants (GAME and MORTIMER 1974 Down; SAEKI et al. 1980 Down; CLEVER et al. 1997 Down), and RAD54 is also required for cDNA conversion of a chromosomal Ty1 element (LIEFSHITZ et al. 1998 Down). We found that transposition is elevated 5-fold in a rad54 mutant (Table 3). We also observed a 12-fold stimulation of transposition in a rad50 mutant (Table 3).

Mutants deficient in DNA ligase show increased levels of Ty1 transposition:
Because of the results obtained with mutants in the members of the RAD52 epistasis group, we hypothesized that unligated 3' ends adjacent to a newly transposed Ty1 element might initiate recombination with a sister chromatid or by single-strand annealing that would result in removal of the element or leave a solo {delta}, respectively. Therefore, increased transposition in cells deficient for homologous recombination could be due to the inability to remove nascent transposition events. On the basis of the hyper-recombination phenotype of a cdc9 mutant (GAME et al. 1979 Down), unligated 3' ends may survive longer in the absence of DNA ligase and may result in an even greater decrease in transposition in recombination-proficient cells. To test these ideas, we determined the level of Ty1his3-AI transposition in congenic strains with either the CDC9 wild-type or the cdc9-1 temperature-sensitive allele (see MATERIALS AND METHODS). Surprisingly, Ty1 transposition increased 38-fold in a cdc9-1 mutant (Table 3). This result suggests that nascent Ty1 transposition events are not removed by recombination.

rad1, rad2, and msh2 mutants do not show increased levels of transposition:
The stimulation of transposition by mutations in DNA repair functions of the RAD52 epistasis group, by a DNA ligase mutant, and by components of TFIIH, including alleles of the NER genes SSL2 (RAD25) and RAD3 (LEE et al. 1998 Down), led us to ask whether increasing transposition was a general phenotype of repair-deficient mutants. Therefore, we mutated the NER genes RAD1 and RAD2, as well as the MMR gene MSH2, and determined the level of Ty1his3-AI transposition. RAD1 is not required for cDNA conversion of a Ty1 element (NEVO-CASPI and KUPIEC 1996 Down), although it is required for cDNA conversion of a chromosomal his3 gene (DERR 1998 Down). We observed wild-type levels of Ty1 transposition in a rad1 null mutant (Figure 1; Table 3). RAD2 is required for the NER incision step (GUDZER et al. 1995 Down), and Rad2p coimmunoprecipitates with Msh2p (BERTRAND et al. 1998 Down). However, rad2 null mutants only exhibit minor defects in mitotic recombination (IVANOV and HABER 1995 Down). A mutation in rad2 did not alter transposition levels from the wild-type strain (Table 3).

Because the presence of DNA sequence heterogeneity among the different chromosomal Ty1 elements suggests that cDNA recombination may be sensitive to the MMR system, we reasoned that mutants in MMR might be more permissive for cDNA recombination and could result in elevated levels of Ty1 transposition or cDNA recombination. However, we observed wild-type levels of His+ formation in a msh2 null mutant (Table 3). In the same experiment, we measured spontaneous resistance to the antimetabolite canavanine and observed a 90-fold increase in the msh2 mutant. These results suggest that MMR does not affect the rate of Ty1 transposition, that the msh2 mutant strain does not contain a secondary msh2 suppressor, and that his3-AI does not revert even in a strain possessing a strong mutator phenotype.

In summary, we find that various DNA-repair-deficient mutants lead to elevated levels of Ty1his3-AI transposition. Three of the mutants are particularly interesting: a rad52 mutant is extremely deficient in mitotic recombination and repair and has a 24-fold increase in Ty1 transposition; a rad57 mutant is only moderately deficient in mitotic recombination and is increased for transposition 21-fold; and finally, a DNA-ligase-deficient cdc9 mutant, which has an elevated level of mitotic recombination, also increases the rate of transposition 38-fold.

Pattern of Ty1HIS3 insertions in a rad52 mutant:
Most Ty1HIS3 insertions occur at multiple sites and reflect simple insertions. However, certain Ty1 insertions are composed of multimeric elements, and their formation was shown to be RAD52 independent (WEINSTOCK et al. 1990 Down; SHARON et al. 1994 Down). To determine whether we were seeing a similar phenomenon or, alternatively, insertion into only one or a few sites, we examined ~100 independent His+ events by Southern blot analysis (in pools of 8) from the wild-type strain JC364 and the rad52 mutant strain DG1520. DNA was digested with PvuII, and Southern hybridizations were performed with a His3-specific probe, such that the hybridization represents the 3' junction of the Ty1His3 insertion (Figure 2). Multimeric arrays would result in a constant product of ~3 kb due to the junction fragment with a tandem Ty1 element. Although a band consistent with multimeric arrays was present in pools of the wild-type strain, this did not represent the majority of events, and there appeared to be even fewer bands migrating at the position expected for a multimeric array in the rad52 mutant (Figure 2). Furthermore, it was possible to count >=85 novel bands for each strain examined, indicating no extreme preference for an intergration or recombination sequence. As expected from the pool size, the genomic Ty1his3-AI element present in all samples hybridized with an approximately eightfold greater intensity than any single new transposition event (Figure 2). Similar results were obtained when ~100 His+ events were examined from rad51 or rad57 null mutant strains (data not shown).



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  		Figure 2. Southern blot analysis of independent His+ transpositional events in wild-type strain JC364 or in a rad52 mutant strain DG1520. DNA was isolated from pools of eight independent His+ colonies, digested with PvuII, separated by agarose gel electrophoresis, and probed with HIS3. The strong band marked his3-AI represents the endogenous Ty1his3AI-270. The arrow points to the expected position for multimeric arrays of Ty1 insertions.

Ty1 mRNA levels in repair-deficient mutants:
To determine whether Ty1 RNA levels were increased in the repair-deficient mutants, the steady-state levels of Ty1 RNA were examined by Northern blot analysis (see MATERIALS AND METHODS). Duplicate Northern blots were hybridized with 32P-labeled Ty1 or HIS3 probes. Both of these probes hybridize with Ty1 RNA, except that the former will hybridize with all Ty1 transcripts, whereas the latter is specific for Ty1his3-AI and Ty1HIS3 transcripts (Figure 3). Both blots were also hybridized with an actin probe as a loading control. We observed a 2.7-fold increase in the Ty1/actin RNA ratio in some of the strains. A smaller difference was noted among the different strains for the His3/actin ratio, which in all cases was <2-fold greater than that observed in the wild-type strain.



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  		Figure 3. Analysis of Ty1 RNA in DNA-repair-deficient strains. Northern blots were probed with either antisense Ty1 and antisense actin ssRNA probes (A) or with sense His3 and antisense actin ssRNA probes (B). Strains JC364, DG1520, yAR294, yAR306, yAR292, and yAR348. Only the relevant differences in genotype are shown. Hybridization signals were quantitated by phosphorimage analysis, and the Ty1/actin or His3/actin ratios were normalized to the wild-type control, which is considered to be 1x.

Ty1 gene expression:
Because Ty1 RNA may be packaged within VLPs and therefore inaccessible to both RNA degradation and translation, a small increase in de novo transcription could lead to more efficient translation. We addressed this question by determining the level of Ty1 gene expression utilizing TyA1/TyB1::lacZ fusion plasmids and by determining the amount of TyA1 protein present in the repair-deficient mutants. ß-Galactosidase levels were measured in whole cell extracts from two different TyB1::lacZ fusion plasmids. All of the strains also harbored a genomic Ty1 element with a TyB1::lacZ fusion construct in the genome (Ty1-146, Table 2). The strains were also transformed with either plasmid pMB38-9mer (wild type), where the TyA1 and TyB1 coding sequences are in their natural configuration, and lacZ translation requires a +1 frameshift near the end of the TyA1 open reading frame (BELCOURT and FARABAUGH 1990 Down), or with plasmid pMB38-9mer (fusion), where the TyA1/TyB1::lacZ is expressed as a single open reading frame (BELCOURT and FARABAUGH 1990 Down). The genomic Ty1-146 element was expressed at extremely low levels (no plasmid, Table 4) and was measured to ensure that it was not contributing significantly to the ß-galactosidase activity derived from the plasmid-borne constructs. No significant differences were observed in any of the recombination repair mutants harboring plasmid pMB38-9mer (fusion). We conclude that the small differences detected in our Northern blot analysis are unlikely to contribute significantly to the translation efficiency of Ty1 RNA in the repair-deficient mutants. We also did not detect a significant difference in ß-galactosidase for the pMB-9mer (wild-type) plasmid. As expected, ß-galactosidase activity observed in pMB-9mer (wild-type) is ~10-fold lower than that observed in pMB-9mer (fusion), due to the programmed frameshifting event required for translation of lacZ (CLARE et al. 1988 Down; BELCOURT and FARABAUGH 1990 Down). Therefore, Ty1 frameshifting does not appear to be affected in the mutant strains. Taken together, these results suggest that the rates of protein synthesis in the repair-deficient strains and in the wild-type strain are similar.


 
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  		Table 4. ß-Galactosidase assays with Tyb::LacZ fusions

Ty proteins in repair-deficient mutants:
Total protein was isolated from both wild-type cells and from repair-deficient cells, separated by SDS-PAGE electrophoresis, and transferred to membranes for Western blot analysis. The blots were first probed with a polyclonal antiserum directed against total Ty1-VLPs, which primarily recognizes TyA1 protein (GARFINKEL et al. 1991 Down). After immunodetection and phosphorimage analysis, the blots were stripped and reprobed with antiserum directed against the histidyl tRNA synthetase (Hts1p) protein, which is abundantly expressed in yeast cells and is not expected to vary among the repair-deficient strains. We calculated the ratio of TyA1 to Hts1p for each of the strains, and divided this by the ratio in the wild-type strain. As a control, we also included an spt3 mutant, which, as expected, has undetectable levels of TyA1 protein, but shows equivalent levels of Hts1p (Figure 4A). Phosphorimage analysis indicates that the level of TyA1 protein remains at the wild-type level in the different repair-deficient strains. Because of the low abundance of TyB1 proteins, we were unable to detect these proteins from total protein extracts.



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  		Figure 4. Western blots of Ty1 proteins in DNA-repair-deficient strains. (A) Electrophoretically separated and transferred aliquots of total protein extracts were probed with antiserum generated against VLPs, which mainly recognizes the TyA p54 and p58 proteins. The blots were then reprobed with antiserum against the abundant heat-shock protein Hts1p. The relative ratio of the signals for anti-Ty to anti-Htsp1 quantitated by ECF and phosphorimage analysis was normalized to 1x for the wild-type strain (JC364). (B) VLP preparations from repair-deficient strains. Genotype is shown only for the relevant DNA repair gene mutated in each strain.

Endogenous Ty1-VLPs were partially purified from total protein extracts from each of the repair-deficient mutants by sucrose sedimentation. Ty1 proteins do not constitute a major component of the sucrose-enriched fractions when purified from uninduced cells; however, the enrichment was sufficient to detect TyB1 proteins. A Western blot of the partially purified VLPs was prepared and was probed with antiserum directed against IN (b2). After immunodetection by ECL, the blot was stripped and was probed with antiserum directed against RT (b8), and immunodetection was also performed using ECL. The blots were then stripped a second time and were probed with anti-TyA1 antiserum, followed by ECL immunodetection. The presence of IN, RT, and TyA1 in VLPs prepared from wild-type, rad50, rad52, rad57, and cdc9 strains is shown in Figure 4B. Note that although the same amount of total protein was loaded for each strain, the relatively minor contribution from VLPs in these partially purified preparations resulted in significant variation of Ty1 proteins from one preparation to another (perhaps dependent upon the efficiency of breakage with glass beads; CONTE et al. 1998 Down). We also measured the reverse transcriptase activity across the sucrose gradient, and found that the amount of activity, although low, accurately reflected the relative amount of VLP proteins present in each of the preparations (data not shown).

Analysis of cDNA levels in repair-deficient mutants:
To determine whether the increased level of transposition in the repair-deficient mutants was due to increased levels of Ty1 cDNA, cells were grown to mid-to-late log phase, and DNA was isolated by a gentle procedure to minimize shearing of the genomic DNA (see MATERIALS AND METHODS). Unincorporated linear Ty1 cDNA is ~6 kb in size and should migrate below the bulk genomic DNA. Undigested DNA was transferred to nylon filters following electrophoresis and the resulting blots were hybridized with 32P-labeled probes for Ty1 or His3 sequences (Figure 5A). After autoradiography and phosphorimage analysis, the blots were stripped and rehybridized with a 32P-labeled 2-µm plasmid probe (Figure 5B). We used 2-µm DNA as an internal standard because it migrates separately from the total genomic DNA, as does Ty1 cDNA. As a control, we also included an spt3 strain, which should contain very little Ty1 cDNA. Our relative proportions of cDNA are calculated as the ratio of His3 to 2-µm signal from each strain, divided by this ratio from the wild-type strain (such that the wild-type strain is equal to 1), and these numbers are shown below each lane (Figure 5B). A similar blot was probed with a 32P-labeled Ty1 probe, stripped, and rehybridized with the 2-µm plasmid probe. The ratio of the Ty1 to 2-µm signal (relative to the wild type) is shown in Figure 5C. Whereas the cDNA levels in the wild-type strain were not significantly above the spt3 negative control, there was a significant amount of cDNA detectable in all of the repair-deficient mutants examined. Therefore, our quantitation probably is a minimum estimate for the fold increase of cDNA detected in the repair-deficient mutants over wild type. We conclude that all of these mutants have significantly increased amounts of Ty1 cDNA.



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  		Figure 5. cDNA quantitation from repair-deficient strains. Southern blot of total undigested DNA isolated from agarose plugs. (A) his3-AI probe. (B) 2-µm plasmid probe. Numbers below B indicate the quantitation of cDNA calculated by dividing the ratio of his3 counts per minute to 2-µm cpm for each strain by the same ratio for the wild-type strain (such that the wild-type ratio is equal to 1x). (C) Quantitation from similar blots, where Ty1 internal sequences were used as the probe. Genotype is shown only for the relevant DNA repair gene mutated in each strain.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

The results presented here permit two conclusions and raise several important issues concerning how Ty1 elements and their yeast host coexist. First, all of the members of the RAD52 epistasis group examined inhibit Ty1 transposition post-translationally by limiting the accumulation of unincorporated cDNA. Second, our results suggest that the MMR pathway does not inhibit Ty1 transposition and reinforce earlier work indicating that the NER pathway also is not inhibitory (LEE et al. 1998 Down). The S. cerevisiae genome contains ~30 dispersed copies of Ty1, most of which are competent for transposition (CURCIO et al. 1988 Down; CURCIO and GARFINKEL 1994 Down; JORDAN and MCDONALD 1998 Down; KIM et al. 1998 Down). Paradoxically, Ty1 RNA is very abundant (ELDER et al. 1983 Down; CURCIO et al. 1990 Down), yet Ty1 transposition is a rare event (CURCIO and GARFINKEL 1991 Down). There are multiple steps during retrotransposition that could be modulated by host functions. In general terms, Ty1 transposition involves transcription of the element, synthesis of Ty1 proteins, packaging of Ty1 RNA and met-tRNA into VLPs, maturation of VLP proteins, reverse transcription to produce a double-stranded linear cDNA, transport of the cDNA into the nucleus, and finally, incorporation of the cDNA into the genome by either Ty1 IN or cellular homologous recombination functions (for reviews see BOEKE and SANDMEYER 1991 Down; SANDMEYER 1992 Down). Host functions that destabilize Ty1 proteins (CONTE et al. 1998 Down), minimize insertional mutagenesis of genes transcribed by RNA polymerase II (LIEBMAN and NEWNAM 1993 Down; HUANG et al. 1999 Down), and prevent the accumulation of Ty1 cDNA (LEE et al. 1998 Down) have been identified.

Given that DNA-repair-deficient mutants are likely to accumulate higher levels of endogenous unrepaired DNA lesions than repair-proficient cells and that Ty1 transcript levels are increased in cells that have been treated with DNA-damaging agents (MCCLANAHAN and MCENTEE 1984 Down, MCCLANAHAN and MCENTEE 1986 Down; ROLFE 1985 Down; RUBY and SZOSTAK 1985 Down; BRADSHAW and MCENTEE 1989 Down), it is formally possible that repair-deficient cells are induced for Ty1 gene expression. For example, rad52 mutant cells constitutively transcribe the DNA damage response gene, DDR2, whose message is normally undetectable in the absence of DNA damage (MCCLANAHAN and MCENTEE 1986 Down). Also, temperature-sensitive mutants of DNA ligase I, cdc9-1, grow slowly (TOMKINSON et al. 1993 Down) and have increased mitotic recombination (GAME et al. 1979 Down) even at the permissive temperature. Both of these phenotypes may result from an increase in nicked DNA created during DNA replication. However, we do not observe a significant change in the RNA transcript levels of either the his3-AI-marked Ty1 or of Ty1 elements in any of the repair-deficient strains examined.

We have examined the accumulation of Ty1 proteins to determine whether members of the RAD52 epistasis group affect protein accumulation. Mutants of the haploid-specific mitogen-activated protein (MAP) kinase fus3 also have elevated rates of transposition (CONTE et al. 1998 Down), and Ty1 proteins are greatly stabilized in the absence of FUS3, suggesting that Fus3 phosphorylation leads either directly or indirectly to degradation of Ty1 proteins. A similar mechanism is probably not operating in the DNA-repair-deficient mutants, because the total amount of Ty1 proteins and the ratio of TyA1/TyB1 proteins remain unchanged in the mutants (Figure 4). However, we have not measured the rates of synthesis of the various proteins, or their half-lives, so we cannot rule out subtle changes in Ty1 protein synthesis or stability.

The finding that Ty1 transposition increases in a cdc9 mutant is surprising and has led us to consider the possibility that Ty1 might transpose to sites of DNA damage. Interestingly, Ty sequences are occasionally found at the sites of DSBs when homologous recombination is blocked either by a rad52 mutation or by lack of a homologous donor sequence (MOORE and HABER 1996 Down; TENG et al. 1996 Down; GARFINKEL 1997 Down), and mutants in the rad52 epistasis group could have elevated levels of spontaneous unrepaired DSBs. However, the capture of Ty sequences at the sites of unrepairable DSBs is rare (MOORE and HABER 1996 Down; TENG et al. 1996 Down), does not include intact Ty elements, and requires the RAD50 gene (TENG et al. 1996 Down). Because we find that a null rad50 mutant also results in an elevated level of Ty1 transposition, it is unlikely that our observed increase in His+ frequency is due to "rescue" of DNA damage by Ty1 elements.

A striking correlation exists between increased transposition in DNA-repair-deficient mutants and increased levels of unincorporated Ty1 cDNA. A similar phenotype is observed in certain mutants of the TFIIH RNA polymerase II complex, which is involved in both RNA polymerase II transcription inititation and in NER (ORPHANIDES et al. 1996 Down; SVEJSTRUP et al. 1996 Down). One allele of the essential gene SSL2/RAD25 has been isolated in a screen for hypertransposition mutants (LEE et al. 1998 Down). Additional mutant alleles of this gene as well as in another component of TFIIH, RAD3, also cause increased Ty1 transposition and Ty1 cDNA accumulation, but do not affect Ty1 RNA or proteins. Because other alleles of SSL2 and RAD3 that are defective for NER do not result in hypertransposition or increased cDNA levels, the NER functions of TFIIH are not required to inhibit Ty1 transposition (LEE et al. 1998 Down). Here, we provide additional evidence that NER functions are not important for inhibiting Ty1 transposition, because null mutants of RAD1 and RAD2, which are required for NER but are not part of the core TFIIH complex, do not cause high levels of Ty1 transposition.

To gain insight into the mechanism by which homologous recombination and DNA repair functions might be affecting the accumulation of Ty1 cDNA, we consider several possible common similarities between a Ty1 cDNA molecule and a recombination or repair substrate. A DNA DSB, produced for example by endonucleases or ionizing radiation, is a potent recombination initiator (GAME and MORTIMER 1974 Down; STRATHERN et al. 1982 Down; RUDIN et al. 1989 Down; MCGILL et al. 1993 Down), whose repair is strongly dependent upon the genes of the RAD52 epistasis group (GAME 1993 Down; SUGAWARA et al. 1995 Down). One feature that a Ty1 cDNA molecule shares with a DSB is the presence of (nontelomeric) DNA ends. Physical analyses of a DNA molecule after the introduction of a DSB in vivo show degradation of the 5' ends of the DNA from the site of the break (STRATHERN et al. 1982 Down; SUN et al. 1989 Down; CAO et al. 1990 Down; HABER 1995 Down). Normal early degradation of the 5' ends from the site of a DSB is still seen in mutants of the rad52 epistasis group, but is generally far more extensive, and recombinant products are formed more slowly (SUGAWARA et al. 1995 Down; IVANOV et al. 1996 Down).

Perhaps there is a competition for cDNA ends between the DNA repair machinery and Ty1 IN. Limited IN DNA-binding activity might be insufficient to protect many of the cDNA ends, and degradation of the ends by the DNA repair machinery would remove sequences required for IN recognition. We propose that even if most cDNA molecules are acted upon by the DNA homologous recombination machinery, most do not result in His+ cells via homologous recombination even though Ty1 cDNA can act as a donor in recombination (MELAMED et al. 1992 Down; DERR and STRATHERN 1993 Down; SHARON et al. 1994 Down; NEVO-CASPI and KUPIEC 1996 Down). Although a broken molecule can act as a donor in a homologous recombination event, such as in one-step gene replacement (ORR-WEAVER et al. 1981 Down; ROTHSTEIN 1983 Down), it is the recipient of information >90% of the time (STRATHERN et al. 1982 Down; MCGILL et al. 1993 Down).

cDNA conversion of a chromosomal Ty1 element is dependent on RAD52, RAD51, and RAD54, but not on RAD57 (LIEFSHITZ et al. 1995 Down). If we assume that the ability to convert a chromosomal Ty1 element is related to the ability of a cDNA to function as a donor of His+ events, then we might expect that in rad52 mutants this pathway would be unable to lead to His+ events, whereas in rad57 mutants His+ events should only be marginally affected. However, we see similar increases in unincorporated cDNA and Ty1his3AI transposition in both rad52 and rad57 mutants. Therefore, cDNA conversion of chromosomal Ty1 elements is probably not contributing to His+ events in our experiments. Furthermore, events resulting in His+ cells in a rad52 mutant are most likely transpositional insertions mediated by IN (SHARON et al. 1994 Down; LIEFSHITZ et al. 1995 Down; NEVO-CASPI and KUPIEC 1997 Down). Although Rad52p and Rad57p may both be components of the same recombination complex, we cannot rule out the possibility that the increased level of cDNA in the absence of Rad52p or Rad57p is caused by different mechanisms. Interestingly, a rad57 null mutant is reasonably competent for homologous recombination and DNA repair at 30°, but is impaired for both recombination and repair at 20° (LOVETT and MORTIMER 1987 Down; KANS and MORTIMER 1991 Down; RATTRAY and SYMINGTON 1995 Down). Ty1 transposition is also a temperature-sensitive process, with a temperature optimum between 15° and 20° (PAQUIN and WILLIAMSON 1984 Down). Perhaps the recombination complex forms inefficiently at 20°, and Rad57p acts by stabilizing the complex. It would be interesting to determine whether the level of Ty1 cDNA is different at 20° and at 30° in a rad57 mutant strain.

We next consider the possibility that many of the cDNA molecules do serve as donors for conversion of chromosomal Ty1 elements, but that encountering the nonhomology provided by the HIS3 gene aborts recombination, leading to a reversal of the initiation event (thus preserving the cDNA molecule). We consider this "recombination inititation/abortion" model unlikely because the increase in unincorporated cDNAs observed in the repair-deficient mutants is not specific for the his3-AI-marked element, but is also observed for unmarked Ty1 cDNAs. Furthermore, Msh2p and Msh3p have been shown to play a role in aborting or resolving certain recombination events (PAQUES and HABER 1997 Down; SUGAWARA et al. 1997 Down). However, a msh2 null mutation does not stimulate Ty1his3-AI transposition. Finally, the RAD1 gene is required for cDNA-mediated conversion of a chromosomal target (DERR 1998 Down) and for some cDNA-mediated conversion events of genomic Ty1 elements (NEVO-CASPI and KUPIEC 1996 Down). If we assume that cDNA conversion of a genomic Ty1 element in our experiments also requires RAD1, then we would expect a large decrease in His+ events in a rad1 mutant. However, we observe that a rad1 null mutant exhibits wild-type levels of Ty1 transposition. Therefore, unproductive or productive cDNA conversion of chromosomal Ty1 elements does not appear to contribute significantly to the His+ events measured here.

We have not directly determined whether the increase in cDNA levels is due to increased cDNA synthesis or increased cDNA stability. Above, we have argued in favor of increased cDNA stability, in keeping with our knowledge about the process of DNA repair. Furthermore, we saw no stimulation in reverse transcriptase protein or activity in VLPs from the different mutants.

Now that the complete sequence of an S. cerevisiae laboratory strain is known, we can understand further the natural history of Ty elements and how transpositional dormancy is established and maintained. Most of the Ty1 elements appear to be functional, and codon bias analysis suggests that they are under selection (JORDAN and MCDONALD 1998 Down, JORDAN and MCDONALD 1999 Down; CURCIO and GARFINKEL 1999 Down). In addition, Ty1 elements have a narrow distribution and/or copy number within the genus Saccharomyces (FINK et al. 1981 Down; K. G. WEINSTOCK, D. J. GARFINKEL and J. STRATHERN, unpublished results), and within wild isolates of S. cerevisiae (EIBEL et al. 1981 Down; WILKE et al. 1992 Down). Together, these results suggest that Saccharomyces has efficient mechanisms to keep Ty1 elements out of the yeast genome and to largely inhibit its transpositional activity once the element is established (CURCIO and GARFINKEL 1999 Down). Perhaps one reason that Saccharomyces has evolved such an efficient DNA repair system is to prevent both horizontal transmission and subsequent uncontrolled retrotransposition of Ty elements.


*  ACKNOWLEDGMENTS

The authors thank Joan Curcio, Dwight Nissley, Jeffrey Strathern, and Bum Soo Lee for discussions, ideas, and for critical review of the manuscript. We also thank Sharon Moore, Lori Rinckel, and Shirong Zhang for sharing reagents and providing technical guidance, and Richard Fredrickson for the artwork. This research was sponsored by the National Cancer Institute, Department of Health and Human Services (DHHS), under contract with ABL. The contents of this publication do not necessarily reflect the views or policies of the DHHS, nor does any mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Manuscript received June 18, 1999; Accepted for publication October 1, 1999.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
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