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Department of Biology, Juniata College, Huntingdon, Pennsylvania 16652
5 Corresponding author: Department of Biology, Juniata College, 1700 Moore St., Huntingdon, PA 16652.
E-mail: keeney{at}juniata.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The yeast Saccharomyces cerevisiae is host to several families of Ty transposable elements. Ty1 consists of two long terminal repeats (LTRs) flanking a region of DNA that encodes the structural proteins and enzymes necessary for replication and integration (VOYTAS and BOEKE 2002). The open reading frames (ORFs) encode Gag and Pol, functionally similar to Gag and Pol of retroviruses. Gag encodes structural proteins and Pol encodes the enzymes integrase (IN), reverse transcriptase (RT), and protease (PR). Transposition begins with the transcription and translation of the element by the host cell machinery. The Gag and Pol regions are translated as polyproteins, which are subsequently processed by PR. Virus-like particles (VLPs) are likely assembled from unprocessed Gag and Gag-Pol and enclose the Ty1 mRNA, primer, PR, RT, and IN. Within these VLPs, reverse transcription takes place, synthesizing the cDNA that is then integrated into the host genome via an IN-mediated integration reaction. Alternatively, cDNA sequences resulting from reverse transcription may be recombined with chromosomal Ty1 sequences in a RAD52-dependent recombination/repair process. The life cycle of a retrotransposon does not include an infectious extracellular stage comparable to that of retroviruses.
In most common lab strains of S. cerevisiae, 
30 copies of the Ty1 element are present. Studies have shown that Ty1 RNA is present as 0.1–0.8% of the total RNA, yet processed Ty1 proteins are difficult to detect when uninduced (CURCIO and GARFINKEL 1991a). Transposition events, therefore, normally occur at very low levels, 10–5–10–7 events per generation (CURCIO and GARFINKEL 1991b). Overexpression of a plasmid Ty1 element by induction with a galactose promoter (GAL1), however, shows a dramatic increase in the appearance of transposition events (10,000–28,000-fold higher) with comparatively little increase in Ty1 RNA and protein levels (220- to 225-fold higher; CURCIO and GARFINKEL 1992). Galactose-induced Ty1 transposition, therefore, overcomes the "transpositional dormancy" that normally characterizes endogenous Ty1 elements and does so by improving the efficiency of Ty1 RNA and protein use, rather than by simply increasing the amounts of these products. This indicates that the transpositional dormancy of endogenous Ty1 elements is most likely regulated by post-translational mechanisms.
There are numerous examples of endogenous host genes involved at multiple levels to limit transpositional activity. Transcriptional expression of endogenous elements has been shown to vary over a 50-fold range, and Ty1 elements located within the rDNA region are silenced (BRYK et al. 1997; MORILLON et al. 2002). Activation of the invasive-filamentous pathway in diploid cells induces Ty1 transcription (MORILLON et al. 2000). Protein processing has been shown to increase with galactose induction of Ty1, suggesting that PR may not be adequately activated without GAL induction (CURCIO and GARFINKEL 1992). The nucleotide excision repair/transcription factor IIH (NER/TFIIH) complex inhibits Ty1 cDNA accumulation and FUS3 destabilizes VLP-associated proteins (LEE et al. 1998; CURCIO and GARFINKEL 1999; CONTE and CURCIO 2000). Chromatin assembly factor I and histone regulatory proteins maintain a chromatin structure that prevents integration of Ty1 cDNA and mutations in the RAD52 epistasis group demonstrate increased transposition and a marked increase in the level of Ty1 cDNA (QIAN et al. 1998; RATTRAY et al. 2000). The nature of these interactions indicates that cellular processes affecting repair and differentiation control levels of transposition in response to environmental signals (CURCIO and GARFINKEL 1999).
Indeed, Ty1 transposition is regulated by changes in the temperature of the environment. Transposition is optimal at 24–28°. Increases in temperature to 33–35° decrease transposition to undetectable levels (PAQUIN and WILLIAMSON 1984; LAWLER et al. 2002). We have previously shown that at high temperatures the Ty1 polyproteins are not cleaved, indicating that PR is no longer active (LAWLER et al. 2002). In this study, we describe a genetic screen used to identify mutations in host genes that allow transposition at high temperature. Following chemical mutagenesis, yeast cells were plated and colonies subsequently identified for transposition at 37°. Several mutant strains were isolated, which demonstrate higher transposition levels than the isogenic wild-type strain at temperatures >34°. We have named the mutants htt, for high-temperature transposition. In this study we describe the cloning and identification of one of the mutants as sir4. SIR4 is a silent information regulator gene implicated in many cellular processes, including cell aging and chromatin silencing, and has previously been shown to regulate integration of the Ty5 retroelement of yeast (ZHU et al. 1999; GARTENBERG 2000; GASSER and COCKELL 2001; XIE et al. 2001). The results presented here are the first implication of SIR4 regulation of Ty1.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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TRP1 strain construction:
The analysis of transposition in spores of mutant crosses was found to be complicated by the genetics of the TRP1 locus. Strain YPH645 contains a wild-type TRP1 locus. The trp1
1 mutation, which is present in our mutant strains, removes a portion of the upstream activating sequence (UAS) of the GAL3 gene, an inducer of the galactose catabolic pathway. As a result, the level of galactose-induced transposition in trp11 strains is slightly reduced. At high temperature, the difference in galactose induction between TRP1 and trp11 strains is even more pronounced, such that the level of transposition in TRP1 stains is significantly greater than that in strains harboring trp11. To make the high-temperature phenotype stronger, the trp11 locus in each of the htt mutant strains was replaced with the wild-type TRP1 sequence. The Trp+ strains JKc1005 and JKc1015 were constructed by transforming strains JKc125 and YH8, respectively, with a 2697-bp PCR fragment derived from a TRP1 strain. The fragment contains the deleted TRP1 region, 700 bp 5' flanking sequence, and 545 bp 3' flanking sequence. PCR was used to confirm that the TRP1 locus was properly regenerated. Primer sequences for PCR were 5'-TTTTAAAGCGGCTGCTT GAG-3' (forward primer) and 5'-AAGGCAGATCAAACTTTCGC-3' (reverse primer).
Library transformation and cloning:
Yeast strain JKc1005 was transformed with a p366 LEU2 CEN library obtained from American Type Culture Collection, Rockville, Maryland (SCHIESTL and GIETZ 1989). Approximately 1.7 x 105 transformants were selected on SC-Leu and screened for restoration of mating. For each transformation plate, a lawn consisting of a MATa his1 strain was replica plated to YPD, followed by a replica plating of a transformation plate to the same YPD plate. The YPD plates were incubated at 30° for 5 hr, allowing sufficient time for mating to occur. Cells were then replica plated to minimal media and incubated at 30° until colonies appeared. Positive colonies from the original transformation plates were tested for the loss of high-temperature transposition.
Sequencing:
The LEU2 library plasmid was rescued into Escherichia coli strain MC1666 (pyrF trpC LeuB) and sequenced using primer JK048 (5'-CACTATCGACTACGCGATCA-3'). To determine the location of the cloned DNA, the resulting sequence was compared to the yeast genome using BLASTN on the Saccharomyces Genome Database website (http://genome-www.stanford.edu/Saccharomyces/). To identify the mutant sir4 allele, genomic DNA was isolated from strain JKc1003. The sir4 locus was amplified by PCR in two 2-kb halves using primers JK125/JK126 (5'-AAAAAAGGAAGCTTCAACCCAC-3'/5'-TCAGTTAGGCTATCATTATCTGAAGA-3') and JK127/128 (5'-CCTTTCAATAAAAGTGAAAGCAAACC-3'/5'-AGAAAAACAGGGTACACTTCGTTAC-3'). The resulting PCR products were cloned into the vector pCR2.1-TOPO vector by TOPO cloning (Invitrogen, Carlsbad, CA). DNA was prepared from resulting clones and the two halves of the sir4-119 allele were sequenced using the following primers: JK125, JK111 (5'-ACTCATTTTTATCAGGAG-3'); JK112 (5'-TAACATCAAAGAAGATCG-3'); JK113 (5'-GAATCCCACATTGATTCG-3'); JK127, JK114 (5'-AGCCAATTTTTTGGAAAC-3'); and JK115 (5'-ACCTTATTGAACAAGGGA-3').
rad52 strain construction:
Plasmid pSM20, containing the rad52::LEU2 allele, was digested with BamHI; digestion was confirmed by gel electrophoresis, yielding two bands: 5 and 4.6 kb. The remaining digest was transformed into yeast (SCHIESTL and GIETZ 1989), and cells were plated to SC-Leu medium to select for integration of the disrupted rad52 allele. Isolated transformants were confirmed by UV-induced growth sensitivity as compared to the isogenic RAD52 parent.
FUS3 plasmid construction and spl2::kanMX4 and fus3::kanMX4 strain construction:
Plasmid pCS1 was constructed by cloning an 1 kbp NotI/HindIII fragment containing FUS3 from pJL70 (kindly provided by Joe Lawler) into the same sites of pRS425 (SIKORSKI and HIETER 1989). The spl2::kanMX4 and fus3::kanMX4 alleles were PCR amplified using genomic DNA from Research Genetics (Birmingham, AL) strains 1964 and 3042, respectively. The primer pair used for spl2::kanMX4 was JK123/JK124 (5'-TTCCTACCCCAATGATGGTT-3'/5'-GTGGCGGTCATCGAAGAT-3') and the primer pair used for fus3::kanMX4 was JK121/JK122 (5'-CGTTCAAAAGAACATACATAAGGA-3'/5'-CACAAG ACAAAAGAAGGGGTAG-3'). The resulting PCR product from each strain was then transformed into strains JKc1005 and JKc1015. Following transformation, the cells were washed in water and then resuspended in 1 ml YPD medium. Cells were grown for 8 hr, at 30°, with shaking, to allow for expression of the kanMX4 gene. Cells were then plated to YPD containing 75 ug/ml G418. Replacement of the wild-type allele was confirmed by PCR of genomic DNA from G418-resistant colonies.
Transposition assays:
For patch assays using plasmids pGTy1H3mhis3AI or pX3, strains were grown as 12 x 12-mm patches on SC-Ura medium to maintain plasmid selection. Cells were then replica plated to galactose medium and incubated at the appropriate temperature for 24–48 hr to induce transposition. Following galactose induction patches were printed to SC-His medium for pGTy1H3mhis3AI assays or to YPD medium, followed by replica plating to 5-FOA and finally to SC-Trp for pX3 assays. For quantitative assays using plasmid pGTy1H3mhis3AI, strains were initially grown as 12 x 12-mm patches on SC-Ura medium to maintain plasmid selection. Cells were then replica plated to galactose medium and incubated at the appropriate temperature for 44–48 hr to induce transposition. Following galactose induction patches were printed to SC-His medium. The cells remaining on the galactose plates were transferred to 10 ml sterile water (dilution 1). A total of 50 µl of this dilution was transferred to 5 ml sterile water (dilution 2). A total of 100 µl of dilution 1 was plated to SC-His medium, except for temperatures >32°, in which case cells in dilution 1 were pelleted, resuspended in 200 µl water, and plated to SC-His. A total of 50 µl of dilution 2 was plated to YPD. Following incubation at 30° (YPD 2 days; SC-His 4 days) resulting colonies were counted. Transposition frequency was calculated by dividing the number of colonies on SC-Hisby the total number of cells plated on SC-His, as determined by the colony number on YPD, factoring in the dilution and original volume of dilution 1 plated.
Cell homogenates:
The cell growth procedure was based on a protocol previously described (MERKULOV et al. 1996). Specifically, cultures were grown at either 22° or 37°. The starting density was A600 of 0.2 and cells were collected when the density reached an A600 of 2 (12 hr at 37° and 36 hr at 22°). Aliquots (1.5 ml) of the culture were collected by centrifugation, resuspended in 40 µl of buffer B [10 mM N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/KOH, pH 8.0, 15 mM KCl, 5 mM EDTA], and frozen at –75°. Aliquots were thawed on ice and the total volume was brought to 200 µl with buffer B. Cold glass beads were added to the meniscus. A total of 40 µl of 100% trichloroacetic acid (TCA) was added and the samples were vortexed at top speed for 4 min. Samples were placed immediately on ice, and 1 ml ice-cold 5% TCA was added. Samples were spun for 20 min, 14,000 x g, in the cold. The liquid was aspirated and the pellet was resuspended in 1 ml cold water. Samples were spun for 10 min as before and the supernatant was aspirated. Proteins were extracted by resuspending the pellet in 150 µl sample buffer (6% SDS, 0.5 M Tris base) and incubating at 50° for 10 min. Samples were spun for 1 min (14,000 x g) and the supernatant was removed to a fresh tube. The extraction process was repeated and the supernatants were pooled. One-third volume of a solution of 0.25 M DTT, 50% glycerol, and 0.2% bromphenol blue was added and samples were spun (14,000 x g) for 3 min. The supernatant was transferred to a fresh tube.
VLP preparation:
Cells were grown and lysed as described (EICHINGER and BOEKE 1988). Extract (7.5 ml) was loaded onto a 70/30/20 (5/5/15 ml) sucrose step gradient and centrifuged for 180 min at 28,000 rpm in a Sorvall AH629 swinging bucket rotor. The remaining extract was saved for use as whole-cell extract. Fractions were collected by puncturing the bottom of the tube and collecting 1.2-ml fractions. To pellet VLPs, peak fractions 4, 5, and 6 were pooled, diluted to 11 ml with buffer B, and pelleted for 1 hr, at 35,000 rpm in a Sorvall A1256 fixed-angle rotor. The pellet was resuspended in 150 µl buffer B.
Immunoblotting:
Whole-cell extracts and purified VLPs were mixed with an equal volume of 2x sample loading buffer (20% v/v glycerol, 0.125 M Tris-Cl pH 6.8, 5% w/v SDS, 10% v/v 14 M ß-mercaptoethanol, 0.2% w/v bromphenol blue) and boiled (3 min) prior to loading on 10% (Gag blots) or 7.5% (Pol blots) SDS gel. Gels were transferred to nitrocellulose (for Gag blots) or polyvinyl difluoride (PVDF) membrane (for Pol blots) in Tris-glycine buffer containing 10% methanol at 24 V for 1 hr. Membranes were blocked in PBS containing 5% nonfat dried milk. Membranes were then probed with antibody as described (MERKULOV et al. 2001). Antibody binding was detected using the appropriate secondary antibody followed by ECL (nitrocellulose) or ECL-Plus (PVDF) reagent and exposed to X-ray film. Anti-Gag (anti-VLP polyclonal serum R2-F) and anti-IN (8B-11 monoclonal antibody) are described elsewhere (EICHINGER and BOEKE 1988; MONOKIAN et al. 1994).
cDNA Southern blotting:
Cells were grown as described (EICHINGER and BOEKE 1988), except that galactose induction was at various temperatures. Cells from 10 ml of galactose-induced culture were frozen and genomic DNA was isolated as described (KEENEY and BOEKE 1994). A total of 10 ul of DNA was digested in a 40-ul reaction containing 1 ul RNase (10 mg/ml). Samples were electrophoretically separated (0.75% agarose gel), transferred to Gene Screen, and hybridized to a 520-bp XhoI-HindIII neo probe from pJEF1105 labeled with the Amersham (Arlington Heights, IL) Bioscience Rediprime II random prime labeling system. The membrane was washed twice for 5 min in 2x SSC at room temperature and once for 30 min in 2x SSC, 0.1% SDS at 65°, and exposed on the Kodak Image Station 2000R using the rad PADD (high signal capture).
Recombination assays:
The plasmid pJK592 (LEU2, HIS3, CEN) was constructed by cloning a 1145-bp PstI/BamHI HIS3 fragment from pJJ217 into the same sites of pRS415 (SIKORSKI and HIETER 1989; JONES and PRAKASH 1990). Undigested plasmid or plasmid digested at the NdeI site within the HIS3 ORF was transformed as described (SCHIESTL and GIETZ 1989). For cotransformation of linear DNA, a 345-bp PCR product containing HIS3 sequences flanking the NdeI cut site was generated using oligonucleotides JK177 (5'-CAGAAAGCCCTAGTAAAGCGT-3') and JK178 (5'-TCCAAACCTTTTTACTCCACG-3') on BamHI-digested pJJ217 plasmid template DNA. Transformants were selected on SC-leucine medium. Integrity of the HIS3 ORF was assessed by replica plating to SC-histidine medium.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The mutants were crossed with an otherwise isogenic MATa strain (JB721) to determine if the htt mutations were recessive or dominant and contained within a single-gene locus. Unfortunately, the strains were found to have a very poor sporulation frequency. The mutant strains were therefore crossed to the nonisogenic strain YPH645, and at least six complete tetrads were obtained for each mutant. All of the mutants were confirmed to be recessive, single-gene mutations and showed comparable levels of high-temperature transposition.
Cloning of htt119 (sir4):
During tetrad analysis, the htt119 mutant was found to have a mating defect that segregated with the htt phenotype. Thus, the mutant gene was cloned via complementation of the mating defect by transformation of a wild-type LEU2 genomic library. Of seven putative positive clones, loss of the LEU2 library plasmid resulted in mating deficiency and restoration of the htt phenotype in all seven clones, indicating that a gene located on the library plasmid was likely responsible for restoration of mating and for repression of the high-temperature transposition phenotype. Rescue of the library plasmid and retransformation into the original htt-119 strain revealed that the restoration of mating and repression of the high-temperature transposition phenotype were indeed associated with the presence of the plasmid and not a chromosomal entity. Each of the rescued plasmids had a similar restriction enzyme digest profile (data not shown).
Sequencing of one of the rescued plasmids revealed genomic sequence from chromosome IV containing five ORFs (Figure 1A). SIR4 was the most likely candidate gene because sir4 mutants are known to be defective in mating (HERSKOWITZ et al. 1992). A unique BamHI site in the plasmid located within the SIR4-coding region was filled in to create a frameshift mutation, and the resulting plasmid was transformed into the htt119 strain. The high-temperature transposition phenotype was maintained (and mating was not restored), indicating that sir4 is indeed the mutated host gene responsible for the high-temperature transposition phenotype in htt-119 (Figure 1B). We refer to our mutant allele as sir4-119.
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Transposition activity is increased in sir4 mutant strains at high temperature:
Transposition was quantitated in two distinct strain backgrounds containing sir4 mutant alleles, using plasmid pGTy1H3mhis3AI, a galactose-inducible element containing a reverse-oriented intron-disrupted his3 marker gene (Figure 2; CURCIO and GARFINKEL 1991b). The intron is spliced following transcription, and subsequent reverse transcription generates Ty1 cDNA containing a functional HIS3 marker gene. The results are shown in Figure 3. Transposition in sir4 strains at high temperature is increased 30-fold over that in wild-type strains. Two distinct sir4 null alleles, sir4 and sir4-119, showed high-temperature transposition in two strain backgrounds, indicating that the effect is not strain or allele dependent. As shown previously, the GRF167 strain has a higher rate of transposition than W303 (LAWLER et al. 2002). Interestingly, in the W303 strain background, the sir4 mutant demonstrates increased levels of transposition at all temperatures.
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90 kD) as well as the full-length Gag-Pol-p199 polyprotein and any processing intermediates (Figure 4). As expected, the amount of Ty1 protein detected at high temperature is greatly reduced as compared to permissive temperature (Figure 4A, lanes 1 and 2 vs. lanes 3 and 4). In comparison to the SIR4 strain, cell homogenates derived from the sir4-119 strain at high temperature show increased levels of mature IN protein (Figure 4A, lane 3 vs. lane 4). We also looked at production and processing of integrase by immunoblotting sucrose gradient-purified VLPs, as well as whole-cell extract (Figure 4B). In whole-cell extract at high temperature, the amount of IN protein detected in the sir4-119 strain is slightly greater than that found in the wild-type strain (Figure 4B, lane 6 vs. lane 8). The Gag-Pol polyprotein is not detected in the whole-cell extracts. In VLPs purified from cells induced at high temperature, the amount of Gag-Pol polyprotein and higher-molecular-weight intermediates in the sir4-119 VLPs are reduced as compared to wild type, while the amount of the lower-molecular-weight intermediates is increased (Figure 4B, lane 10 vs. lane 12). The mature IN protein in the sir4-119 mutant strain at high temperature is slightly increased as compared to wild type in whole-cell extracts, and is less so in purified VLPs. Although protein processing is dramatically reduced at high temperature, these results suggest that both production and processing of the Ty1 Gag-Pol polyprotein are slightly increased in the sir4-119 strain, as compared to wild type, at high temperature.
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Microarray data also revealed that expression of SPL2, which encodes a protein with similarity to cyclin-dependent kinase inhibitors, is increased 100-fold in the absence of sir4. If high-temperature transposition is due to Sir4p-mediated upregulation of this gene, then deletion of SPL2 would be expected to suppress high-temperature transposition. Isogenic spl2 and spl2 sir4-119 strains were generated by direct replacement of the spl2 ORF with kanMX4. The high-temperature phenotype is maintained in the spl2 sir4-119 strains and spl2 deletion alone has no effect on transposition as compared to the wild-type control (data not shown). These results indicate that neither fus3 nor spl2 is involved in the sir4-mediated increase in transposition activity at high temperature.
Ty1 cDNA production is not increased by high temperature or sir4 deletion:
We have previously shown that the level of cDNA production decreases as temperature increases (LAWLER et al. 2002). We therefore assessed the level of Ty1 cDNA in whole-cell extracts of galactose-induced wild-type and sir4 mutant cells at increasing temperatures (Figure 5). The decrease in Ty1 cDNA with increasing temperature is the same in wild-type and sir4 strains. Thus, the increase in transposition in sir4 mutant strains is not due to an increase in Ty1 cDNA production.
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mating types, respectively, which are kept silenced by a Sir4p-containing chromatin complex. In sir4 mutant strains, HMR and HML are unsilenced, thus allowing expression of both a and mating factors, and resulting in a pseudodiploid state. Homologous recombination of induced chromosomal breaks is slightly increased in MATa/ diploids as compared to MATa/a diploids (LEE et al. 1999; CLIKEMAN et al. 2001). Thus, it is possible that the increase in His+ events seen at high temperature in sir4 strains is a result of an increase in homologous recombination due to the induced "pseudodiploid state" in sir4 mutants. We used a plasmid transformation assay to measure HR and NHEJ activity in wild-type and sir4 strains at 30 and 36° (TSUKAMOTO et al. 1997; BOULTON and JACKSON 1998). A CEN plasmid (pJK592) containing the selectable markers LEU2 and HIS3 was transformed into wild-type and sir4 strains at 30° and 36°, selecting for Leu+ colonies. Both yeast strains harbor the his3200 deletion, such that very little homology exists between the HIS3 plasmid sequence and the genome (107 bp 5' and 28 bp 3' of plasmid HIS3 sequences are present at the his3200 chromosomal locus). An outline of the experimental design and the results are shown in Figure 7. To assess NHEJ, the plasmid was digested with the enzyme NdeI, which cuts within the HIS3 coding sequence, and then transformed. As expected, sir4 strains were deficient in NHEJ, at both 30° and 36° (TSUKAMOTO et al. 1997; ASTROM et al. 1999). To assess HR, NdeI-digested plasmid was cotransformed with a PCR-generated HIS3 fragment that spans the NdeI cut site. The HR assay also includes NHEJ activity, but the level of HR activity is minimally 15-fold that of NHEJ, such that the contribution of NHEJ is not significant. As expected, HR is much more efficient than NHEJ in both wild-type and sir4 mutant strains. HR is modestly increased (2-fold) at high temperature in the wild-type strain, but not the sir4 strain. Thus, neither cell-type nor temperature induction of the general cellular HR pathway in sir4 strains is responsible for the high-temperature phenotype. It is notable that transformation efficiency is increased at least twofold in both wild-type and mutant strains at 36° (data not shown).
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strains deleted for HMR were quantitated for His+ cell formation at high temperature. The deletion of the a-factor-containing HMR locus removes the pseudodiploid state, and these strains regain the ability to mate (Figure 8A). In this particular assay, His+ events occurring in the sir4 mutant at 36° were 14.3-fold higher than those in wild type (Figure 8A, far right column). In a sir4 hmr mutant, His+ events were increased only 5-fold above those in wild type, suggesting that removal of the pseudodiploid state decreases the high-temperature recombination activity. Given that the sir4 mutant had no effect on recombination of a non-Ty1 substrate (Figure 7), this result suggests that the recombination activity may be specific for Ty1 cDNA. Interestingly, a SIR4 hmr strain, which we expected to be comparable to wild type, showed a 4.5-fold increase in His+ events as compared to the wild type SIR4 HMR strain. This surprising result indicates that the HMR locus alone inhibits high-temperature His+ cell formation.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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15- to 30-fold increase in apparent transposition over wild-type strains at high temperature. It is notable that in the W303 strain background, which has an inherently lower level of transposition activity, deletion of sir4 resulted in increased transposition activity at permissive temperature as well. SIR4 is a silent information regulator gene implicated in many cellular processes including cell aging and chromatin silencing (reviewed in GARTENBERG 2000; GASSER and COCKELL 2001). SIR4 acts as a regulator of many genes through the creation of silent chromatin, in complex with SIR2 and SIR3. These three proteins assemble at silencer regions with the aid of SIR1, ORC (the origin recognition complex), and RAP1 (MOAZED et al. 1997). Interestingly, the yeast element Ty5 integrates preferentially to the silenced mating loci and telomeres in yeast, via interaction of Ty5 integrase to Sir4p (XIE et al. 2001). Ty1, however, targets to Pol III promoters, not silenced chromatin (DEVINE and BOEKE 1996). It is unknown whether Ty1 integrase interacts directly with Sir4p, or if IN plays any role in Ty1 recombination. Ty1 IN does contain a nuclear localization sequence (NLS), indicating that it could play an active role in chromosomal recombination of Ty1 cDNA, even in the absence of functional VLPs (MOORE et al. 1998).
In the Ty1 assay system that we used to quantitate transposition, the selectable marker can be transferred to the host genome by IN-mediated recombination or by homologous recombination to Ty1 or marker sequences present in the yeast cell. The latter activity requires the RAD52 gene product, which is required for homology-dependent recombination in yeast. The increase in apparent transposition in sir4 strains at high temperature is rad52 dependent, as assayed using a splicing-dependent HIS3-marked Ty1 element, indicating that the increase is due to recombination of HIS3-containing cDNA products, rather than to IN-mediated integration of Ty1 cDNA. However, there is a slight but significant increase in true transposition (RAD52-independent) events in the sir4 mutant strain (as compared to wild type) at permissive temperature (Figure 6), suggesting that Sir4p may also play a role in regulating proper integration.
We found that the proportion of recombination of Ty1 cDNA relative to integration increases in both wild-type and sir4 mutant strains at high temperature (Table 2). Sharon et al. have previously shown that a block in true IN-mediated transposition events via a mutation in the IN protein increases the rate of homologous recombination (SHARON et al. 1994). Consistent with this observation, the recombination data in this article show that at high temperature, when IN-mediated transposition is blocked (by a processing defect), an underlying recombination pathway is revealed. By an unknown molecular mechanism, the absence of Sir4p increases the relative frequency of recombination, resulting in a high-temperature transposition phenotype. We have shown that the increase in Ty1 recombination is not due to a temperature-induced increase in global recombination, suggesting that the increase in recombination at high temperature may be specific for Ty1 cDNA substrates. We favor three possible mechanisms by which a mutation in SIR4 could increase recombination at high temperature.
SIR4 inhibits Ty1 processing or destabilizes Ty1 cDNA:
We have previously shown that high temperature greatly reduces the production and PR-mediated processing of Ty1 proteins (LAWLER et al. 2002). In this study, we found that production of processed IN appears to be slightly restored in sir4 mutant strains. Recombination to Ty sequences requires the generation of at least partial Ty1 cDNA products. In this study, we confirm that the level of Ty1 cDNA decreases rapidly as temperature increases in both wild-type and sir4 mutant strains. It is possible that partial cDNA products are produced at high temperature, which may not appear as a distinct band by Southern blotting. A slight increase in Gag-Pol polyprotein processing in the absence of Sir4p could, via increased RT activity, result in a slight increase in partial cDNA products, providing sufficient substrate to give the level of increased recombination observed in sir4 mutant strains. It may be significant that the difference in processing is more notable in whole-cell homogenates or extracts than in purified VLPs, since it is unknown to what extent the VLP structure is needed for the recombination process. Alternatively, the absence of Sir4p could, directly or indirectly, increase the stability of partial Ty1 cDNAs, thus allowing more time for recombination events.
Cellular recombination is induced by a pseudodiploid state resulting from the absence of Sir4p:
Both HR and NHEJ are subject to mating-type control in yeast (LEE et al. 1999; CLIKEMAN et al. 2001). HR-mediated repair of a defined double-strand break is increased in MATa/ diploid cells, as compared to a homozygous diploid cell (MATa/MATa). NHEJ-mediated recombination, however, decreases 10-fold in heterozygous (MATa/) diploid cells, as compared to a non-a/ diploid (mat/MAT) strain (ASTROM et al. 1999; LEE et al. 1999). Sir4p is central to the maintenance of silencing of the HML and HMR mating-type cassettes, so in the absence of Sir4p, both MATa- and MAT-factor genes are expressed, resulting in a pseudodiploid, nonmating cellular state. Thus, it is possible that the increase in His+ events at high temperature is due to an increase in HR induced by pseudodiploidy. We tested this hypothesis by deletion of the hmr mating-type cassette in a sir4 mutant strain, thus eliminating the expression of a-factor genes in the sir4 mutant (Figure 8). We do indeed see an approximate threefold reduction of the high-temperature transposition phenotype, indicating some mating-type control of this recombination activity. Interestingly, this does not account for all of the high-temperature activity. Quite surprisingly, the frequency of His+ events at high temperature increased (4-fold) in the SIR4 hmr control strain. Although HMR is expected to be silenced in the presence of Sir4p, there may be basal level expression of a-factor that affects transposition or recombination activity directly or affects expression of haploid-specific genes that regulate transposition or recombination activity. It is also possible that Sir4p-mediated MAT silencing is weakened at high temperature.
Transposition of the Ty1 element is known to be subject to cell-type regulation at permissive temperature. Transcription of Ty1 is decreased 10-fold in diploids, mediated via a DNA sequence within Ty1 that binds the a1/2 regulatory protein (ERREDE et al. 1985, 1987). Transposition activity is also slightly reduced in diploid (a/) cells, but not in proportion to the decrease in mRNA production(PAQUIN and WILLIAMSON 1986). In the strain used in this study, deletion of hmr and sir4 had no effect on transposition activity at permissive temperature (Figure 8B).
SIR4 maintains a chromatin structure that inhibits Ty1 cDNA recombination:
Since deletion of Sir4p increases recombination at high temperature, we should consider how structural changes to chromatin in the absence of Sir4p affect this process, perhaps opening silenced regions to recombination. Many chromosomal structural changes take place in the absence of Sir4p. Sir2p, Sir3p, and Sir4p are considered the structural components of silenced chromatin, and the absence of Sir4p greatly disrupts normal formation of silenced chromatin. Since Sir4p is found mainly in complex with Sir2p and Sir3p, we also assayed for transposition in strains mutated for these genes and in combination with sir4. Neither sir2 nor sir3 strains demonstrate the high temperature transposition phenotype. The phenotype in sir4 strains is abolished in combination with either sir2 or sir3. This result suggests that in the absence of Sir4p, the remainder of the Sir complex is mediating the increase in recombination seen at high temperature.
No one model presented here is exclusive of the others. Indeed it is likely that the model by which Sir4p suppresses recombination is complex. Future experiments will endeavor to sort out these possibilities and attempt to determine if the increase in recombination in sir4 mutants is due to cellular effects brought about by temperature or simply due to the fact that IN-mediated transposition is blocked at high temperature. If recombination increases in the absence of sir4 because IN-mediated transposition is blocked, then we would expect to see increased recombination of Ty1 elements harboring mutations in the IN protein in sir4 mutant strains. Future studies will also address the identification of genomic recombination sites in sir4 mutant strains and the possible interaction of IN with Sir4p.
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2 Present address: Biochemistry, Cellular and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
3 Present address: Curriculum in Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.
4 Present address: Molecular Cell Biology Program, Washington University School of Medicine, St. Louis, MO 63110.
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LITERATURE CITED |
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