The Rad27 (Fen-1) Nuclease Inhibits Ty1 Mobility in Saccharomyces cerevisiae

Corresponding author: David J. Garfinkel, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201., garfinke{at}ncifcrf.gov (E-mail)

Communicating editor: S. SANDMEYER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.

THE retrotransposon Ty1 is the most abundant mobile genetic element in Saccharomyces cerevisiae. These elements are present in 30 copies per haploid genome and are structurally and functionally related to retroviruses (reviewed by VOYTAS and BOEKE 2002 ; Fig 1A). Ty1 elements are flanked by long terminal repeats (LTRs) and are transcribed from end to end, resulting in RNA that serves as template for both translation and reverse transcription. Translation results in synthesis of TyA1, which encodes a gag-like capsid protein, and TyB1, which encodes the enzymatic proteins protease, reverse transcriptase/ribonuclease H, and integrase. The structural proteins assemble into a virus-like particle (VLP) within which reverse transcription takes place. The resulting linear double-stranded cDNA enters the genome through integrase-mediated integration at a new chromosomal site or, to a lesser degree, by homologous recombination with genomic elements. The cDNA recombination pathway is more active when the integrase-mediated pathway is blocked. Introduction of the Ty1 cDNA into the genome by integration or by homologous recombination is termed Ty1 mobility.

View larger version (31K): In this window In a new window Download PPT slide   Figure 1. Life cycle of Ty1. (A) Ty1 elements in the genome are normally transcribed and a full-length Ty1 RNA is made. The Ty1 RNA serves as template for both translation and reverse transcription. Translation of Ty1 RNA results in synthesis of proteins that are required for transposition. These include the TyA1-gag protein, which forms the structural component of the virus-like particle (VLP) and the enzymatic proteins protease, integrase, and reverse transcriptase/ribonuclease H that are required for cDNA synthesis and integration into the genome. Reverse transcription takes place within the VLPs and a full-length cDNA is made. The cDNA enters the genome predominantly through integrase-mediated integration into a new chromosomal site or, to a lesser degree, by recombination with preexisting genomic Ty1 elements. (B) A phenotypic assay for Ty1 mobility. The tagged element consists of Ty1 containing the reporter gene, his3-AI, where the HIS3 gene (boxed) is interrupted by an artificial intron (AI). The arrows indicate the direction of transcription of his3-AI. The transcript is shown as a wavy line and vertical lines in the transcript indicate the splicing of the AI. When this spliced transcript undergoes reverse transcription and integration, a functional HIS3 is recreated and the cells become His+. This assay provides a phenotypic selection of a single Ty1 element undergoing retrotransposition or cDNA recombination, termed cDNA mobility.

Ty1 retrotransposition is potentially mutagenic since these elements can transpose and mutate essentially any gene (SMITH et al. 1995 ). Further, homologous recombination between Ty elements can lead to chromosomal rearrangements, thereby affecting the integrity of the genome (VOYTAS and BOEKE 2002 ). However, yeast cells minimize such events by modulating several steps in the Ty1 life cycle. Host genes have been identified that affect Ty1 transcription (WINSTON et al. 1984 ), programmed +1 frameshifting that is required to synthesize the TyA1-TyB1 fusion protein (reviewed by FARABAUGH 1995 ), Ty1 protein processing and VLP maturation (CURCIO and GARFINKEL 1992 ; CONTE et al. 1998 ), target site preference (JI et al. 1993 ; DEVINE and BOEKE 1996 ; reviewed by BOEKE and DEVINE 1998 ), and cDNA stability (LEE et al. 1998 , LEE et al. 2000 ). Recently, SGS1 has been shown to inhibit Ty1 mobility by preventing transposition of multimeric Ty1 elements (BRYK et al. 2001 ).

In this work we examine the effects of the host gene RAD27 and other members of the RAD2 nuclease family on Ty1 element mobility. RAD27, the yeast homolog of the mammalian gene FEN1, is related to the RAD2 family of structure-specific nucleases that are involved in DNA metabolism and repair (reviewed by FRIEDBERG 1991 and PRAKASH et al. 1993 ; REAGAN et al. 1995 ). The members of the RAD2 nuclease family, EXO1, DIN7, RAD27, YEN1, and RAD2 are conserved from humans to bacteriophages (reviewed by LIEBER 1997 ) and mutations in these genes can result in increased risk of disease (TISHKOFF et al. 1997 ). For example, mutations in human XP-G (RAD2 homolog) cause xeroderma pigmentosum (reviewed by DE BOER and HOEIJMAKERS 2000 ), and variants in EXO1 may be associated with hereditary nonpolyposis colorectal cancer (WU et al. 2001 ).

Rad27/Fen1 is a 5'-3' exonuclease and a 5'-flap endonuclease that plays an important role in DNA replication, repair, and recombination (LIEBER 1997 ). The nuclease activity of Rad27/Fen1 removes RNA primers made during lagging strand DNA synthesis (reviewed by BAMBARA et al. 1997 ). Rad27/Fen1 participates in DNA repair through removal of damaged DNA by the base-excision repair pathway (KLUNGLAND and LINDAHL 1997 ; JOHNSON et al. 1998 ; KIM et al. 1998 ) and also prevents short sequence recombination (SSR), a process that can lead to genome rearrangements (NEGRITTO et al. 2001 ). Consistent with its involvement in DNA metabolism, rad27 deletion mutants are sensitive to DNA damaging agents (REAGAN et al. 1995 ; XIE et al. 2001 ). Mutations in RAD27 result in a potent mutator phenotype and display a variety of genomic rearrangements (CHEN and KOLODNER 1999 ; GREENE et al. 1999 ; XIE et al. 2001 ). Deletion of FEN1/RAD27 also results in expansion of di- and trinucleotide repeats that have been shown to be the underlying cause of several human disorders (SCHWEITZER and LIVINGSTON 1998 ; WHITE et al. 1999 ). Our results indicate a novel role for RAD27 in inhibiting Ty1 mobility, which is important for maintaining genome stability in yeast.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Construction of mutants:
The exo1 mutant BLY187 was constructed by one-step gene disruption of the EXO1 gene in strain DG1657 (MATa ura3-167 his3-200 trp1-hisG leu2-hisG Ty1-270his3-AI Ty1-588neo Ty1-146[tyb1::lacZ]) using plasmid pSH164 (kindly provided by S. Holbeck) digested with NdeI. The din7 mutant BLY210, an isogenic din7::TRP1 derivative of the DIN7 strain DG1657, was constructed by one-step gene replacement with a 2.3-kb NdeI/SphI fragment from plasmid pBL26 (a pUC19 derivative that contains the din7::TRP1 allele). These gene disruptions were verified by Southern analysis. The rad27 deletion strain BLY184 was constructed in DG1657 by single-step gene disruption using plasmid pBL22 (a pUC19 derivative carrying rad27::LEU2) digested with HindIII and NdeI. The wild-type and yen1 mutant strains W303-1A and LSY485-2C (kindly provided by L. Symington) were transformed with pBDG954, a centromere-plasmid containing Ty1neo-AI (the original plasmid, pBJC546, was kindly provided by M. J. Curcio) to generate strains DG2250 and DG2248, respectively. The rad27 mutants DG2102 (rad27-G67S), DG2103 (rad27-G240D), ANU115 (rad27-324), and the wild-type strain DG2101 were constructed by digesting plasmids pEAI143, pEAI144, pEAI142, and pEAI141 (kindly provided by E. Alani) with BglII (XIE et al. 2001 ), followed by transformation of the parental strain DG1657. Leu⁺ transformants were selected and rad27 mutants were identified by their mutator phenotype at the CAN1 locus and by temperature sensitivity. The rad27-E158D mutant was made in two steps. First, the ClaI/BstXI fragment carrying rad27-E158D mutation from plasmid pLAY362 (NEGRITTO et al. 2001 ; kindly provided by A. Bailis) replaced the wild-type RAD27 fragment present in pEAI141 to give rise to pANU101. Second, plasmid pANU101 was digested with BglII and introduced into strain DG1657. The correct Leu⁺ transformant containing rad27-E158D, ANU105, was identified by its mutator phenotype at CAN1 and then sequenced to confirm the presence of the E158D mutation. The rad27 mutant strains used in the cDNA recombination assay (ANU116, ANU117, ANU118, ANU119, ANU120, and DG2180) were constructed essentially in the same manner as above except that the starting strain was DG2179 (MAT his3-200 ura3-167 leu2-hisG trp1-hisG).

Ty1 mobility:
To detect spontaneous Ty1 insertion events in strains bearing the Ty1his3-AI reporter, cells were streaked for single colonies on YPD plates and incubated at 20° for 5 days. The cells were then replica plated onto synthetic complete medium lacking histidine (SC-His) and incubated at 30° for 3–4 days. Quantitative Ty1his3-AI insertion rates were determined as described previously (CURCIO and GARFINKEL 1991 ).

Ty1 insertions at SUF16:
Spontaneous Ty1 insertions upstream of the SUF16 (glycine tRNA) locus on chromosome III were detected after growing individual colonies from wild-type and rad27 mutant strains on YPD plates at 20° for 7 days. Three colonies from each strain were then inoculated into 10 ml of YPD broth and grown for 2 days at 20°. Total genomic DNA was isolated from each culture as described by HOFFMAN and WINSTON 1987 and analyzed by PCR. The primers SNR33 OUT (5'-TTTTAGAGTGACACCATCGTAC-3', specific to the SNR33 gene adjacent to the 3' end of SUF16 on chromosome III), and TYB OUT (5'-GAACATTGCTGATGTGATGACA-3', specific to Ty1) were used in PCR reactions to amplify Ty1 insertions, as described previously (LEE et al. 2000 ). A sample of the PCR reaction was analyzed by agarose gel electrophoresis, transferred to Hybond-N membrane, and subjected to Southern hybridization using a ³²P-labeled Ty1 LTR probe. To ensure that the genomic DNA was PCR competent, DNA preparations were analyzed by PCR using primers that bracket SUF16 (SNR33 OUT and YCR016W OUT 5'-GATCATCATCTATTAGATTGGA-3').

Northern analysis:
Total RNA was isolated as described by SCHMITT et al. 1990 from wild-type and rad27 mutant strains grown to late log phase in 10 ml of YPD broth at 20°. The RNA samples were run on a 1% agarose/formaldehyde gel and transferred to nitrocellulose membrane. The ³²P-labeled DNA probes were made by randomly primed DNA synthesis (Amersham, Piscataway, NJ). The Ty1his3-AI probe was made by digesting plasmid pOY1 (LEE et al. 1998 ) with PstI and gel purifying a 0.5-kb fragment. A 3.6-kb PvuII fragment from pOY1 was used to make the Ty1 probe. The PYK1 probe was made from a 1.4-kb EcoRI-XbaI fragment from pBDG502. Hybridization analysis was performed as described previously (LEE and CULBERTSON 1995 ; LEE et al. 1998 ), and the signals were quantified using a Typhoon 8600 phosphorimager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant 1.2 software.

Western analysis:
Total protein was isolated from wild-type and rad27 mutant strains after growth in YPD broth at 20°, as described by LEE et al. 1998 . Protein concentrations were determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Proteins were separated on a 10% SDS-polyacrylamide gel (Invitrogen, San Diego) and transferred to Immobilon-P membrane (Millipore, Bedford, MA) using a semidry electroblotter. The membrane was incubated with polyclonal antibodies to Ty1-VLPs for 2 hr. Detection was performed using an ECF Western blotting kit (Amersham), and signals were quantified by phosphorimaging according to the supplier's recommendations. The membrane was then stripped of the Ty1-VLP antibody and incubated with a polyclonal antibody to Hts1 (histidinyl tRNA synthetase, kindly provided by T. Mason) and processed as described above.

Southern analysis of Ty1 cDNA:
A single colony from each strain was inoculated in 5 ml of YPD broth and grown to late log phase at 20°. A 5-µl aliquot of the culture was inoculated into 25 ml of fresh YPD and grown for an additional 2 days at 20°. Total genomic DNA was isolated from these cultures. The DNA samples were digested with PvuII, separated on a 0.8% agarose gel, and transferred to Hybond-N membrane. Southern hybridization was performed using a ³²P-labeled DNA probe derived from the Ty1 PvuII-SnaBI fragment of Ty1-H3. The intensity of the cDNA fragments was determined by phosphorimage analysis and normalized to four conserved chromosomal Ty1 junction fragments, as described by LEE et al. 1998 .

Detection of Ty1 multimeric arrays by PCR analysis:
An assay developed by BRYK et al. 2001 was used essentially as described. Wild-type and rad27 mutant strains carrying a Ty1his3-AI element were streaked for single colonies on YPD plates and grown at 20° for 2–3 days. The cells were then printed to SC-His plates and incubated for 2–3 days at 30° to isolate His⁺ prototrophs that sustained a Ty1HIS3 insertion. Ten independent His⁺ prototrophs from each strain were clonally purified and grown in 5 ml YPD broth at 30° overnight. Total genomic DNA was isolated and the DNA samples were analyzed by PCR using HIS3 OUT (5'-GTACTAGAGGAGGCCAAGAG-3') and TYA OUT (5'-TCTCTGGAACAGCTGATGAAG-3') primers. The genomic DNA was also subjected to PCR analysis using primers flanking the FPR1 gene to ensure that the DNA was PCR competent.

cDNA recombination:
A cDNA recombination assay developed by BRYK et al. 2001 was used essentially as described. The wild-type strain ANU116 and the rad27 mutant strains ANU117, ANU118, ANU119, ANU120, and DG2180 were transformed with the integrating plasmid pBJC573 linearized wih PacI, which results in the insertion of Ty1his3-AI and the URA3 gene flanked by a 1.2-kb direct repeat from the BIK1-HIS4 region on chromosome III. Ura⁺ transformants were grown as patches on SC-Ura plates at 30°, and the resulting patches were replica plated to two YPD plates, one of which was incubated at 20° and the other at 30° for 3 days. The plates were then printed to SC-Ura-His plates and grown at 30° for 2 days. No His⁺ Ura⁺ colonies were observed from YPD plates grown at 30°, suggesting that the His⁺ Ura⁺ colonies that arose following incubation at 20° were independent. The Ura⁺ His⁺ colonies were grown as small patches on YPD plates, printed to 5-fluoroorotic acid (5-FOA)-His plates and incubated at 30° for 3 days. The fraction of Ura⁺ His⁺ patches that failed to grow on 5-FOA-His plates was then determined.

Ty1 cDNA stability:
The Ty1 cDNA stability assay developed by LEE et al. 2000 was slightly modified for this study. A single colony from each strain was inoculated in 80 ml of YPD broth and grown for 2 days at 20°. A 40-ml sample of the culture was pelleted, washed, and resuspended in 80 ml of fresh YPD. Further dilution of the above culture was made by inoculating 40 ml of the culture in 1 liter of fresh YPD broth containing 600 µg/ml of the reverse transcriptase inhibitor phosphonoformic acid (PFA; Sigma, St. Louis). This concentration of PFA was found to inhibit cDNA synthesis without affecting cell growth. Immediately after addition of PFA, 100 ml of the culture was pelleted, washed, and stored at -70°. Subsequently, 100 ml aliquots were removed after 1, 2, 4, 6, and 8 hr of growth at 20°. The cells taken at each time point were quickly pelleted and stored at -70°. Total genomic DNA was isolated from each sample, digested with PvuII, and processed for Southern analysis as described above. Hybridization signals were not heavily influenced by outgrowth of cells because all cells required at least 5 hr to double in density (DG1657, 5 hr; BLY184, 6.7 hr; ANU115, 5.4 hr). Signals were quantitated by phosphorimage analysis as described by LEE et al. 2000 . A plot of the percentage of Ty1 cDNA remaining after each elapsed time point relative to the amount at zero time was plotted on a log scale. The half-life of cDNA was calculated from the slope of the best-fit line using Cricket Graph software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

RAD27 inhibits Ty1 mobility:
Our previous work suggested that the DNA helicases Rad3 and Ssl2 inhibit Ty1 transposition by destabilizing Ty1 cDNA (LEE et al. 1998 , LEE et al. 2000 ). Since Rad3 and Ssl2 probably do not degrade Ty1 cDNA directly, we tested the RAD2 family of structure-specific nucleases for their effects on Ty1 mobility. These genes were chosen because of their role in DNA replication and repair (LIEBER 1997 ) and short sequence recombination (NEGRITTO et al. 2001 ). We examined the effects of the null alleles of the five members of the RAD2 family on Ty1 mobility using the Ty1 transposition reporter gene, his3-AI, described in Fig 1B. We previously showed that disruption of RAD2 had no effect on Ty1 mobility (RATTRAY et al. 2000 ). Deletion of EXO1, DIN7, and YEN1 did not result in an increase in Ty1 mobility (Fig 2). However, deleting RAD27 significantly increased Ty1 mobility, as monitored by the increase in the number of His⁺ papillae appearing on SC-His plates.

View larger version (74K): In this window In a new window Download PPT slide   Figure 2. Effect of the RAD2 gene family on Ty1 mobility. (A) Null mutants of exo1 (BLY187), din7 (BLY210), rad27 (BLY184), and the wild-type parent (DG1657) were monitored for Ty1 mobility using Ty1his3-AI. (B) A null mutant of yen1 (LSY485-2C) and the wild type (W303-1A) were assayed using Ty1neo-AI Cell patches were grown on YPD (A) or SC-Ura (B) plates at 20° for 4 days and then replica plated to SC-His (A) or YPD + G418 (B). These plates were incubated at 30° for 3 days and then photographed.

Effect of rad27 mutants on Ty1 mobility:
Since deletion of RAD27 increased Ty1 mobility (Fig 2), we analyzed several RAD27 point mutants that have been examined previously for their roles in mutation avoidance, flap cleavage, repeat-tract instability (XIE et al. 2001 ), or short sequence recombination (NEGRITTO et al. 2001 ). The rad27 point mutations, G67S and G240D, fall within the nuclease domains that are highly conserved in both prokaryotes and eukaryotes (SHEN et al. 1997 ; HOSFIELD et al. 1998 ), and their biochemical activities have been studied (XIE et al. 2001 ; KAO et al. 2002 ). The G67S mutant has a weak exonuclease but near wild-type levels of single- and double-flap endonuclease activities, while the G240D mutant is devoid of exonuclease activity but has significant double-flap endonuclease activity. The rad27-324 mutant has a frameshift mutation at codon 324 that truncates the C-terminal 58 amino acids. This mutant has DNA repair defects and mutator phenotype at the CAN1 locus similar to those of the null mutant (XIE et al. 2001 ), but has not been biochemically characterized. The rad27-E158D mutant has a conserved glutamate residue changed to an aspartate and retains partial flap endonuclease activity, but no exonuclease activity (FRANK et al. 1998 ; NEGRITTO et al. 2001 ), and partially complements the elevated SSR phenotype of a rad27 null mutant (NEGRITTO et al. 2001 ).

We studied the effects of these rad27 mutants on Ty1 mobility by monitoring the rate of His⁺ colony formation using a chromosomally marked Ty1 element carrying his3-AI. All rad27 mutants increased Ty1 mobility relative to the wild-type strain DG2101 (Table 1). The point mutations rad27-G67S, rad27-G240D, and rad27-E158D increased Ty1 mobility from 4- to 22-fold relative to the wild-type strain. The null mutant rad27::LEU2 showed an increase in mobility similar to that of the point mutant rad27-G67S. The rad27-324 frameshift mutant had the maximum increase in Ty1 mobility (198-fold) compared to the point mutants and also the null mutant. Since rad27-324 appeared to be more severe than the null mutant in terms of increased Ty1 mobility, we determined if this mutant was dominant to RAD27. To perform the dominance test, we constructed diploid strains bearing the wild type and mutant alleles and determined whether the wild-type RAD27 could complement the phenotypes of the mutant rad27 alleles when compared to a set of homozygous control strains. Our results indicate that the mutator, temperature sensitivity, and Ty1 mobility phenotypes exhibited by all five rad27 mutants are recessive, since they are effectively complemented by RAD27 in the diploid strains (data not shown). Finally, we constructed double mutants containing the rad3-G595R, which has been previously shown to increase SSR and Ty1 retrotransposition (LEE et al. 2000 ), and the rad27 alleles for epistasis analysis. As is true of many double mutants containing a mutation in RAD27 and a mutation in another gene involved in DNA repair (SYMINGTON 1998 ; TONG et al. 2001 ), all of the rad27 rad3-G595R double mutants grew very poorly and were not pursued further.

Ty1 insertion at the SUF16 locus increases in rad27 mutants:
Ty1 elements preferentially target genomic regions upstream of tRNA genes (JI et al. 1993 ; DEVINE and BOEKE 1996 ). To determine if the rad27 mutations affected target site selection, we monitored spontaneous Ty1 insertion events using a qualitative PCR assay (LEE et al. 1998 ) at one such tRNA (SUF16) on chromosome III (Fig 3A). Our results showed an elevated level of insertion at the target site (SUF16) in all the rad27 mutants relative to wild type as observed by the intensity of the PCR products that hybridized with a radiolabeled Ty1 LTR probe (Fig 3B). The maximum level of Ty1 transposition was observed with the rad27-324 mutant. The highly intense signal observed with rad27-G240D suggests that a Ty1 insertion occurred early during cell growth and propagated through subsequent generations, creating a "jackpot" event. As a negative control, genomic DNA from an isogenic spt3-101 strain, in which transcription of Ty1 is severely reduced (WINSTON et al. 1984 ), was analyzed for insertions near SUF16. The hybridization patterns reflect the window of insertion of the Ty elements ranging from 500 to 1800 bp upstream of SUF16, which has been observed previously (LEE et al. 1998 ). These results suggest that the normal target site preferences are maintained in rad27 mutants and that the level of insertion (as monitored by the intensity of the hybridization fragments) parallels the transposition rate observed in these mutants (Table 1).

View larger version (58K): In this window In a new window Download PPT slide   Figure 3. Spontaneous Ty1 insertions upstream of SUF16. (A) Schematic representation of the SUF16 locus on chromosome III. Ty elements are known to preferentially insert upstream of the glycine tRNA gene SUF16. The arrows indicate the PCR primers, SNR33 OUT and TYB OUT, with homologies to the SNR33 gene and TYB1, respectively. The insertion patterns for various strains reflect the hotspots for Ty1 integration upstream of SUF16. (B) PCR analysis of DNA from three colonies of each strain. The PCR fragments were resolved on a 1.2% agarose gel, transferred to Hybond-N membrane and subjected to a Southern hybridization using a 32P-labeled LTR probe. The genotype of each strain is indicated. Size markers are alongside the figure.

Ty1 transcript level in rad27 mutants:
Northern analysis was performed to determine if the rad27 mutations increased Ty1 transposition by altering the level of Ty1 or Ty1his3-AI transcripts. Total RNA was isolated from wild-type, rad27::LEU2, rad27-G67S, rad27-G240D, rad27-E158D, and rad27-324 strains and subjected to Northern blot hybridization using ³²P-labeled probes specific to Ty1 or his3-AI (Fig 4). The Ty1 probe detects all Ty1 element transcripts while the his3-AI probe detects only the marked Ty1 element transcript. The level of Ty1 or Ty1his3-AI RNA was normalized to that of the PYK1 transcript and ratios were determined by phosphorimage analysis. The rad27-G67S, rad27-G240D, rad27-E158D, and rad27-324 mutants showed no significant increase in the levels of Ty1 or Ty1his3-AI transcripts relative to the wild-type strain. The rad27::LEU2 null mutant, however, had an increase in the level of Ty1 RNA (3-fold) and Ty1his3-AI RNA (1.5-fold). Taken together, these results suggest that RAD27 inhibits Ty1 mobility predominantly at a post-transcriptional step.

View larger version (35K): In this window In a new window Download PPT slide   Figure 4. Level of Ty1 RNA in the rad27 mutants. Northern analysis of total RNA isolated from various rad27 strains with Ty1 and PYK1 (A) or his3-AI and PYK1 radiolabeled probes (B). The Ty1 probe detects all Ty1 transcripts while the his3-AI probe hybridizes only with the marked Ty1his3-AI RNA. The signals were quantitated by phosphorimage analysis and normalized to the PYK1 transcript signal. The numbers below each lane indicate the fold increase in the total Ty1 RNA (A) or Ty1his3-AI RNA (B) relative to the wild-type strain.

RAD27 inhibits Ty1 transposition at a post-translational step:
We next determined whether RAD27 inhibited Ty1 transposition by altering the level of endogenous TyA1-gag protein, which can be detected in unfractionated cell extracts. Total protein was extracted from wild-type, rad27::LEU2, rad27-G67S, rad27-G240D, rad27-E158D, and rad27-324 strains and subjected to quantitative Western analysis using anti-Ty1 VLP antibodies to detect TyA1 protein (Fig 5). The blot was then stripped of Ty1 VLP antibodies and reprobed with antibodies to the Hts1 protein (cytoplasmic and mitochondrial histidinyl tRNA synthetase). The amount of TyA1 protein in the wild-type and mutant rad27 strains was determined by normalizing to the level of Hts1 protein by phosphorimage analysis. As a positive control, purified Ty1-VLPs (kindly provided by S. Moore) were probed with similar antibodies. Protein samples from an isogenic spt3-101 strain served as a negative control and had undetectable levels of TyA1 protein. The amount of endogenous TyA1 remained essentially unaltered in the rad27 mutants as compared to the wild-type strain. These results suggest that RAD27 inhibits Ty1 mobility at a post-translational step.

View larger version (41K): In this window In a new window Download PPT slide   Figure 5. Level of TyA1 protein in the rad27 mutants. Total protein extracted from wild-type or rad27 mutant strains was separated on a 10% SDS-polyacrylamide gel, immunoblotted to Immobilon-P membrane, and probed with antibodies against VLP to detect TyA1 or the histidinyl tRNA synthetase, Hts1. Immunodetection was performed using the ECF Western blotting kit and the signals were quantitated by phosphorimage analysis. The numbers below each lane indicate the ratio of TyA1 to Hts1 signal for each mutant over that of the wild type.

Ty1 cDNA increases in rad27 mutants:
To determine if RAD27 inhibited Ty1 transposition by affecting the level of unincorporated Ty1 cDNA, total genomic DNA was isolated from wild-type, rad27-G67S, rad27-G240D, rad27-E158D, rad27-324, and rad27::LEU2 strains and digested with PvuII. PvuII cleavage produces a 2-kb Ty1 fragment that is indicative of unintegrated Ty1 cDNA (Fig 6, top). The PvuII-digested samples were then subjected to Southern hybridization using a ³²P-labeled PvuII-SnaBI fragment of Ty1. This probe hybridizes with unincorporated Ty1 cDNA fragments of 2 kb and also with chromosomal Ty1 elements, thereby generating a variety of larger fragments consisting of Ty1 elements joined to genomic DNA. The amount of unincorporated Ty1 cDNA was determined by phosphorimage analysis of the blot and normalized to four conserved chromosomal Ty1 junction fragments (Fig 6, bottom).

View larger version (25K): In this window In a new window Download PPT slide   Figure 6. Ty1 cDNA increases in the rad27 mutants. The figure at the top depicts the 2-kb cDNA fragment of Ty1 that is released after PvuII digestion of total yeast DNA. The solid box represents the PvuII/SnaBI restriction fragment of Ty1 that was used as probe in the Southern hybridization. Total DNA was prepared from wild-type and rad27 mutant strains, digested with PvuII, and subjected to Southern hybridization using a 32P-labeled probe derived from TyB1 described above. The position of the 2-kb cDNA fragment is shown by the lower arrow. The four conserved chromosomal junction fragments used for normalization of the cDNA fragment are indicated by the dots alongside the figure. The top arrow on the right refers to the 2.2-kb fragment that was intensified in the rad27::LEU2 mutant. Two possible mechanisms by which the 2.2-kb PvuII fragment could have arisen are indicated. In the first case (top), the 2.2-kb fragment released after PvuII (p) digestion is from a multimeric Ty1 element, while in the second case the 2.2-kb PvuII fragment is from a single Ty1 LTR circle.

All rad27 mutants showed an increase in the levels of Ty1 cDNA that ranged from a modest 1.3-fold up to 5-fold relative to the wild-type strain. As expected, we could not detect any Ty1 cDNA in the spt3-101 strain. The rad27-324 mutant showed the maximum increase (5-fold) in Ty1 cDNA followed by the rad27::LEU2 mutant. We also observed that the increase in the cDNA levels roughly parallels the increased Ty1 mobility in these mutants.

Ty1 multimeric arrays in rad27 mutants:
We consistently observed an intense 2.2-kb PvuII-generated fragment above the unincorporated Ty1 cDNA fragment in the rad27::LEU2 strain (Fig 6). The size of the 2.2-kb fragment suggests two possible mechanisms by which it could be formed. First, the fragment could have resulted from a multimeric Ty1 element insertion upon digestion with PvuII. Such multimeric Ty1 elements have previously been observed at HML in a wild-type yeast strain (WEINSTOCK et al. 1990 ) and at an elevated level in an sgs1 null mutant (BRYK et al. 2001 ). Second, the 2.2-kb PvuII fragment could have been be formed by digestion of a circular Ty1 element containing a single LTR. Although circular forms of unintegrated retroviral DNA are routinely observed in infected cells, a circular form of Ty1 cDNA does not accumulate to an appreciable amount within VLPs isolated from yeast cells undergoing a high level of Ty1 transposition (EICHINGER and BOEKE 1988 ).

To determine if increased multimeric Ty1 elements are present in rad27 mutants, especially in the null mutant, we isolated 10 independent Ty1HIS3 events from wild-type and rad27 mutant cells containing a genomic Ty1his3-AI element. Total genomic DNA was analyzed by PCR to determine if the Ty1HIS3 element was present as part of a multimer (BRYK et al. 2001 ). Note that the initial Ty1his3-AI element is not multimeric. Primers specific to HIS3 and TYA1 were used to detect a Ty1 multimer consisting of a tagged element joined to another Ty1 element (Fig 7A). A PCR fragment of 570 bp is indicative of a single LTR Ty1 multimer while a 905-bp fragment suggests a 2-LTR Ty1 multimer. Our results indicate that only the null mutant has an increased fraction of Ty1 multimers (9/10) and all contain a single LTR (Fig 7B). The rad27-G67S, rad27-G240D, rad27-E158D, and rad27-324 mutants did not show an increase in the number of Ty1 multimers relative to the wild-type strain. This result was confirmed by subjecting the same 10 His⁺ strains to Southern analysis after digestion by PvuII and hybridization with a HIS3 probe (data not shown).

View larger version (16K): In this window In a new window Download PPT slide   Figure 7. Ty1 multimer analysis in the rad27 mutants. (A) A Ty1 multimer within a chromosome is depicted at the top. For simplicity, two Ty1 elements sharing a single LTR between the upstream Ty1HIS3 element and another Ty1 element are shown. PCR primers homologous with the HIS3 gene and to the TyA1 region are indicated. The size of the PCR fragment amplified by these primers is 570 bp. (B) Total yeast DNA isolated from 10 independent His+ cultures from each strain was analyzed by PCR and the number of multimers observed for each strain is shown.

Deletion of RAD27 leads to increased recombination between Ty1 cDNA and genomic Ty1 elements:
To test the possibility that the Ty1 multimers observed in the rad27 deletion strain were correlated with an increase in Ty1 cDNA recombination, we determined the frequency of recombination between Ty1HIS3 cDNA and an introduced genomic Ty1his3-AI element (BRYK et al. 2001 ). A plasmid consisting of a Ty1his3-AI element, the URA3 gene, and sequences upstream of HIS4 was integrated into the wild-type and rad27 mutant strains at HIS4 on chromosome III. Transcription, splicing, and reverse transcription of Ty1his3-AI results in Ty1HIS3 cDNA with a functional HIS3 gene. Recombination between the Ty1HIS3 cDNA and genomic Ty1his3-AI results in conversion of the genomic Ty1his3-AI to Ty1HIS3 adjacent to URA3. Selecting for loss of URA3 by growing the cells in 5-FOA results in concomitant loss of the HIS3 gene due to recombination between the flanking direct repeats of HIS4 sequence. The fraction of the His⁺ Ura⁺ colonies that became His^- Ura^- as a result of selection for loss of URA3 was determined to detect the cDNA recombinants (Table 2). The rad27-G67S, rad27-G240D, rad27-E158D, and rad27-324 mutants did not show any significant increase in the number of His^- Ura^- colonies relative to the wild-type strain. The rad27 deletion mutant, however, had a significant (P 0.005) increase in the number of His^- Ura^- colonies relative to wild type (7 vs. 1.8%). These results suggest that RAD27 inhibits recombination between Ty1 cDNA and genomic Ty1 elements and that Ty1 multimers may arise by homologous recombination.

Stability of Ty1 cDNA is increased in rad27 mutants:
The increased level of Ty1 cDNA observed in rad27 mutants could be caused by increased synthesis or stability prior to integration into the genome. Either mechanism would lead to an increase in the level of Ty1 cDNA and transposition, since Ty1 cDNA is a limiting component required for high levels of transposition in vitro (EICHINGER and BOEKE 1990 ). Therefore, we measured the decay rates of unincorporated Ty1 cDNA in wild type and rad27 mutants after inhibiting reverse transcription with the nonnucleotide reverse transcriptase inhibitor, phosphonoformic acid (PFA; BERGDAHL et al. 1998 ; LEE et al. 2000 ). We chose rad27-324 and rad27::LEU2 for this experiment because these mutants had the maximum increase in Ty1 cDNA relative to the other rad27 mutants. The wild-type, rad27-324, and rad27::LEU2 strains were initially grown in different concentrations of PFA (100 µg/ml to 1 mg/ml) to determine the drug concentration that fully inhibited Ty1 cDNA accumulation without affecting cell growth (data not shown). When the wild type and the rad27 mutants were grown in 600 µg/ml of PFA for 48 hr, Ty1 cDNA was nearly undetectable (Fig 8A, lanes 1–4). The spt3-101 mutant was used as a negative control and had, as expected, an undetectable level of Ty1 cDNA. We next measured the cDNA decay rates of wild type and the rad27 mutants by treating them with 600 µg/ml PFA. An aliquot of cells from each strain was removed at different time points (0–8 hr) after addition of PFA and total genomic DNA was isolated. After digestion with PvuII, the genomic DNA samples were subjected to Southern analysis using a ³²P-labeled Ty1 probe (Fig 8A). The cDNA fragments were analyzed as described earlier (Fig 6) and the half-lives were determined (see MATERIALS AND METHODS; Fig 8B). The half-lives of rad27 mutants were increased nearly fivefold (444 min for rad27-324 and 451 min for rad27::LEU2) relative to the wild-type strain (93 min).

View larger version (61K): In this window In a new window Download PPT slide   Figure 8. Stability of Ty1 cDNA from RAD27 wild-type, rad27-324, and rad27::LEU2 strains after treatment with the reverse transcriptase inhibitor phosphonoformic acid (PFA) (A). Total genomic DNA isolated from wild-type, rad27-324, and rad27::LEU2 strains grown in the presence of PFA (600 µg/ml) to midlog phase was digested with PvuII, separated on a 0.8% agarose gel, and subjected to Southern hybridization with a 32P-labeled Ty1 probe (lanes 2, 3, and 4). As a control, PvuII-digested DNA from an spt3-101 strain (lane 1) not treated with PFA is shown. Lanes 5–22 represent decay rates of Ty1 cDNA after treatment of the indicated strains with PFA for various time periods from 0 to 480 min. Lanes 5–10, 11–16, and 17–22 represent the decay rates for RAD27 wild-type, rad27-324, and rad27::LEU2 strains, respectively. The 2-kb cDNA fragment is indicated by the arrow and the Ty1 chromosomal junction fragments that were used for normalization of the cDNA level are noted by the dots alongside the figure. (B) A logarithmic plot of the cDNA decay rates from the above blot. The percentage of Ty1 cDNA remaining after growth in PFA for various time points relative to the cDNA amount at 0 time is plotted. The half-lives of the Ty1 cDNA in RAD27 wild-type, rad27-324, and rad27::LEU2 strains were determined from the best-fit slope of the line.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

From our analysis of members of the RAD2 nuclease family, the results presented here indicate an important role for the structure-specific nuclease Rad27 in inhibiting Ty1 mobility. We determined whether members of the RAD2 nuclease family modulate Ty1 mobility for two reasons. The RAD2 nucleases recognize specific nucleic acid structures; therefore, we hypothesized that these nucleases may affect Ty1 transposition, possibly by recognizing Ty1 reverse transcription or gapped integration intermediates as substrates. In addition, NEGRITTO et al. 2001 have shown that RAD27 inhibits short sequence recombination, a process that is related to the stability of Ty1 cDNA through the action of the NER/TFIIH helicase genes RAD3 and SSL2 (LEE et al. 2000 ). Our results indicate that among the RAD2 nuclease family, only RAD27 markedly affects Ty1 mobility by altering the stability of Ty1 cDNA.

To understand how RAD27 inhibits the Ty1 mobility (Fig 1), we have determined if several genetically and biochemically defined rad27 point mutations, as well as a rad27 null mutation, alter transpositional integration at the SUF16 locus, Ty1 RNA and TyA1 protein level, cDNA accumulation and recombination, and Ty1 multimer formation. Our work indicates that there is not a simple correspondence between the severity of different rad27-mediated DNA repair and nuclease defects and Ty1 retrotransposition. For example, the rad27-G240D mutant has a more severe exonuclease and single-flap endonuclease defect than rad27-G67S (XIE et al. 2001 ), yet rad27-G240D has a more modest increase in Ty1 mobility (5-fold) than rad27-G67S (22-fold). Mutations that specifically destroy the endonuclease activity but do not affect the flap exonuclease activity of RAD27 would help determine whether both nuclease activities are required to inhibit Ty1 mobility. The most striking mutant analyzed in our study is rad27-324, which has not been characterized biochemically and resembles a null mutant in phenotypic analyses (XIE et al. 2001 ). Surprisingly, rad27-324 increases Ty1 mobility much more (198-fold) than the rad27::LEU2 null mutation does (22-fold), yet rad27-324 appears to be recessive to wild type with respect to Ty1 mobility and also its mutator phenotype at CAN1. A dominant rad27 mutant with defects in both nuclease activities has been reported by GARY et al. 1999A . It would be interesting to analyze the effects of this mutation on Ty1 mobility.

Although the relationship between the biochemical and genetic phenotypes of various rad27 point mutations appears complex, only the rad27 null mutation significantly increases the level of cDNA recombination and multimer formation as well as retrotransposition. This result strongly suggests that the presence of any of the mutant Rad27 proteins suppresses cDNA recombination and multimer formation. How can this occur? One idea put forth by XIE et al. 2001 to explain the variable biochemical activities and genetic phenotypes observed for the rad27-G67S and rad27-G240D mutants is that Rad27 might play a structural role in maintaining genome stability in addition to its catalytic functions. In support of this idea, Rad27 has been shown to interact with DNA replication proteins (see below), and large complexes containing both recombination and DNA repair proteins are present in eukaryotic cells (WANG et al. 2000 ). Furthermore, a systematic genomic screen has shown strong genetic interactions between RAD27 and many DNA repair and genome stability genes (TONG et al. 2001 ). The alteration of key components could affect these complexes, allowing Ty1 mobility to increase. We may also learn more about the relationship between RAD27 and Ty1 mobility by determining the level of Rad27 protein in the point mutants. Alternatively, the rad27 mutations could affect Ty1 mobility indirectly by altering the level or activity of other replication, repair, or recombination proteins, perhaps by disrupting the cell cycle (VALLEN and CROSS 1995 ).

Like rad3-G595R and ssl2-rtt (LEE et al. 1998 , LEE et al. 2000 ), Ty1 integration at the Ty1 hotspot SUF16 (JI et al. 1993 ; DEVINE and BOEKE 1996 ) increases to varying degrees in the rad27 mutants. However, since the Ty1 insertion pattern remains unchanged, target site specificity is not determined by RAD27. RAD27 mutants have a variety of genome instability defects, including an increase in the rate of spontaneous mutation, DNA repeat-tract instability, and chromosome loss (VALLEN and CROSS 1995 ; TISHKOFF et al. 1997 ; PARENTEAU and WELLINGER 1999 ; XIE et al. 2001 ). Increased Ty1 transcription and transposition also occurs in response to DNA damage caused by chemical mutagens or UV radiation (MCCLANAHAN and MCENTEE 1984 ; ROLFE et al. 1986 ; BRADSHAW and MCENTEE 1989 ; STALEVA and VENKOV 2001 ). Given the mutator phenotype of rad27 mutants, an increase in Ty1 mobility may be the result from an increase in Ty1 RNA level. However, our results suggest that the Ty1 or Ty1his3-AI RNA level remains about the same in the rad27 point mutants or the rad27-324 frameshift mutant relative to the wild-type strain. There is an increase in the Ty1 RNA level (threefold) in the rad27::LEU2 deletion mutant that could be a response to DNA damage. However, TyA1 protein level remains essentially unchanged in the rad27 mutants. Taken together, our results suggest that RAD27 inhibits Ty1 mobility at a post-translational step.

EICHINGER and BOEKE 1990 made the interesting observation that Ty1 cDNA is limiting for integration in a cell free system using purified VLPs. Mutations in DNA repair and recombination genes, such as those in the RAD52 epistasis group (RATTRAY et al. 2000 ; SCHOLES et al. 2001 ), RAD3 and SSL2 (LEE et al. 1998 , LEE et al. 2000 ), and as shown here RAD27, have an increased level of unincorporated Ty1 cDNA. Therefore, Ty1 cDNA level may also be limiting for Ty1 mobility in vivo. We have shown that the rad27::LEU2 and rad27-324 mutations increase the steady-state level of Ty1 cDNA by about fivefold. We then used PFA inhibition of Ty1 reverse transcriptase (LEE et al. 2000 ) to show that the half-life of Ty1 cDNA also increases about fivefold in the rad27::LEU2 or rad27-324 mutants. These results suggest that Rad27 can influence the stability of Ty1 cDNA, perhaps by degrading partially reverse-transcribed molecules containing flaps or RNA/DNA hybrids when they enter the nucleus. A DNA flap created by an internal plus strand initiation site has been implicated in transport and integration of HIV-1 (ZENNOU et al. 2000 ). Ty1 elements also contain an internal priming site (POCHART et al. 1993 ; HEYMAN et al. 1995 ), but a similar flap structure has not been detected. Furthermore, it has been reported that a significant fraction of cDNA associated with VLPs isolated from cells expressing a specific Ty1 element is composed of incompletely reverse-transcribed molecules containing RNA/DNA hybrids (MULLER et al. 1991 ), although this result is probably element specific (EICHINGER and BOEKE 1988 ). Since we are measuring the level of cDNA produced by all Ty1 elements in the genome, it is likely that a variety of reverse transcription intermediates could be present. Not all mutations that increase Ty1 transposition post-translationally, however, cause an increase in cDNA accumulation. Deletion of SGS1 results in an elevated level of Ty1 mobility without a concomitant increase in cDNA (BRYK et al. 2001 ). Epistasis tests between rad27 and sgs1 null mutations suggest these genes function in different genetic pathways to modulate Ty1 mobility (data not shown).

Rad27 may also be involved in repair synthesis across the gap created after attachment of the 3' ends of Ty1 cDNA to host DNA, since Ty1 integrase makes a 5-bp staggered cleavage at a target site (VOYTAS and BOEKE 2002 ). Fen-1 is able to remove the 2-base 5'-flap that is generated during retroviral integration using model substrates in vitro (BRIN et al. 2000 ; YODER and BUSHMAN 2000 ). However, since Ty1 integration incorporates a blunt-ended cDNA into the genome (BRAITERMAN and BOEKE 1994 ; MOORE et al. 1995 ), a 5'-flap should not be present in the gapped integration product. It is also difficult to envision how a requirement for Rad27 in repairing a gapped integration intermediate would lead to an increase in Ty1 mobility in the absence of Rad27. Therefore, we do not favor the idea that Rad27 acts at this step in the process of Ty1 retrotransposition.

Our work has revealed interesting allele-specific phenotypes conferred by the rad27 mutations. In particular, the rad27 null mutant contains a Ty1 PvuII fragment that is consistent with the presence of a multimeric array (WEINSTOCK et al. 1990 ; SHARON et al. 1994 ). To detect the tail-to-head joint molecule characteristic of an integrated Ty1 dimer, we have performed PCR analysis of spontaneous Ty1HIS3 insertions using one primer homologous with HIS3 and a second from the 5' end of TYA1 (BRYK et al. 2001 ). Surprisingly, almost every Ty1HIS3 insertion (9/10) analyzed is part of a multimer in the rad27::LEU2 mutant, but multimeric arrays are not elevated in the other rad27 mutants. Theoretically, we expect to recover only half the possible multimers if they are all dimeric and composed of one marked insertion. The observation that 90% of the Ty1HIS3 insertions are multimeric suggests that these arrays contain only marked elements and/or they are larger than dimers. Southern analysis indicates that the Ty1 multimers obtained in the null mutant have not simply recombined with the preexisting Ty1his3-AI element, but are dispersed elsewhere in the genome (data not shown). Clearly, RAD27 is essential for suppressing Ty1 multimer formation, a process that can add many Ty1 elements to the genome (WEINSTOCK et al. 1990 ). Increased multimer formation is also observed when retroviral or Ty1 integration is blocked (HAGINO-YAMAGISHI et al. 1987 ; SHARON et al. 1994 ) or, to a more limited degree, in an sgs1 mutant (BRYK et al. 2001 ).

Multimeric elements likely arise by homologous recombination between LTRs of different cDNA molecules prior to integration into the genome or by recombination of the cDNA molecules with genomic Ty1 elements (WEINSTOCK et al. 1990 ; SHARON et al. 1994 ; BRYK et al. 2001 ). We have investigated the possibility that elevated Ty1 cDNA recombination in rad27::LEU2 is correlated with increased multimer formation by measuring the frequency of recombination between Ty1 cDNA and a specific marked genomic Ty1 element. Our results show that the level of cDNA recombination increases significantly in the rad27::LEU2 null mutant when compared to the wild-type strain or the other rad27 mutants including the rad27-324 mutant. This result is consistent with the notion that the multimers observed in the complete absence of Rad27 protein can result from recombination between Ty1 cDNA and a genomic element. Whether Ty1 multimers always arise from homologous recombination with a genomic element remains to be determined. The fact that de novo Ty1 insertions occur more frequently upstream of SUF16 and maintain the wild-type pattern in a rad27::LEU2 null mutant suggests that multimers may also form ectopically by homologous recombination and then insert via Ty1 integrase in the absence of RAD27. However, even the defective Rad27 proteins Rad27-G67S, -G240D, -E158D, and -324 apparently block Ty1 cDNA recombination as well as multimer formation. In contrast, Ty1 multimer formation in an sgs1 mutant results from intermolecular recombination between unincorporated cDNA molecules (BRYK et al. 2001 ).

The mechanism underlying the allele-specific effects of rad27-324 on Ty1 mobility is interesting to consider. One idea is that the Rad27-324 protein may not enter the nucleus, since the frameshift mutation truncates part of a putative nuclear localization signal (NLS) at the C terminus of the protein. The mammalian Rad27 homolog, Fen-1, has a functional bipartite NLS, KRKxxxxxxxxKKK located at residues 354–367 (QIU et al. 2001 ), while Rad27 has a putative bipartite NLS, KKFxxxxxxxxxxxLKK at residues 317–333 (from PSORT II server, unpublished result). Our results suggest, however, that a Rad27-324/green fluorescent protein fusion protein remains nuclear localized (data not shown). Another idea is that Rad27-324 is altered in binding to additional proteins that depend upon Rad27 to gain access to Ty1 cDNA or that stimulate Rad27 activities. The C-terminal residues 337–350 of Rad27 are involved in binding to proliferating cell nuclear antigen (PCNA; GARY et al. 1999A , GARY et al. 1999B ). PCNA is a processivity factor that binds to and enhances the catalytic activities of Fen-1/Rad27 during DNA synthesis (LI et al. 1995 ) and DNA repair (GARY et al. 1999B ). The rad27-324 mutant might be defective in binding PCNA since the mutation truncates the PCNA-binding motif and this could affect its enzymatic functions that are required to inhibit Ty1 mobility. Interactions with other proteins such as the Dna2 helicase (BUDD and CAMPBELL 1997 ) might also be affected in rad27-324. It would be interesting to analyze the biochemical activities of Rad27-324, as well as to determine the effect that Rad27 interactors have on Ty1 mobility.

In summary, our results indicate an important and novel role for RAD27 in inhibiting Ty1 mobility by affecting the fate of cDNA. Since RAD27 functions in the nucleus, it may act once the Ty1 cDNA enters the nucleus as part of a preintegration complex, but prior to integration. The increased stability of Ty1 cDNA in rad27 mutants would then allow the cDNA to enter the genome by integrase-mediated integration or homologous recombination with preexisting elements. In addition, intermolecular recombination events would generate multimeric Ty1 insertions if Rad27 is absent. Understanding how RAD27 is integrated into the growing number of Ty1 host defense genes (SCHOLES et al. 2001 ) will be necessary to understand fully how Ty1 elements and their yeast host coexist.

	ACKNOWLEDGMENTS

We gratefully acknowledge the comments of M. J. Curcio, S. Moore, and A. Rattray. We also thank E. Alani, A. Bailis, S. Holbeck, M. J. Curcio, T. Mason, S. Moore, and L. Symington for strains and reagents. This research was sponsored by the Center for Cancer Research of the National Cancer Institute, Department of Health and Human Services. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement from the United States Government.

Manuscript received July 19, 2002; Accepted for publication October 15, 2002.

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