INTRODUCTION

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Ty1 is a retrotransposon of Saccharomyces cerevisiae which is structurally and functionally similar to retroviruses (for review, see references 3 and 18). Transcription of a genomic element results in the formation of intracellular virus-like particles (VLPs) which contain the element-encoded catalytic enzymes required for the process of transposition: protease, reverse transcriptase, and integrase (IN) as well as Ty1 RNA and a cellular tRNA^Met required to prime reverse transcription. The resulting full-length cDNA copy is correctly inserted into a new genomic location by IN-mediated strand transfer. Correct Ty1 integration events are characterized by a 5-bp target site duplication (TSD) without rearrangement or loss of sequence adjacent to the insertion site and by the coupled joining of both termini of the element to a single insertion site (concerted integration).

Both Ty1 VLPs and recombinant IN are active in a physical assay which monitors the insertion of a radioactively labeled long terminal repeat (LTR)-based oligoduplex into an identical target molecule (29). Although this assay demonstrates strand exchange activity of both recombinant IN and VLP-associated IN, it bears limited similarity to transposition in vivo. A transposition assay has been developed which detects the integration of supF-marked Ty1 elements into bacteriophage lambda and demonstrates that purified VLPs are sufficient for correct integration events (13). Gel electrophoresis and electron microscopy have been used to show VLP-mediated insertion of an exogenous linear donor molecule with Ty1 LTR-like termini into both linear and circular target molecules (4). A subset of products analyzed from these insertions resemble correct integration events. Devine and Boeke (11) have exploited the integration activity of Ty1 VLPs to insert a selectable artificial transposon into random sites of target DNA of unknown sequence as an aid to DNA mapping and sequencing.

Although correct integration has been demonstrated and characterized for retroviral INs, including human immunodeficiency virus 1 (HIV-1) (8, 9, 20, 22), Rous sarcoma virus (RSV) (28), avian sarcoma virus (22, 24), and avian myeloblastosis virus (AMV) (17, 27, 36, 37), the ability of an LTR-retrotransposon IN to carry out correct integration outside the context of the VLP has not been established. Using a genetic assay (11), we demonstrate that IN requires no additional VLP-associated proteins for correct integration of a donor substrate bearing Ty1 LTR sequences at each end into a supercoiled circular plasmid target. We have characterized this activity in regard to efficiency compared to VLPs, target site preference, divalent cation preference, and utilization of donor molecules containing non-Ty1 LTR ends. Our survey of mutated ends has shown that while not all sequences are acceptable IN substrates, a variety of mutant substrates undergo correct integration. In addition, Southern hybridization of the reaction reveals products consistent with correct integration events.

	MATERIALS AND METHODS

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Purification of Ty1 VLPs and recombinant Ty1 IN. Ty1 VLPs were isolated from strain GRY458 using established methods (13, 19). The yeast system for the ectopic expression of Ty1 IN has been described previously (29). This study required several IN and VLP preparations, which led to some variability in concentration and activity. However, experiments involving quantitative comparisons were carried out with the same IN and VLP preparations.

Purification of donor DNA fragments. An 864-bp fragment containing the dihydrofolate reductase gene, which confers resistance to the antibiotic trimethoprim (TMP), was used as a donor in the integration assay (11). Half XmnI sites (GAANN/NNTTC) on each end allowed the introduction of the terminal 5'-AC-3' nucleotides of the Ty1 U3 LTR in the NN position followed by the two XmnI-specific Ts. The resulting sequence comprises a U3-like terminus at each end of the donor fragment. This donor fragment, which was purchased from PE Applied Biosystems (Foster City, Calif.), was cloned into the XmnI site of the plasmid pUC19. This construct was subsequently used as a template for PCR amplification of donor fragments. Typically, PCRs were carried out for 30 cycles using 2.8 U of Expand high-fidelity DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) and 5 ng of template for each 100-µl reaction mixture. Primers and deoxynucleoside triphosphates were removed from the reactions by Wizard PCR Prep (Promega, Madison, Wis.). The product was restricted with XmnI, which generated phosphorylated 5' ends. The digested fragment was loaded onto a 1% low-melting-point agarose (Life Technologies, Rockville, Md.) gel and resolved at 6.5 V/cm² for 15 h in the presence of 5 µg of ethidium bromide/ml. Excised DNA fragments were purified by QiaexII (Qiagen, Valencia, Calif.), and the concentrations were determined spectrophotometrically. In certain experiments, amplification using phosphorylated primers allowed inclusion of additional LTR or mutated sequences and eliminated the requirement for XmnI digestion and gel purification (see Table 2 for primer combinations). To avoid untemplated nucleotides at the termini of donor molecules made by the phosphorylated primer method, Vent DNA polymerase (New England Biolabs, Beverly, Mass.) was used in the PCR.

Integration conditions. The standard 20-µl reaction mixture contained 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol, 5% polyethylene glycol 8000, 200 ng of donor fragment (0.35 pM), 1 µg of target DNA (0.2 pM), and either Ty1 IN or Ty1 VLPs. Reaction mixtures were incubated at 30°C for 1 h, after which 5 µl of stop mix (0.25 M EDTA, 1% sodium dodecyl sulfate, 5 µg of proteinase K/ml [EM Science, Gibbstown, N.J.]) was added. The reaction mixtures were then incubated at 65°C for 30 min. Ammonium acetate was added to a final concentration of 0.33 M, and the DNA was precipitated by the addition of 2.5 volumes of cold ethanol. The DNA pellet was washed once in 70% ethanol, dried, and resuspended in 20 µl of H₂O. An aliquot (3 µl for the experiment shown in Fig. 1, 6 µl for all other experiments) was introduced into 40 µl of HB101 cells at an approximate density of 2 × 10¹⁰ cells/ml by electroporation (1.8 kV, 200  resistance, 25 µF capacitance) with electroporation cuvettes having a gap size of 1 mm (BTX, San Diego, Calif.). After incubation at 37°C for 1 h, cells were diluted appropriately and plated on L agar plates containing either 100 µg of TMP/ml and 15 µg of tetracycline (TET) or TET only. Typically, three dilutions of the cell suspension were each plated in triplicate on TMP-plus-TET plates to select colonies containing integrant plasmids. Two dilutions were each plated in triplicate on TET-only plates to determine electroporation efficiency. After overnight incubation at 37°C, colonies were counted and integration efficiency was calculated as the number of TMP-and TET-resistant colonies divided by the number of TET-only-resistant colonies.

Sequencing. ABI Prism (PE Applied Biosystems) sequencing reactions were carried out according to the manufacturer's instructions.

Physical analysis of reaction products. Reactions for Southern analysis were carried out as described above except that the volumes were doubled and contained 460 ng of donor molecule (0.8 pM) and 5 µg of pUC19 as the target plasmid (2.8 pM). Following the 1-h incubation period, the reaction was stopped by the introduction of EDTA, to a final concentration of 63 mM, and 1 µg of proteinase K (EM Science) per ml in 10 mM Tris-HCl (pH 8.0) with 1 mM EDTA (TE) and incubated at 37°C for 30 min. The DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol precipitated. Following precipitation, the pellet was air dried and resuspended in 30 µl of TE. Three microliters of the resuspended DNA was digested with either StyI or PacI (New England Biolabs). Undigested controls were incubated in restriction enzyme buffer in the absence of enzyme. Following incubation at 37°C for 1.5 h, DNA molecules were resolved electrophoretically on a 1% agarose gel at 0.16 V/cm² for 14 h. The donor fragment was labeled with [-³²P]dCTP by Megaprime (Amersham-Pharmacia) and used as a probe in Southern hybridizations. Phosphorimaging was performed using a Storm860 and ImageQuant software, version 1.1 (Molecular Dynamics, Sunnyvale, Calif.).

	RESULTS

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Comparison of recombinant IN and VLPs and reaction conditions. Integration experiments were performed to compare recombinant IN and VLPs in the genetic assay with respect to integration efficiency and target site preference (Fig. 1). To estimate the efficiency of recombinant IN and VLPs (Fig. 1A), equal volumes of protein were added to the reaction mixture (x axis a). Although the total protein in the recombinant IN extract (x axis b) was less than that in the VLP extract (x axis c), the estimated IN concentration in VLPs (x axis d) was similar to that of recombinant IN. This estimate was based on an immunoblot comparison of samples of recombinant IN of a known concentration to VLPs containing an unknown concentration of IN. ImageQuant analysis indicated that about 1/16 of total VLP protein consisted of IN.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Comparison of recombinant Ty1 IN and Ty1 VLPs in the genetic selection assay. (A) Integration efficiencies with varying concentrations of recombinant IN or VLP-associated IN, measured as number of TMP-plus-TET-resistant colonies divided by number of TET-resistant colonies. The x axes indicate the volume of protein extract added to the reaction (a), the concentration of Ty1 IN protein (b), the total protein in the VLP sample (c), and the estimated concentration of Ty1 IN contained in VLPs (d). Points represent the mean of three replicate reactions. Vertical bars indicate one standard deviation from the mean. (B) Target sites mapped by sequence analysis for recombinant Ty1 IN (outside the circle) and Ty1 VLP (inside the circle). Only correct integrations are indicated.

Divalent cation requirement. Using an electrophoretic analysis method for concerted integration, Braiterman and Boeke (4) have demonstrated that Ty1 VLPs are more active in the presence of Mg²⁺ than in the presence of Mn²⁺. We quantitated the integration efficiency of recombinant IN in the presence of Mg²⁺ or Mn²⁺ (Fig. 2A) and found that the overall integration efficiencies were similar. However, sequence analysis of 39 integrant plasmids from Mg²⁺ colonies and 38 integrant plasmids from Mn²⁺ colonies revealed that in this experiment 74% of the Mg²⁺-derived products were correct integration events while only 45% of the Mn²⁺-derived products were correct. The most frequent class of aberrant integrations observed with either cation was integration of each end at separate sites in the target.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   (A) Effect of the divalent cations Mn2+ and Mg2+ in the genetic assay. Points represent the mean of two separate experiments, each having two replicates. Vertical bars represent one standard deviation from the mean. Ty1 IN concentration is 0.7 µg at each point. (B) Assay for nonspecific endonuclease activity of Ty1 IN as detected by the conversion of RFI to RFII circles in the presence of no cation (lanes 2 and 3), Mn2+ (lanes 4 and 5), or Mg2+ (lanes 6 and 7). Lane 1 contained pUC19 only, in TE. Other reaction mixtures contained pUC19 in reaction buffer with 1.2 µg of Ty1 IN (lanes 3, 5, and 7) or pUC19 in reaction buffer only (lanes 2, 4, and 6).

Physical analysis of reaction products. To confirm that integrants arose from an in vitro IN-mediated reaction rather than a bacterial cell recombination or DNA repair mechanism, integration products were visualized by Southern analysis using pUC19 as the plasmid target and the donor fragment as a hybridization probe (Fig. 3). After terminating the reaction, aliquots were digested with either StyI or PacI, which recognizes a single site at bp 267 or 132, respectively, in the donor molecule but no sites in the target plasmid. An additional aliquot was incubated in restriction enzyme buffer in the absence of enzyme.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   Southern analysis of integration products. (A) Diagram of products expected and the fragments resulting from digestion with either StyI or PacI. Marks on integrated donor molecules indicate approximate positions of restriction sites. (B) Electrophoretic analysis of in vitro integration products or with varying concentrations of IN and either undigested (Lanes 1, 3, 5, 7, and 9) or digested with StyI (Lanes 2, 4, and 6) or PacI (Lanes 8 and 10). Positions of expected digestion fragments are indicated by letters corresponding to the indicated lengths in panel A. Molecular weight standards on the left side of the figure were derived from the positions of bacteriophage -HindIII digest and 1 Kb-Plus ladder (Life Technologies).

Genetic analysis of donor molecules with mutated ends. Using the oligoduplex assay for IN activity, we have previously characterized the ability of molecules with mutated ends to serve as substrates for Ty1 IN (30). Although an in vitro concerted integration reaction is not identical to an in vivo transposition event, it does resemble in vivo integration more closely than the oligoduplex assay. Wild-type (WT) U3 and mutated donors (Table 2) were used in the genetic selection assay with both IN and VLPs. Because the U5 sequence has been shown by a physical assay to be inhibitory for VLP-catalyzed integration (5), a donor molecule containing a 4-bp WT U3 end and a 4-bp WT U5 end was tested to determine if the U5 sequence affected integration efficiency. Vora et al. (35) have reported that the fifth position of the U5 RSV LTR inhibits activity of AMV IN and RSV IN, and that when this position is mutated to become a U3-like end, IN activities increase. To determine if Ty1 LTR sequences beyond the 4-bp termini altered donor utilization, an 8-bp U3/U5 donor was tested. The TGCC U3 substitution is of particular importance because this mutation prevents in vivo transposition of an overexpressed Ty1 element (32). The terminal sequences involving rearrangements such as TGGT reversal, TGGG U3/GTCC U3, TA U3/TA U3 strand flip, and all A-T ends were compared to the results obtained with these termini in the oligoduplex assay (30), and one substrate with nonphosphorylated ends was tested as a comparison to a similar substrate used previously with VLPs (14).

Physical analysis of donor molecules with mutant ends. Products generated by integration of selected mutant donor molecules were also examined by Southern analysis using a ³²P-labeled donor as previously described (Fig. 4). Although donors carrying mutant ends gave rise to the same types of products as the WT substrate, the TGGT U3 mutant was lower overall in amount of product generated and was extremely deficient in the RFII product. This result, combined with the low integration efficiency, suggests that the RFII product is primarily, if not exclusively, responsible for TMP- plus TET-resistant colonies recovered in Escherichia coli. The U3/U3 4-bp WT donor showed the same pattern as observed previously (Fig. 3B). Interestingly, the TGCC U3/U5 WT substrate, which is defective for in vivo transposition, showed product formation similar to that observed with the U3/U3 WT substrate. The U3/U5 4-bp WT substrate, while exhibiting both the RFII and linearized products in the undigested reaction, showed an altered digestion pattern from the U3/U3 WT donor. Although the StyI 3,880-bp fragment and the PacI 4,150-bp fragment were diminished, the 3,550-bp fragment of each digest was present as well as the smaller fragmentsStyI, 3,220 bp and PacI, 2,950 bp. This result suggests that the U3 end was preferentially utilized for bimolecular integration.

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Physical assay of reactions carried out with recombinant Ty1 IN and selected mutant donors. S, digestion with StyI; P, digestion with PacI. Products expected are identical to those illustrated in Fig. 3 and are indicated on the gel.

	DISCUSSION

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Our results demonstrate that purified recombinant Ty1 IN can integrate an exogenously added linear donor into a supercoiled target plasmid correctly. Most integration events are concerted and contain a 5-bp TSD. We have used both a genetic selection and a physical assay to characterize integration products. The genetic assay allows recovery of integrant plasmids for further analysis while the physical assay provides a direct visualization of products without the intervening step of introducing the products into bacterial cells.

The genetic assay has been used to compare IN and VLPs with respect to quantitative efficiency, frequency of correct integration events, and target site selection. A comparison of similar concentrations of recombinant IN and VLPs shows VLPs to be fivefold more active than recombinant IN in the genetic assay (Fig. 1A). The interpretation of this result, however, is not straightforward since it compares activities of Ty1 INs which are in different microenvironments and which have been purified by different methods. Improvements in protein purification methods may eventually reduce this difference in activity. The observation that addition of TYA1 to recombinant IN enhances integration activity by twofold suggests that TYA1 plays a role in IN stability but is not entirely competent to perform this function when added in trans. This is not surprising considering that IN within the VLP is associated with TYA1 during protein maturation, whereas recombinant IN is expressed outside the context of the particle. Additionally, we cannot rule out an as-yet-unidentified accessory factor which may copurify with VLPs. Sequencing analysis of plasmids recovered from antibiotic-resistant colonies shows about the same percentage of correct integrations regardless of whether recombinant IN or VLP-IN is used. The sequencing analysis (Fig. 1B) also shows that highly preferred sites for either IN are absent when purified DNA is used as a target. However, Ty1 transposition in vivo shows target site selection near genes transcribed by RNA polymerase III (12). A naked DNA target which lacks chromatin structure as well as accessory proteins associated with RNA polymerase III transcription might not be expected to exhibit target site preferences.

We have characterized the divalent cation preference of Ty1 IN (Fig. 2). The presence of a divalent cation, Mn²⁺ or Mg²⁺, is required for catalytic activity of INs (15, 24, 25). Additionally, divalent cations have been shown to influence structural conformation of IN (2, 39). Although initial characterization of retroviral INs using the oligoduplex assay have been reported to require Mn²⁺ for optimal activity (10, 15, 24), Engelman and Craigie (15) have shown that HIV-1 IN can utilize either cation under varied reaction conditions. Vora et al. (37) and Fitzgerald et al. (17) characterized the cation requirement of AMV IN and demonstrated that, although Mn²⁺ efficiently promotes donor-to-donor insertions and unimolecular half-site insertions into a circular target, Mg²⁺ is more efficient for bimolecular concerted integration into a circular target.

Here, we report that correct concerted integrations catalyzed by Ty1 IN are more efficient with Mg²⁺ than with Mn²⁺. Although similar numbers of TMP- plus TET-resistant colonies result when either cation is present in the reaction mixture (Fig. 2A), sequencing analysis reveals that only 45% of the Mn²⁺-derived integrants are correct integrations, compared with 74% when Mg²⁺ is present. Engelman and Craigie (15) have found that HIV-1 IN displays more nonspecific nuclease activity in the presence of Mn²⁺ than with Mg²⁺. Such an activity might play a role in nonconcerted integration by creating precleaved insertion sites in the target or by linearizing the target plasmid. VLP-catalyzed integration yields a significant class of products in which the donor is integrated near the end of a linear target (4). We have tested recombinant Ty1 IN for an alteration in nuclease activity in the presence of both cations under our standard reaction conditions and have observed no profound difference in the conversion of RFI to RFII circles with either cation (Fig. 2B). Consequently, the increased proportion of nonconcerted Mn²⁺-promoted events does not appear to be due to nuclease activity on the target plasmid. However, we cannot rule out that Mn²⁺ interacts with the target in a different way or that Mn²⁺ interacts with other components of the reaction, permitting less stringent integration.

To visualize IN-catalyzed products directly, we used Southern analysis with a radiolabeled donor fragment (Fig. 3). This analysis revealed two predominant products. The faster migrating product is consistent with a 4,414-bp linear fragment resulting from a bimolecular integration (Fig. 3A). The slower migrating product is consistent with an RFII circle containing one donor fragment per target plasmid, as indicated by an RFII electrophoretic marker. A comparison of the TGGT U3/TGGT U3 donor with the WT U3/U3 donor by using genetic and Southern analyses suggests that this product represents primarily bimolecular concerted events. This mutated donor shows a severe deficiency in both TMP-resistant colony formation and in RFII product in the Southern analysis. Other mutated donors which are used proficiently in the genetic assay exhibit near-WT amounts of the RFII product. This result suggests that the RFII product is primarily responsible for colony formation in the genetic assay. Although half-site insertions also result in RFII products, these events seem unlikely to give rise to colonies which resemble IN-mediated insertions in the genetic assay.

The possibility also exists that colonies arise from electroporation of the bimolecular concerted linearized product. Since the donor fragments initially insert in a concerted manner, sequence analysis of this fragment might still reveal a target site duplication. However, since either end of each donor can insert into the target, the sequencing analysis should have revealed some plasmids which lack a donor-target junction. If the linear molecules recircularized in the bacterial cells, sequencing would have revealed donor-donor junctions rather than donor-target junctions. No aberrant sequences of this type were detected. Even though we used a recA strain of E. coli as the bacterial host in the genetic assay, we cannot rule out the possibility that other recombination pathways convert bimolecular linearized products into a form resembling RFIIs. However, the results shown in Fig. 4 compared with the results of the genetic assay suggest that this is not a common event. We base this conclusion particularly on the relative amount of bimolecular linear product observed with the TGGT U3/TGGT U3 substrate compared to the U3/U3 4-bp substrate. Although the amounts of linear product are similar, the results of the genetic assay show the TGGT U3/TGGT U3 substrate to be reduced by greater than 2 orders of magnitude compared to the U3/U3 4-bp substrate. If a significant number of recombination events yielded colonies in the genetic assay, we would have expected to observe more quantitative similarity between these two substrates.

In a similar assay using exogenous donor and Ty1 VLPs, Braiterman and Boeke (5) have evaluated several terminal and subterminal donor mutations. Their results show that VLP-mediated integration is tolerant of a wide range of mutations. In addition, their results suggest that the U3 terminus is preferred and that the U5 terminus is inhibitory. Although our results for the genetic assay show no reduction in integration efficiency when one of the U3 termini is replaced by 4 bp of U5 sequence, Southern analysis indicates that the U3 end is preferred in bimolecular concerted events. U3 is also the preferred end for AMV (16, 21, 36, 37), whereas U5 is the preferred end for HIV-1 (7, 20, 26, 33), human foamy virus (31), and feline immunodeficiency virus (34). Fitzgerald et al. (17) have suggested that the least effective terminus is that which is also involved in another element function and therefore is constrained from evolving into the most effective substrate for integration. The demonstrated U3-over-U5 preference of Ty1 is consistent with this hypothesis since the U5 terminus is also part of the TYA1 coding sequence.

Vora et al. (35) have shown that the fifth position of the RSV U5 LTR is responsible for a three- to fivefold preference of U3 ends over U5 ends by RSV IN and AMV IN. Since our U3 WT/U5 4-bp WT donor showed no reduction in utilization, we also analyzed a U3 WT/U5 WT donor containing 8 bp of each LTR. Additional LTR sequences did not result in further impairment of U3 WT/U5 WT donor utilization by either IN or VLP. Rather, the 8-bp LTR donor showed a two- to threefold enhancement in integration efficiency compared to the 4-bp donor. These differences in subterminal donor preferences may be attributed to two differences between the RSV LTR and the Ty1 LTR. The fifth position in the RSV LTR is the first nonidentical U3/U5 nucleotide, whereas the third position of the Ty1 LTR is the first nonidentical position. Additionally, the RSV LTR undergoes IN-mediated 3' dinucleotide cleavage prior to integration (23), whereas Ty1 LTRs do not (30). Consequently, the critical fifth position of the RSV LTR may be analogous to the third position of the Ty1 LTR. If so, then the important nonidentical nucleotide is included in the 4-bp donor, and enhanced activity of the 8-bp donor may be due to additional subterminal LTR sequences. Braiterman and Boeke (5) have shown that subterminal mutations have a profound effect on VLP-mediated integration. Wilhelm et al. (38) reported that a subterminal mutation in the U3 LTR that changes the WT TGG at positions 4 to 6 to CAT abolishes Ty1 transposition in vivo.

Several of the termini examined in this study have been evaluated in an oligoduplex assay (30) which showed that Ty1 IN does not utilize oligoduplexes having G or C termini. The data presented here are broadly consistent with those results. However, the difference between G-C termini and A-T termini is not as distinct in the genetic assay as in the oligoduplex assay. Mutations that exhibit more severe effects in the oligoduplex assay than in a concerted integration reaction or in vivo integration have also been reported for retroviruses (1, 35).

Our results indicate that recombinant Ty1 IN is necessary and sufficient for catalyzing correct integration in vitro. Both recombinant IN and VLPs show a wide range of tolerance for non-LTR termini of an exogenously added donor, including a terminal mutation which has been shown to be ineffective for in vivo transposition (32). This result suggests that mechanisms which define specificity for correct ends for transposition may act differently with exogenous donors than with the endogenous element. Experiments which utilize the endogenous element in a two-ended integration assay may indicate whether IN is only promiscuous with exogenous donor or whether an element or host-encoded specificity factor is required to fully reconstitute donor preference in vitro.