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J Virol. Apr 2002; 76(7): 3240–3247.
PMCID: PMC136051

Ty5 gag Mutations Increase Retrotransposition and Suggest a Role for Hydrogen Bonding in the Function of the Nucleocapsid Zinc Finger

Abstract

The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.

Retrotransposons and retroviruses (collectively referred to as retroelements) replicate through an mRNA intermediate. Retroelements have a conserved genomic organization. They encode Gag and Pol polyproteins from an open reading frame(s) between two long terminal repeats (LTRs). In the retroviruses, Gag is processed into capsid (CA), nucleocapsid (NCp), and matrix proteins, which assemble into a virus particle; most retrotransposons encode CA and NCp homologues. The retroelement Pol polyprotein has the enzymatic functions required for replication, including protease (PR), integrase (IN), and reverse transcriptase (RT) activities. RT synthesizes a cDNA copy of the retroelement, and IN inserts the cDNA into the chromosome of the host.

Retrotransposons serve as important models for understanding retroelement replication. This is particularly true of the yeast retrotransposons, namely, those of Schizosaccharomyces pombe (Tf1) and Saccharomyces cerevisiae (Ty1, Ty3, and Ty5), where considerable genetic resources and tools are available to facilitate their study. Among the yeast retrotransposons, Ty5 has become an important model for understanding integration specificity, because of its preference for integrating into silent regions of the yeast genome (37-39). Ty5 is unusual among retroelements in that it encodes a single open reading frame that is cleaved by PR into PR, RT, and IN and two forms of Gag (Gag-p27, and Gag-p37) (15). The 10-kDa fragment released when p37 is processed to p27 is likely NCp. Ty5 is also one of a small group of retrotransposons (the hemiviruses) that prime reverse transcription with a half-tRNA (5, 17). Studies of Ty5, however, have been hampered by its low transposition frequency (~10−5) compared to other yeast retrotransposons (e.g., ~10−2 for Ty1) (9, 39). The Ty5 element used in all studies to date (Ty5-6p) comes from Saccharomyces paradoxus, a sibling species of S. cerevisiae (39). All S. cerevisiae elements are either solo LTRs or degenerate elements (20, 40).

Although Ty5-6p is transposition competent, considering its low transposition frequency, it may carry mutations that impede high-efficiency replication. Alternatively, Ty5 could be partially incompatible with the cellular environment of its surrogate host, S. cerevisiae, and differences in host factors between species might negatively impact Ty5 transposition. In this regard, a number of host genes have been identified that affect retrotransposition of yeast elements, such as Ty1 (8, 35). High-frequency transposition is important for undertaking genetic and biochemical studies directed at understanding unique aspects of Ty5 biology. In this study, we screened for Ty5 mutants with increased transposition frequency. In addition to making Ty5 a more tractable model system, we felt that mutations that increase transposition offer the opportunity to understand better the element-host relationship and to identify amino acids critical for Ty5 replication.

MATERIALS AND METHODS

Strain and plasmid construction.

Ty5 transposition was measured in the S. cerevisiae strains YPH499 (MATa ura3-52 lys2-801 ade2-101 trp1Δ63 his3Δ200 leu2Δ1), W303 (MATα ade2-1 can1-100 his3-11 leu2,3,112 trp1-1 ura3-1), and their rad52::TRP1 derivatives. pXW97 and pXW98 are GAL-Ty5 plasmids recovered from the library screen (see below) that show elevated transposition. pDR3 through pDR6 are derivatives generated by swapping a XhoI-BspMII fragment with a wild-type GAL-Ty5 fragment. pDR4 and pDR5 carry the D252N and Y68C mutations, respectively. To make the double mutant, the XhoI-HpaI fragment of pDR5 was cloned into pSP72 (Promega) to generate pDR10. The BamHI-SphI fragments of pDR4 and pDR10 were swapped to generate a clone (pDR12) with both mutations. The XhoI-HpaI fragment from pDR12 was excised and used to replace the wild-type fragment of Ty5 in pNK254. This generated the doubly mutant plasmid pDR14. Other plasmids were generated as follows:

(i) PCR-based mutagenesis was used to insert N-terminal epitope tags (RGSH6; Qiagen) into wild-type Ty5 and the single mutants (2).

Two overlapping primers were used: DVO557 (5′-ATG-AGA-GGA-TCG-CAT-CAC-CAT-CAC-CAT-CAC-ACA-TAT-AAG-CTA-GAT-CG-3′) and DVO558 (5′-GTG-ATG-GTG-ATG-GTG-ATG-CGA-TCC-TCT-CAT-AAT-GTT-GTA-AGT-TTA-TTG-G-3′). This resulted in the plasmids pNK520 (wild-type Ty5), pXG21 (Y68C), and pXG23 (D252N). As previously described, insertion of the RGSH6 tag at the N terminus still allows for Ty5 transposition (15).

(ii) pXG50 carries a Ty5 element with the D252R mutation and was constructed by PCR mutagenesis using overlapping primers DVO1509 (5′-GGG-GCT-CGG-CAT-CGC-TTA-AGC-3′) and DVO1510 (5′-GCG-ATG-CCG-AGC-CCC-ACA-AAT-3′) (2)

Plasmids with Ty5 double mutants include pXG48 (Y68C and D252R) and pXG49 (Y68C and D252K) and were constructed by PCR mutagenesis using pDR5 (Y68C) as a template and primers DVO1509, DVO1510, and DVO1511 (5′-GGG-GCT-AAG-CAT-CGC-TTA-AGC-3′) and DVO1512 (5′-GCG-ATG-CTT-AGC-CCC-ACA-AAT-3′).

(iii) The competitor template for cDNA quantification by PCR was generated by first cloning a 820-bp KpnI-SacII fragment from Ty5 into pBluescript (Stratagene) to generate pXG24.

pXG28 was constructed by deleting a 60-bp XmaI fragment from pXG24.

Mutagenesis and library screening.

The GAL-Ty5 plasmid (pNK254) was mutagenized by growing for 2 days in the Escherichia coli strain XL-1 Red, which has mutations in multiple DNA repair pathways (Stratagene) (12). This resulted in approximately 1 or 2 base changes per 3 kb of Ty5 DNA (D. Rowley and D. F. Voytas, unpublished data). The mutagenized library was transformed into YPH499, and patch assays were performed with independent transformants to assess transposition (39). Of 3,000 transformants evaluated, only two showed noticeably higher levels of transposition. Plasmids from these transformants (pXW97 and pXW98) were purified and retransformed into YPH499 and W303 to confirm that the plasmids conferred the increase in transposition.

Assays for integration, recombination, and target specificity.

Quantitative transposition assays were conducted as previously described (39), with the exception that the induction of transposition on galactose media was carried out for 3 days. The total number of His+ cells generated by Ty5 after growth on galactose includes both integration and cDNA recombination events. To calculate the frequency of integration, transposition assays were carried out in a rad52Δ strain to eliminate recombination events (19). To determine the relative levels of integration and recombination, 100 His+ colonies were randomly selected from plates of synthetic complete media lacking histidine (SC-H), patched to new SC-H plates, and allowed to grow for 3 days at 30°C. Cell patches were replica plated onto SC-H plates with 5-fluoroorotic acid (5-FOA). The percentage of integration was calculated as the number of colonies that grew on SC-H-5-FOA plates divided by the number of colonies on SC-H plates. Our assay that measures targeting of Ty5 to a plasmid-borne HMR locus was carried out as previously described (12).

Protein preparation and immunoblot analysis.

The conditions used for cell growth and the induction of Ty5 transcription were as previously described (15). Harvested cells were disrupted by the glass bead method (2). The supernatant was collected from the cell lysate after centrifugation (20,000 × g, 60 min, 4°C). The remaining pellet was extracted with sample loading buffer (2). An equivalent volume of supernatant and pellet was used to compare proteins in the soluble and insoluble fractions. Proteins were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and were electrophoretically transferred to nitrocellulose membranes (NitroBind; Micron Separations, Inc.). The protocols for transfer and Western blot analysis were as previously described (15). All Western blots used the RGSH6 monoclonal antibody (Qiagen).

Assaying Ty5 cDNA.

Yeast total DNA was purified by the glass bead method from cells grown to an optical density of 3.0. All DNA was quantified spectrophotometrically (2). PCR primers 1 and 2 were DVO208 (5′-CAG-CCG-GAA-TGC-TTG-GCC-A-3′) and DVO200 (5′-CAT-TAC-CCA-TAT-CAT-GCT-3′), respectively. Serially diluted competitor template was added to reactions with the same amount of yeast DNA. PCR conditions were as follows: 94°C, 1 min; 54°C, 1 min; and 72°C, 1 min for 30 cycles. After electrophoresis of the PCRs, bands in each lane were quantified using National Institutes of Health image software (version 1.62; http://rsb.info.nih.gov/nih-image/). Lanes were selected for calculating Ty5 cDNA levels in which amounts of products derived from cDNA and competitor were nearly equal. cDNA levels were considered to be the amount of competitor DNA added to that reaction, with adjustments made for slight differences in amounts of the two products.

Calculating hydrogen bonding potential.

Hydrogen bonding potential refers to the number of observed hydrogen bonds between a given amino acid and all four DNA bases (24). For each retroelement in the Ty1/copia group, including the wild-type and mutant Ty5 elements, the hydrogen bonding potential was summed for amino acids in each interval of the CX2CX3-4HX4C motif (i.e., the C-C, C-H, and H-C intervals). When the hydrogen bonding potential of the Ty1/copia group was considered as a whole, the average potential for each interval was calculated.

RESULTS

Two mutations in gag increase Ty5 transposition frequency.

The S. paradoxus Ty5 element (Ty5-6p) transposes in S. cerevisiae at frequencies 1,000-fold lower than for Ty1 (39). To test whether there are mutations in Ty5-6p that negatively impact transposition, we screened for Ty5 mutants with increased transposition frequencies. Plasmids with a Ty5 element were mutagenized and transformed into the YPH499 strain of S. cerevisiae. Transposition was assayed for more than 3,000 independent transformants. Our transposition assay measures the frequency by which His+ cells are generated after inducing transcription of a GAL-Ty5 by growth on galactose. Ty5 carries a nonfunctional his3 marker gene interrupted by an artificial intron (his3-AI), and a functional HIS3 gene is generated upon reverse transcription of spliced Ty5 mRNA (9, 39). A His+ phenotype results when Ty5 cDNA enters a target DNA molecule by integration or recombination.

Two mutants, pXW98 and pXW97, were identified with approximately sixfold-higher transposition frequencies (Fig. (Fig.1).1). To identify the mutations responsible for the increase, restriction fragments from pXW98 and pXW97 were swapped with the wild-type element Ty5-6p. Transposition of the chimeric elements was retested, and the mutations were localized to a 3-kb XhoI-BspMII fragment encompassing the 5′ half of Ty5. DNA sequencing revealed a single base change in pXW98 that resulted in a missense mutation, D252N. This mutation is located just before the conserved H residue in the CCHC zinc finger domain of Gag (CX2CX3HX4C). This zinc finger is the defining feature of NCp proteins (34). In pXW97, a single mutation resulted in the missense mutation Y68C. We combined the two mutations into one Ty5 element and found that it transposed approximately 36-fold higher than the wild type. Because the fold increase in transposition of the double mutant is the product of the fold increase of the two single mutants, it is likely that the mutations affect different steps of Ty5 transposition.

FIG. 1.
Two gag mutations (Y68C and D252N) increase Ty5 transposition frequency. Ty5 elements are drawn schematically. Arrowheads represent the LTRs; the wild-type element pNK252 is white, and the mutant elements are black or gray. The region swapped in pDR3, ...

Effects of gag mutations on integration and recombination.

We were interested in identifying the steps of transposition affected by the two single gag mutations. One possibility is that the mutations affect interactions with a host factor(s) critical for transposition. We previously noted severalfold-lower levels of transposition in the lab strain W303 than in YPH499. This strain difference was also observed for the Ty5 mutants (Table (Table1),1), suggesting that the genetic differences between strains act independently from the gag mutations. Because our transposition assay measures the frequency of His+ cells, which includes both integration and recombination events, we quantified integration in strains with mutations in the recombination-and-repair gene RAD52. Homologous recombination by Ty5 cDNA does not occur in rad52Δ strains (18, 19). The frequency of His+ cells, therefore, represents the integration frequency. Our results showed that, for each of the three Ty5 mutants, the fold increase in His+ frequency (integration plus recombination) in wild-type strains was comparable to the fold increase in integration in the rad52Δ strains (Table (Table1).1). This suggests that the increase in the frequency of His+ cells observed in the mutants is not due to an increase in the efficiency of integration.

TABLE 1.
Transposition frequencies of wild-type and mutant Ty5 elements in different yeast strains

We next specifically tested the effect of the gag mutations on Ty5 cDNA recombination. Because chromosomal Ty5 elements are degenerate, Ty5 cDNA almost always recombines with the plasmid-borne donor element (18, 19). This replaces the his3-AI marker with the wild-type HIS3 gene and confers a His+ phenotype. The plasmid with the donor element also carries a URA3 gene which prevents growth in the presence of 5-FOA. Recombinants, therefore, can be identified by their His+ 5-FOAs phenotype. By this selection strategy, we calculated the percentage recombination for each of the mutants in two different wild-type strains (Fig. (Fig.2).2). The results showed no distinguishable difference in recombination between the wild type and mutants. Integration and recombination, therefore, contribute almost equally to the increase in His+ frequency. It is likely that the mutants affect steps prior to integration and recombination.

FIG. 2.
The effect of Ty5 mutations on integration and recombination. Bars in the graph represent the percentage of integration or recombination relative to the total His+ frequency for each Ty5 genotype. Gray and white bars denote integration in YPH499 ...

Effects of gag mutations on cDNA levels.

Because the gag mutations cause an increase in both integration and recombination, they may act by increasing cDNA synthesis. We were previously unable to detect Ty5 cDNA by Southern hybridization experiments (N. Ke and Voytas, unpublished data), and so we developed a more sensitive, PCR-based assay. This assay was designed to amplify Ty5 cDNA and not DNA from the plasmid-borne donor element. To do this, we took advantage of the artificial intron, which is present in the donor element but not in the cDNA. One PCR primer spans the artificial intron and therefore can only anneal to cDNA and not to Ty5 DNA (primer 1, Fig. Fig.3A).3A). Amplification with this primer and a second, downstream primer (primer 2) should yield a 580-bp product using cDNA as a template. Strains with GAL-Ty5 were grown in the presence or absence of galactose to test the specificity of the PCR assay. Ty5 cDNA was detected only in DNA isolated from strains with GAL-Ty5 and after galactose induction (Fig. (Fig.3B).3B). We also did not observe a product when the PCR assay was used with Ty5 donor plasmid as a template (data not shown). A control DNA template (the competitor template described below) was added to all reaction mixtures to ensure that the PCRs were working.

FIG. 3.
A PCR assay for cDNA quantification. (A) The Ty5 donor element and Ty5 cDNA differ by the presence of an artificial intron (AI) in the HIS3 marker gene. Note that HIS3 is inserted in the opposite orientation relative to Ty5. Primer 1 (DVO208) spans the ...

To quantify relative levels of Ty5 cDNA, we designed a second template to use in competitive PCR experiments. The competitor template can be amplified by both primers and yields a product 60 bp shorter than the product from cDNA (Fig. (Fig.3A).3A). The amount of cDNA in a sample should equal the amount of added competitor when the amount of PCR product generated from both templates is equal. We showed that this was the case in control experiments using our competitor and a cloned copy of Ty5 cDNA (data not shown). PCRs were carried out with total genomic DNA prepared from induced cells with wild-type or mutant Ty5 elements (Fig. (Fig.4A).4A). Ty5 cDNA was quantified from reactions in which the products of the two templates were equal. The zinc finger mutant increased Ty5 cDNA 2.8-fold, the Gag mutant increased cDNA 2.2-fold, and the double mutant increased cDNA levels 4.2-fold (Fig. (Fig.4B).4B). These results indicate that both mutants increase transposition by affecting Ty5 cDNA levels.

FIG. 4.
The effect of Ty5 mutations on cDNA levels. (A) Results of quantitative, competitive PCR assays using DNA extracted from strains with wild-type or mutant Ty5 elements. The pXG28 competitor control (ranging from 0.01 to 0.08 ng) was added to each PCR. ...

Effects of gag mutations on protein processing and solubility.

Wild-type Ty5 Gag is processed by PR into 27- and 37-kDa proteins (15). Ty5 Gag is also largely insoluble and typically requires ionic detergents to go into solution. To monitor Gag processing and solubility, an epitope tag (RGSH6) was inserted into the Gag-Pol N terminus of wild-type and mutant Ty5 elements. The addition of this epitope tag still allows for transposition (15). Gag processing in the single mutants was comparable to that in the wild type, as measured in immunoblot experiments by the ratios of the 27- and 37-kDa species (Fig. (Fig.5).5). The solubility of the mutants was also comparable to that of the wild type, as evidenced by the levels of Gag in the soluble and insoluble (pellet) fractions (Fig. (Fig.5).5). We extracted Gag from the pellets with various concentrations of urea (from 1 to 8 M), and both the wild-type and mutant proteins were solubilized to approximately the same extent by the various urea concentrations (data not shown). We therefore concluded that the Y68C and D252N mutations did not significantly affect Gag processing and solubility.

FIG. 5.
Ty5 Gag expression, processing, and solubility. An epitope tag (RGSH6) was inserted into the N terminus of Gag-Pol in wild-type (WT) and mutant Ty5 elements. Cells were lysed by the glass bead method, and the lysate was separated by centrifugation. The ...

Effects of gag mutations on target bias.

We previously demonstrated that a short domain in the IN C terminus is required for Ty5 to integrate into silent regions of the yeast genome (12, 37). Mutations in this domain also decrease transposition frequency ~4-fold. We were curious as to whether the gag mutations identified here that increase transposition frequency also affect target choice. Target bias was measured by our assay that monitors integration into a plasmid with an HMR locus, a preferred Ty5 target (12). Plasmid insertions give rise to white colonies relative to the red or red-sectored colonies that result from chromosomal integration events. A change in the percentage of white colonies reflects an alteration in target specificity. The Y68C and D252N mutations did not change the target bias compared to wild type (Fig. (Fig.66).

FIG. 6.
Target specificity of the Ty5 mutants. A plasmid-based targeting assay was used to measure Ty5 integration specificity (12). The plasmid carries an HMR locus, one of Ty5's preferred integration sites (39). No significant difference in target specificity ...

Features of the zinc finger important for transposition.

In sequence comparisons of NCp zinc fingers, one significant difference between Ty5 and related Ty1/copia retrotransposons is that Ty5 has 3 (rather than 4) amino acids in the C-H interval of the CCHC motif (Fig. (Fig.7A).7A). Furthermore, in most retrotransposons (18 of 22 or 82%), G is located just before H; this is not the case for Ty5. We were curious to know whether the D252N mutation compensated either for the difference in the spatial organization of the Ty5 zinc finger or for the absence of the nearly invariant G residue. We therefore inserted G before H in a wild-type Ty5 element so that the zinc finger domain matched the evolutionarily conserved consensus sequence. The G insertion did not affect Ty5 transposition frequency (data not shown).

FIG. 7.
Features of the NCp zinc finger domain of Ty5 and other Ty1/copia retrotransposons. (A) Amino acid sequence alignment of the NCp CCHC motif of Ty5 and 22 other retrotransposons in the Ty1/copia group. The arrowhead indicates residue 252 in the Ty5 zinc ...

A second significant difference between Ty5 and the other retrotransposons concerns the potential for hydrogen bonding between the zinc finger motif and nucleic acids (Fig. (Fig.7B).7B). The average potential for hydrogen bond formation was calculated for each interval in the finger motif. The Ty5 zinc finger has significantly less capacity for hydrogen bonding than do the other retrotransposons. This is especially true for the C-C and C-H intervals. The D252N mutation significantly increases the potential to form hydrogen bonds. In addition, the acidic properties of D in wild-type Ty5 might repel nucleic acids, whereas N is neutral. To test whether the chemical properties of the zinc finger affect its biological activity, we created a D252R mutation. Among amino acids, R has the highest hydrogen bonding potential. The D252R mutation increased Ty5 transposition about sevenfold (Table (Table2).2). The double mutant—D252R, Y68C—increased Ty5 transposition about 33-fold. We also generated a D252K mutation which, when combined with Y68C, caused an approximately 40-fold increase in transposition. These results suggest that D inhibits the normal function of the Ty5 zinc finger motif, likely by preventing interactions with nucleic acids. Similar to our observation with the D252N, Y68C double mutant, the fold increase in transposition for other double mutants was the multiple of the fold increase for each single mutant. This indicates that the mutations in the zinc finger domain affect transposition independently of the Y68C mutation.

TABLE 2.
Transposition frequencies of NCp zinc finger mutants

DISCUSSION

Most eukaryotic genomes harbor retrotransposon families ranging from those that are highly successful to those that are likely extinct. We previously characterized the number and diversity of Ty5 elements in various S. cerevisiae strains, most of which have several degenerate insertions. For example, seven Ty5 insertions are present in the completed sequence of strain S288C, all of which are either truncated elements, solo LTRs, or LTR fragments (20). The number of these degenerate insertions suggests that Ty5 elements were once active in S. cerevisiae. In the closely related species, S. paradoxus, many strains have a few, apparently full-length elements. Of two such elements that were sequenced, only one, Ty5-6p, showed transposition activity after being expressed in S. cerevisiae (39). However, the transposition frequency of Ty5-6p was 1,000-fold lower than that of Ty1. We wanted to understand the reasons for this low transposition activity.

Host-encoded factors are important regulators of retrotransposition. A number of host genes, for example, affect transposition of the yeast Ty1 elements (8, 35). Host factors also influence Ty5 transposition. Genetic differences between the S. cerevisiae strains YPH499 and W303 result in a 10-fold difference in Ty5 transposition frequency. Because the functional Ty5-6p element was recovered from S. paradoxus, it is possible that S. cerevisiae host factors negatively impact transposition. However, in preliminary experiments, we found that Ty5 transposition frequencies are comparable in these two yeast species (X. Gao and Voytas, unpublished data). Ty5-6p may itself be inefficient in transposition. We tested this latter hypothesis by identifying Ty5 mutations that increase transposition. We hoped that, by identifying residues that negatively affect transposition, we could gain insight into mechanisms of Ty5 transposition or interactions between this retroelement and its host's cellular environment.

gag mutations increase cDNA synthesis.

Two Ty5 Gag mutants (Y68C and D252N) were identified that increase transposition approximately sixfold. Our transposition assay measures the His+ phenotype generated when Ty5 cDNA enters the S. cerevisiae genome. cDNA has two pathways to insert into target DNA molecules: it can integrate into the chromosome using the Ty5-encoded IN, or it can recombine with the Ty5 element on the donor plasmid (18, 19). The recombination pathway requires the host gene RAD52 (18). We first tested which of these pathways was affected by the gag mutations. A comparable fold increase in transposition frequency was found in both wild-type and rad52Δ S. cerevisiae strains, and this was observed for each Gag mutant compared to wild-type Ty5. In addition, the percentage of integration and recombination relative to the total number of His+ cells did not change. This suggests that the mutations did not alter the efficiency of either the integration or recombination pathways. It is very likely that steps prior to integration and recombination (e.g., cDNA synthesis) were affected by the mutations. An effect on cDNA synthesis is supported by the observation that protein levels and processing, as well as integration specificity, were unchanged by the mutations.

We have not been able to detect Ty5 cDNA by Southern hybridization (Ke and Voytas, unpublished), suggesting that very low amounts of cDNA are present in cells expressing Ty5. This might be one reason for Ty5's low transposition activity. A more sensitive PCR method, therefore, was developed to detect Ty5 cDNA. The specificity of the assay was confirmed by several controls: (i) the PCR assay failed to amplify the Ty5 donor plasmid. (ii) The assay also failed to amplify DNA extracted from yeast strains in which Ty5 transcription was not induced. These first two controls ruled out the possibility of nonspecific amplification from Ty5 in the donor plasmid. (iii) Amplification was not simply due to growth on galactose, which induces Ty5 transcription, as extracts from galactose-grown strains without Ty5 failed to yield an amplification product. (iv) An internal control was amplified in each reaction, indicating that PCR was taking place. PCR products originating from cDNA were only amplified from genomic DNA extracted from yeast strains with Ty5 that had been grown on galactose. (v) The PCR products were of the size predicted for templates from which the artificial intron was removed. Using a competitor template, we were able to determine that the D252N mutation caused a 2.8-fold increase in cDNA levels. Y68C caused a 2.2-fold increase, whereas the double mutant increased cDNA 4.2-fold. Our direct physical measurements of cDNA, therefore, support the idea that cDNA levels are increased in the mutants. Note that the increase in levels of cDNA is not commensurate with the observed increase in transposition. This may be because the assays measure different steps in replication or because there are limitations in the sensitivity of the PCR assay. However, the results of both assays are consistent.

RT carries out cDNA synthesis; however, cDNA levels do not depend on RT alone. Other factors encoded by the retrotransposon and host cell are part of the replication complex and can affect cDNA levels. NCp, for example, participates in cDNA synthesis, and one of the Ty5 mutations, D252N, is located in the zinc finger of NCp. This suggests that Ty5 NCp carries out a role in cDNA synthesis similar to the roles of NCps from other retroelements (34). As described below, the D252N mutation might optimize the zinc finger domain of NCp for nucleic acid binding, making it more efficient in RNA template packaging, tRNA primer annealing, or strand transfer and thereby increasing its effectiveness in cDNA synthesis.

The Y68C mutation is located near the N terminus of Gag. The role of this mutation in cDNA synthesis is less clear. One possibility is that it positively affects virus-like particle formation, thereby increasing the total amount of cDNA synthesized. However, this mutation did not alter the processing or solubility of Ty5 Gag. The Y68C mutation replaces an aromatic, polar uncharged residue (Y) with a hydrophobic residue (C). This could change the local structure of Gag. Posttranslational modifications could also be affected: the hydroxyl group of Y could be phosphorylated or sulfated, whereas C can undergo cysteinylation, oxidation, and glutathionylation or can form disulfide bonds. Changes in posttranslational modifications might make Gag better at particle formation and thereby result in the synthesis of more cDNA.

Host factors that are part of the retroelement replication complex are still not well defined. Strain differences between YPH499 and W303 result in a severalfold difference in Ty5 transposition. Because all three Ty5 mutants displayed the same fold difference in transposition between the two strains, host factor differences and the mutations in gag appear to act at independent steps.

Mutations in the zinc finger implicate a role for hydrogen bonding in NCp function.

NCp is the primary protein in the retroelement nucleocore. NCp binds tightly to both the genomic RNA of retroelements and their tRNA primers (3). Binding is carried out by one or two highly conserved CCHC-type zinc fingers. The zinc fingers are flanked by basic amino acids that also interact with template and primer RNAs. In Ty5, the consensus finger motif differs slightly from most retroelements (C X2C X3H X4C versus C X2C X3GHX4C). Some retroelements like Ty1 of S. cerevisiae do not have a conserved zinc finger; rather, three stretches of basic amino acids in the C terminus of Gag perform the required nucleic acid chaperon activity (7). Thus, although there are exceptions, the use of zinc fingers is the most widespread means of interacting with nucleic acids. The nucleic acid binding activity of NCp is important for a number of steps in replication, including RNA dimerization (3, 11, 27), primer and template RNA packaging (4), annealing of the tRNA primer to the template RNA (6, 22, 28), initiating reverse transcription (7, 29), transferring strong-stop DNA (1, 7, 10, 14), and ensuring fidelity of cDNA synthesis (13).

The D252N mutation is located just before the conserved H in the zinc finger domain of Ty5 NCp. This mutation, therefore, occurs in the interval in which spacing differs in Ty5 from that in other retroelements. In particular, the conserved G, which is located just before H in the zinc finger of most retrotransposons and retroviruses, is missing in Ty5 (Fig. (Fig.7A7A and reference 31). We suspected that the D252N mutation might in some way make the Ty5 zinc finger resemble and function like other retroelement zinc fingers that have the G insertion. To test this, we inserted a G before H in Ty5 NCp; however, this consensus zinc finger did not increase Ty5 transposition (Y. Chin and Voytas, unpublished data). The effect of the D252N mutation, therefore, is likely not due to local structural changes, and the conserved G does not appear to have a critical role in substrate binding, at least in Ty5 NCp.

An alternative explanation for the increase in transposition of the D252N mutation is that it changed a chemical property of the zinc finger. Ty5 has only one R and N in the H-C interval (Fig. (Fig.7A).7A). The zinc fingers of other Ty1/copia retrotransposons often have in their C-C and C-H intervals R, K or N, which are 3 amino acids with strong potential to form hydrogen bonds with nucleic acids. The hydrogen bonding potential of the Ty5 zinc finger is only 64.6% of that of other Ty1/copia retroelements (calculated as the sum of Ty5 hydrogen bonding potential across all intervals divided by the sum of hydrogen bonding potential in the Ty1/copia group elements) (Fig. (Fig.7B).7B). The D252N mutation increases the hydrogen bonding potential in the C-H interval to 71.3%. This might strengthen the zinc finger's ability to bind RNA, and consequently, the D252N mutation might make RNA template packaging and cDNA synthesis more efficient.

To test our hypothesis that hydrogen bonding plays a role in NCp function, we mutated D to R as an alternative means of increasing the hydrogen bonding potential in the C-H interval. As predicted, the D252R mutation increased Ty5 transposition about sevenfold, and the D252R, Y68C double mutant transposed 33-fold more efficiently. Another double mutation, D252K, Y68C, which also has a higher hydrogen bonding potential in the zinc finger, showed a 40-fold increase in Ty5 transposition. The transposition increase caused by these D substitutions is likely due to hydrogen bonding with bases and not phosphate group contacts. R and K, which are basic, should make stronger phosphate contacts than neutral amino acids such as N. If phosphate contacts were important, the D252R and D252K mutations should have the most impact on zinc finger function. However, all three substitutions increased Ty5 transposition by the same magnitude. The D in the wild-type Ty5 zinc finger provides no potential to hydrogen bond with G and U bases and a very low bonding potential for C and A bases. Its acidic property might even repel nucleic acids. The transposition increase caused by replacing D with other strong hydrogen bonding residues suggests an important role of hydrogen bonding in forming complexes between the zinc finger and RNA.

Relationship of the Ty5 zinc finger to other zinc finger motifs.

Our observations regarding the Ty5 NCp zinc finger have bearing on the function of other zinc finger motifs. These include the CCHH motifs (C X2-5C X12H X3-5H) found in proteins such as the mouse transcription factor Zif268 and the CCCC motifs (C X2C X13C X2C) found in proteins such as the glucocorticoid receptor (16, 21). These motifs have large middle intervals of 12 or 13 amino acids, compared to the 3 or 4 amino acids in the CCHC motif, and their C-terminal halves form α-helices (23, 26). In contrast, the crystal structure of retroviral NCp zinc fingers does not reveal any obvious secondary structure (25, 30, 32). This structural difference might distinguish the retroviral zinc fingers from other finger domains and reflect a role in binding single- versus double-stranded nucleic acids.

Amino acids in the CCHH and CCCC motifs also hydrogen bond to nucleic acids. In the CCHH zinc finger of Zif268 (26), hydrogen bonding occurs predominantly between the α-helix of the zinc finger and the G-rich DNA strand in the major groove. Nine of 12 of these interactions are hydrogen bonds with bases, and five of them involve interactions between R and guanine. Therefore, hydrogen bonding between residues within the zinc finger and nucleic acids are important in the function of both the CCHH and retroelement CCHC motifs. Other studies of CCHC zinc fingers of retroviral NCp revealed that flanking basic amino acids are also important for function (33). These basic residues may contact phosphates in the DNA, as has been shown for basic residues in the linker region of the CCHH-type zinc finger (36). It is interesting that there is a conserved D in the C-H interval of the three zinc finger repeats of Zif268 and other CCHH finger motifs (16). Although D has a low hydrogen bonding potential with nucleic acids, D plays a role in specifically binding adenine or cytosine residues in CCHH fingers (36). In contrast, nucleic acid binding by NCp zinc fingers is relatively nonspecific and of weaker affinity than the other finger motifs (21). The D in C-H interval that was mutated in Ty5 is not conserved in the retroviruses (31) or in retrotransposon zinc fingers (Fig (Fig7).7). The D at this position in Ty5 likely has a minimal role in DNA binding.

Previous work has implicated amino acids flanking the zinc finger in binding nucleic acids. Our study suggests that the hydrogen bonding potential of amino acids inside the CCHC motif also play an important role in zinc finger function. Although there are structural differences between the zinc fingers of cellular enzymes and the retroviral NCp zinc finger, the underlying mechanism of nucleic acid binding is likely conserved. Our findings suggest ways to increase the binding affinity of zinc finger domains, which is one of the present challenges in engineering nucleic acid binding proteins.

Acknowledgments

We thank Yvette Chin for generating the Ty5 G insertion mutant.

This work was supported by NIH grant GM51425 and by Hatch Act and State of Iowa funds.

Footnotes

This is journal paper no. J-19637 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 3383.

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