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J. Biol. Chem., Vol. 278, Issue 48, 47678-47684, November 28, 2003

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Extension and Cleavage of the Polypurine Tract Plus-strand Primer by Ty1 Reverse Transcriptase^*

François-Xavier Wilhelm, Marcelle Wilhelm, and Abram Gabriel¶||

From the Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France and the ^¶Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854

Received for publication, May 16, 2003 , and in revised form, September 17, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
During reverse transcription of the plus-strand RNA genome of long terminal repeat retroelements, the RNase H activity of reverse transcriptase (RT)¹ degrades the genomic RNA while the polymerase activity synthesizes minus-strand DNA (Fig. 1) (1–7). For plus-strand DNA synthesis, RNase H generates the polypurine tract (PPT) primer and cleaves the minus-strand tRNA primer. The manner in which this enzyme carries out these multiple functions is not well understood, but two modes of RNase H activity can be distinguished as follows: (i) a polymerase-dependent mode that accompanies DNA synthesis and is positioned by the polymerase active site binding to the recessed nascent DNA 3' end and (ii) a polymerase-independent mode that occurs without DNA synthesis. Wisniewski et al. (5–7) have suggested that the polymerase-independent mode of RNA cleavage by HIV-1 RT is oriented by the polymerase domain binding to the recessed 5' end of RNA fragments paired to the DNA. Both modes of RNase H activity are necessary to remove the RNA after minus-strand DNA synthesis. During DNA synthesis, the RNA template is only partially digested because of a greater rate of polymerization versus RNA template cleavage (8–10). This implies that polymerase-dependent RNase H activity is not sufficient to completely degrade all of the template RNA. The fragments, which remain bound to the minus-strand DNA, must be digested further by the polymerase-independent activity of RNase H. Specific purine-rich fragments called PPTs are more resistant to RNase H digestion and function as RNA primers for plus-strand DNA synthesis (1, 2). Precise cleavage of the RNA at the PPT/U3 junction by RNase H and extension of the PPT primer by the polymerase activity of RT are critical for production of integration-competent elements. We previously showed that a recombinant Ty1 RT can recapitulate in vitro key processes that control proper replication of the Ty1 element in vivo (11). Using wild type RNA/DNA duplexes, we showed that the RNase H activity of Ty1 RT is able to digest the template RNA at the PPT/U3 border (i.e. between nt –1 and +1, see Fig. 2 for numbering) during elongation of minus-strand DNA. The polymerase-independent RNase H activity cleaves the RNA into fragments of 8–10 nt in size. In this paper, we show that the RNA can be further degraded to generate smaller cleavage products of 6 nt in length.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Simplified model for retroelement reverse transcription. Thin lines represent the template mRNA; thick lines with arrow-heads represent the newly synthesized DNA. A, the minus-strand tRNA primer anneals to the primer binding site (pbs) of the genomic RNA. B, the minus-strand strong-stop DNA is synthesized. The 5' end of the genomic RNA is degraded by RNase H. C, minus-strand strong-stop DNA is translocated to the 3' end of the genomic RNA. D, the translocated nascent minus-strand DNA is extended. The genomic RNA template is degraded and removed with the exception of the PPT fragments. E, plus-strand DNA synthesis is initiated from the PPT fragment. F, the nascent plus-strand strong-stop DNA is translocated to the 3' end of the minus-strand DNA. G, reverse transcriptase completes DNA synthesis.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Internal RNase H cleavage of the PPT primer. A, the sequence of the DNA template and three RNA primers (–9/–1, –9/+3, and –9/+7) used in B. Ribonucleotides are shown in lowercase letters, and deoxyribonucleotides are shown in uppercase letters. The star at the 5' end of the RNA primer represents the 32P label. B, the template hybridized to the –9/–1 primer shown in A was incubated with wild type Ty1 RT (RH+) for 0.5–8 min. C, the template hybridized to primer –9/–1, –9/+3, or –9/+7 was incubated with wild type Ty1 RT (RH+) or RNase H– (RH–) Ty1 RT for 10 min.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Expression and Purification of Recombinant Ty1 RT—The wild-type Ty1 recombinant RT and the D468S RNase H active site mutant version (12) of the wild-type construct contained six histidine residues at their amino termini. They were expressed in Escherichia coli and purified by Ni²⁺-nitroloacetic acid-agarose (Qiagen) affinity chromatography as described previously (13)

RNA and DNA Oligonucleotides and RNA Labeling—The RNA oligoribonucleotides were chemically synthesized in our laboratory with a DNA-RNA Applied Biosystem Synthesizer (Model 392) or purchased from Thermo Hybaid, the Interactiva Division (Ulm, Germany). The DNA oligodeoxyribonucleotides were purchased from Thermo Hybaid. The chemically synthesized RNAs were labeled at their 5' termini with ³²P using [-³²P]ATP and T4 polynucleotide kinase.

Primer-template Annealing, RT, and RNase H Assays—To prepare the RNA/DNA duplexes, a 3–5 molar excess of template DNA was hybridized to the primer RNA in a buffer containing 50 mM Tris-HCl, pH 7.8, 15 mM NaCl, and 8 mM -mercaptoethanol. The mixture was heated at 90 °C for 1 min and then incubated for 10 min at 70 °C, 10 min at 42 °C, and 10 min at room temperature. After the addition of the Ty1 RT (at a 2:5 RT to primer-template molar ratio), the samples are incubated at 23 °C for the time indicated in the legends of the figures. RNase H cleavage and/or RT polymerization were initiated with MgCl₂ at a final concentration of 20 mM. The reactions were stopped by the addition of formamide sample buffer containing 25 mM EDTA. The products of the reaction were separated on denaturing 20% polyacrylamide gels and visualized by autoradiography. Quantification of gel bands was performed using a Fuji PhosphorImager.

To prepare the internally labeled 9R/9D primer, the 9R primer was annealed to a template with a 9-nt extension. The 9R primer was extended with the Klenow fragment of E. coli DNA polymerase I in the presence of dATP, dTTP, and [-³²P]dGTP. The labeled primer was electrophoretically separated, extracted, precipitated, and resuspended to obtain a purified product.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Interaction between Ty1 RT and the Plus-strand Primer/Template Initiation Complex—In Ty1, the primer for plusstrand DNA synthesis is a 9-nt oligoribonucleotide with the –9/–1 sequence uggguggua (14–16). Compared with retroviral PPTs, this sequence is unusual in that it is so short (9 nt compared with 18–23 nt for retroviral PPTs) and contains three pyrimidines at positions –2, –5, and –9. To mimic the plus-strand Ty1 primer-template initiation complex, a 9-nt PPT oligoribonucleotide corresponding to the Ty1 PPT was annealed to a 35-nt DNA template containing flanking upstream sequences from the Ty1-untranslated region and downstream sequences from the Ty1 U3 region (Fig. 2A). We have previously shown that the 9-nt PPT primer is relatively resistant to RNase H cleavage when this hybrid substrate is incubated with Ty1 RT (11). However, we observed partial cleavage of the PPT primer 6 nt from the 5' end of the primer between nt –3 and –4 (Fig. 2B). As shown in Fig. 2C, a –1/+1 cleavage occurs rapidly with primers containing different 3' extensions beyond +1, whereas a second cut between –3 and –4 occurs as well. Both cuts are absent when an RNase H minus (RH–) Ty1 RT is used (Fig. 2C). Wisniewski et al. (5–7) have proposed that similar cleavage events, observed using HIV-1 RT, represent a polymerase-independent mode of RNase H activity positioned by the RT binding at the 5' end of the recessed RNA. Although we cannot determine from our data the positioning responsible for this cleavage event, it appears that the internal cleavage of the PPT does occur in our in vitro system.

In the polymerization mode, the polymerase domain of RT binds the RNA primer at its 3' end. Because 3' binding means progression of replication while internal cleavage could generate incorrect termini, it was of interest to analyze the relative efficiency of these two outcomes. We annealed a 48-nt template including 14 nt from the Ty1-untranslated region and 25 nt from the Ty1 U3 downstream region to a 9R/9D chimeric primer containing the nine RNA residues of the PPT and nine DNA residues (internally labeled with radioactive dGTP at positions +2, +5, and +6 as described under "Experimental Procedures") complementary to 9 nt of the U3 sequence. This model substrate is a probable intermediate during plus-strand DNA replication. The primer-template was incubated with either wild type or RNase H– Ty1 RT in the presence of all four dNTPs (Fig. 3). With the RNase H– enzyme, the major elongation product was a 9R/25D band corresponding to the run-off species (Fig. 3, lanes 3 and 4). This product is not prominent, in line with previous data from our laboratories and others (for example, see Ref. 17) that RNA primers are not used efficiently in vitro. The pattern obtained with the wild type enzyme, which has both RNase H and polymerase activity, is more complex because elongation is superimposed upon primer removal and internal primer cleavage. In this case, no 9R/25D run-off band was observed (Fig. 3, lanes 1 and 2), indicating the marked propensity of cleavage of the primer RNA by RNase H. Instead, a 25D band is present corresponding to the run-off product with the primer removed. Two stronger low molecular weight bands (9D and 3R/9D) were also observed. These bands probably result from the polymerase-independent activity of Ty1 RT, which cleaves the chimeric 9R/9D primer at the PPT/U3 junction (9D band) and within the RNA moiety of the chimeric 9R/9D primer (3R/9D band). The 9D band could also be generated during elongation of plus-strand DNA by the polymerase-dependent RNase H activity. Thus, there are multiple outcomes depending on the nature of the binding of RT to its substrate. The enzyme, which binds to the 3' end of the primer, is able to initiate plus-strand synthesis. With the wild type enzyme, the elongated product is rapidly cleaved at the DNA/RNA interface (i.e. the PPT/U3 junction). A large proportion of the enzyme, however, prefers to cleave the RNA within the PPT sequence. In the case of Fig. 3, it appears that only <8% of the product is elongated, whereas 87% is cleaved. If this were to occur in vivo, it would limit the production of plus-strand strong-stop DNA.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Elongation and cleavage of a PPT containing substrate. A, the sequence of the primer-template used in B, a 32P-labeled chimeric 9R/9D primer was annealed to a 48-nt DNA template. B, the substrate in A was incubated for 10 or 30 min with wild type (RH+) or RNase H minus (RH–) Ty1 RT. We have checked that the undigested 32P-labeled chimeric 9R/9D primer and a 32P-labeled 9D oligonucleotide co-migrate with the 9R/9D and 9D bands and that after alkali treatment, the 3R/9D, 9R/9D, and 9R/25D bands were hydrolyzed but that the 9D and 25D bands were not (data not shown). C, schematic representation of elongation and cleavage events. RNase H cuts are indicated by arrows.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Elongation and cleavage during initial synthesis of plus-strand DNA. A, sequences of the extended P/T7 to P/T16 primer templates generated by DNA synthesis using the uggguggua primer and various lengths templates (P/T7 to P/T16). The DNA is internally labeled at G residues. B, products of RT-catalyzed DNA synthesis and RNase H cleavage on substrates shown in A. The upper bands on the autoradiogram (9R/7D to 9R/17D) are the uncleaved extension products. The lower bands (9D to 17D) are the cleaved newly synthesized DNA strands. Closed circles represent each major synthesis or cleavage products.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Cleavage of the RNA/DNA junction by a RT molecule that is not involved in DNA synthesis. A, sequence of the expected synthesis products of the primer-template used in C. B, schematic representation of elongation and cleavage events. As in Fig. 3, the primer was not labeled but the reaction mixture contained [-32P]dGTP so that the newly synthesized DNA was internally labeled. C, lane RH+, the PPT primer annealed to the DNA template was elongated with the wild type enzyme. A 9R/16D run-off product and a cleaved 16D product are observed. Lane RH–, the primer-template was incubated with RNase H– RT, only the 9R/16D run-off product is observed. Lane RH–/RH+, an aliquot of the reaction product shown in lane RH– was removed and mixed with wild type enzyme. The RT-associated RNase H activity of the wild type enzyme cleaves the RNA primer from newly synthesized DNA. 70% of the run-off 9R/16D band is cleaved, giving rise to the 16D band. Lane M is a 32P-end-labeled 16D control oligonucleotide.

To confirm that the n + 1 bands represents non-templated base addition, we carried out extension with P/T16 in the presence or absence of dideoxynucleotides (Fig. 4B, lanes 6 and 7). In one reaction (lane 6), the four dNTPs were included in the nucleotide mixture, whereas in the second reaction (lane 7), ddCTP was included along with dATP, dTTP, and dGTP. A comparison of these two lanes shows that the run-off bands with n + 1 residues added to the primer and the cleaved fragment of DNA containing n + 1 residues were not observed in lane 7 because the last residue of the newly synthesized DNA is a dideoxynucleotide, which cannot be elongated by terminal transferase.

To confirm that cleavage does not occur after 7 nt have been added to the PPT primer, we compared elongation and cleavage on two primer-templates, P/T7 and P/T16, using wild type and RNase H– Ty1 RT (Fig. 5). In these experiments, the primer RNA is 5' end-labeled. With P/T7, extended product accumulates with time and the pattern of elongation is the same regardless of the presence or absence of RNase H activity. For P/T16, the accumulation of product with time is observed in the absence of RNase H. In its presence, only a low and constant level of product is observed, consistent with an equilibrium between extension and cleavage of the labeled primer.

View larger version (87K): [in this window] [in a new window]   FIG. 5. A time course of elongation and cleavage of initially synthesized plus-strand DNA using a 5' end-labeled primer. A, sequence of the expected synthesis products of the two primer-templates P/T7 and P/T16 used in B. The star at the 5' end of the RNA primer represents the 32P label. B, products of RT-catalyzed DNA synthesis and RNase H cleavage on substrates shown in A. Lanes 1–8 show the time course experiment with P/T7 (lanes 1–4) and P/T16 (lanes 5–8) after incubation with wild type Ty1 RT (RH+). Lanes 9–16 show the time course experiment with P/T7 (lanes 9–12) and P/T16 (lanes 13–16) after incubation with RNase H– Ty1 RT (RH–). The bottom of the gel, which is overexposed, is not shown. For the wild type enzyme, which has RNase H activity, cleavage between nt –3 and –4 of the primer was detected in lanes 1–8.

We know from our previous work (11, 13) that the polymerase-dependent cleavage remains at a fixed distance of 14 nt from the 3' end of the elongated molecule. The observation that the PPT/U3 junction is already cleaved after 9 nucleotides have been added to the primer (Fig. 4) suggests that this cleavage could be accomplished by a second RT molecule not directly involved in DNA synthesis. Alternatively, the distance between the polymerase and RNase H active sites may be more dynamic in the case of the PPT sequence, allowing RNase H cleavage after only the addition of 9 nt. To test whether a second RT not involved in elongation could cleave the partially extended primer, we elongated the –9/–1 primer on template T16 using the RNase H– enzyme (Fig. 6, lane 2). An aliquot of the reaction product was then mixed with wild type enzyme. Both samples were then separated on a sequencing gel. As shown in Fig. 6, lane 3, after the addition of the wild type enzyme, 70% of the product synthesized by the RNase H– enzyme is cleaved by the RT-associated RNase H activity of the wild type enzyme. These data show that cleavage can indeed be accomplished by a RT molecule that is not involved in DNA synthesis and are consistent with results obtained with HIV-1 RT (19). This finding suggests a model in which a second RT molecule binds to the 5' end of the extended primer and is possibly recruited to that location to remove the RNA primer when the growing strand of the newly synthesized DNA has been sufficiently extended. Although we do not know that a second RT molecule is involved in the in vivo cleavage of Ty1 replication intermediates within virus-like particles, 15–30 RT molecules are probably present within a virus-like particles and could take part in such reaction (20).

Extension of the Cleaved PPT Primer at a Nick—We have observed that initiation of plus-strand DNA synthesis is rapidly followed by the cleavage of the PPT primer between nt –1 and nt +1. This results in a nick between the primer and the nascent plus-strand DNA. We next asked whether this cleaved RNA could be re-utilized as a primer for a second round of plus-strand synthesis in the presence of downstream DNA. A model heteroduplex was generated in which a template DNA was annealed to the –9/–1 RNA primer with or without +1/+32 downstream DNA (Fig. 7A). These substrates were incubated with Ty1 RT in a reaction mixture containing [-³²P]dGTP. In the absence of downstream DNA, the primer is readily elongated (Fig. 7B, lanes 4–6, large black and white arrow). In contrast, the primer is very poorly extended in the presence of downstream DNA (Fig. 7B, lanes 1–3). The band indicated by a small arrow is a 32-nt downstream DNA that has been elongated by one radioactive dGTP by the terminal transferase activity of Ty1 RT. A lack of strand displacement activity of Ty1 RT cannot explain our observed result since extension and strand displacement do take place when gaps of 4 or 8 nt are introduced between the primer and the downstream DNA (Fig. 7C, lanes 2 and 3, large black and white arrow). Thus, it appears that the Ty1 RT requires a recessed primer end or at least a gap beyond the primer end to initiate polymerization.

View larger version (67K): [in this window] [in a new window]   FIG. 7. Extension of the PPT primer in the presence of downstream DNA. A, sequence of the primer-templates with or without downstream DNA. B, template DNA annealed to the –9/–1 RNA primer with or without a 32-nt downstream DNA was incubated for 4, 10, or 30 min with Ty1 RT in a reaction mixture containing [-32P]dGTP. In the absence of downstream DNA, a strong run-off band is observed (lanes 4–6). The primer is very poorly extended in the presence of downstream DNA (lanes 1–3). The band indicated by an arrow is the 32-nt downstream DNA, which has been elongated by one radioactive dGTP by the terminal transferase activity of Ty1 RT. C, autoradiography of the reaction products obtained after 20-min incubation of the primer-template annealed to downstream DNA molecules of 32, 28, or 24 nt. Run-off bands are detected when gaps of 4 or 8 nt are introduced between the primer and the downstream DNA (large black and white arrow). The bands indicated by small arrows are the 32-, 28-, and 24-nt downstream DNA molecules that have been elongated by one radioactive dGTP by the terminal transferase activity of Ty1 RT.

View larger version (66K): [in this window] [in a new window]   FIG. 8. Strand displacement activity of Ty1 RT. A, sequence of the primer-template with or without downstream DNA. 5' end-labeling of the primer is indicated by a star. A 5-fold molar excess of template DNA was mixed with 5' endlabeled primer. Downstream DNA was present in 5-fold molar excess over template. This ensures that all of the template molecules are bound to the downstream DNA B, template DNA annealed to the 5' end-labeled DNA primer with or without downstream DNA was incubated for 0.5–20 min with Ty1 RT. A run-off band is observed for both substrates. In the presence of downstream DNA, a pausing site is observed.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

In this work, we have further examined the interaction between Ty1 RT and the plus-strand initiation complex. We have also investigated the interplay between progress of plus-strand synthesis and primer removal. We show that Ty1 RT is able to bind a recessed RNA primer on a longer template molecule, leading to either 3' extension or primer cleavage. We see that subsequent cleavage of the extended product is efficient but that extension of the cleaved product is not observed. The lack of extension of the PPT in the presence of a nick suggests that these structures are not efficiently bound by RT in our in vitro system.

Plus-strand DNA synthesis is initiated by the binding of an RT molecule to the 3' end of the PPT. Concomitantly with DNA synthesis, the elongated product is cleaved at the PPT/U3 junction to remove the primer. We show that this occurs after nine or more DNA residues have been added to the primer. Our data suggest that this cleavage can be accomplished by an RT molecule that is not involved in DNA synthesis and that only after the enzyme bound to the growing DNA strand is far enough away can a second molecule bind and cleave the RNA/DNA junction. Alternatively, although we have previously shown that during minus-strand DNA synthesis the polymerase-dependent cleavage remains at a fixed distance of 14 nt from the 3' end of the elongated molecule, the distance between the polymerase and RNase H active sites may be more dynamic in the case of the PPT sequence, allowing RNase H cleavage after only the addition of 9 nt.

Finally, we have shown that, although Ty1 RT has a strand displacement activity, the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first strong-stop DNA has been synthesized and cleaved at the PPT/U3 junction. This result, which is consistent with very recent data obtained with Moloney murine leukemia RT by Shultz et al. (22), does not support a plus-strand primer-recycling model proposed by Lauermann and Boeke (23). That model takes into account the fact that the primer tRNA sequence is not inherited during Ty1 retrotransposition and proposes that after a first round of strong-stop DNA synthesis, the PPT is re-used to initiate a second round of synthesis. It is possible that the nucleocapsid activity associated with the Gag1 (TyA1) protein (24) or other factors, which are not present in our in vitro experiments, are used in vivo to allow recycling of the PPT. This will be the subject of further investigations.

	FOOTNOTES

 
^* This work was supported in part by Grant 9589 from the Association pour la Recherche contre le Cancer, by a CNRS grant (Poste DRA 4935) (to A. G.), and by National Institutes of Health Grant GM60534. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: IBMC, 15 rue R. Descartes, 67084 Strasbourg, France. Tel.: 33-3-88417006; Fax: 33-3-602218; E-mail: wilhelm{at}ibmc.u-strasbg.fr. ||To whom correspondence may be addressed: Dept. of Molecular Biology and Biochemistry, Rutgers University, CABM 306, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5097; Fax: 732-235-4880; E-mail: gabriel{at}cabm.rutgers.edu.

¹ The abbreviations used are: RT, reverse transcriptase; nt, nucleotide(s); PPT, polypurine tract; HIV-1, human immunodeficiency virus, type 1; RH–, RNase H minus; R, ribonucleotide; D, deoxyribonucleotide; P/T, primer/template.

² M. Pandey and A. Gabriel, manuscript in preparation.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 

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