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• W2080498601 abstract "Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination. Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination. human immunodeficiency virus nucleotides reverse transcription nucleocapsid protein stem-loop By reshuffling large regions of genetically distinct genomes, recombination speeds up the rate of evolution (1Coffin J.M. Curr. Top. Microbiol. Immunol. 1992; 176: 143-164Crossref PubMed Scopus (180) Google Scholar, 2Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6900-6903Crossref PubMed Scopus (268) Google Scholar). Recombination constitutes the most frequent genomic aberration in retroviruses; its frequency of occurrence is equal to the cumulative frequency of all the other types of mutations (3Negroni M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google Scholar). The most intensively studied member of this group of viruses, the human immunodeficiency virus type 1 (HIV-1),1 illustrates the impact of recombination on the dynamics of retroviral evolution. In this case, at the very least 10% of the circulating strains have been generated by genetic recombination among different HIV-1 subtypes (4Sharp P.M. Bailes E. Robertson D.L. Gao F. Hahn B.H. Biol. Bull. 1999; 196: 338-342Crossref PubMed Scopus (71) Google Scholar). In retroviruses, recombination occurs mainly during reverse transcription (5Hu W.S. Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1556-1560Crossref PubMed Scopus (382) Google Scholar). Each viral particle contains two copies of single-stranded positive genomic RNA (6Vogt V.M. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). If two different variants of a virus infect the same cell, as recently documented for HIV-1 (7Jung A. Maier R. Vartanian J.P. Bocharov G. Jung V. Fischer U. Meese E. Wain-Hobson S. Meyerhans A. Nature. 2002; 418: 144Crossref PubMed Scopus (337) Google Scholar), the viral progeny will be constituted by homozygous and heterozygous virions. Recombination can then occur when a heterozygous virus infects a new cell. Indeed, during synthesis of the (−) DNA strand the reverse transcriptase (RT) can switch template and, guided by the local sequence homology, transfer DNA synthesis from one genomic RNA molecule (the donor) onto the other (acceptor RNA). In a heterozygous virion this process, known as copy choice, leads to genetic recombination (8Vogt P.K. Silvestri L. Possible Episomes in Eukaryotes. North Holland, 1973: 35-41Google Scholar). Despite the dramatic impact of recombination on the evolution of retroviruses, the underlying mechanisms are not yet understood. Based on the observation that the purification of viral RNA from retroviral particles led to the isolation of fragmented molecules, it was suggested that the genomic RNA is not intact within the viral particle. It was therefore proposed that the switch would be a consequence of a block of reverse transcription caused by a break on the RNA, a model called “forced copy choice” (9Coffin J.M. J. Gen. Virol. 1979; 42: 1-26Crossref PubMed Scopus (314) Google Scholar). In this case the stalling of the RT would constitute the crucial step of the process by allowing an extensive degradation of the RNA template by the RNase H activity carried by the RT itself, as demonstrated for (−)DNA strong stop strand transfer (10Telesnitsky A. Goff S.P. EMBO J. 1993; 12: 4433-4438Crossref PubMed Scopus (57) Google Scholar). The resulting single-stranded DNA would then be available for annealing onto the complementary sequence provided by the acceptor RNA. A similar situation can be encountered if stalling is generated by strong pause occurring during reverse transcription of an intact template. Indeed, a prominent pause site detected duringin vitro reverse transcription of a stretch of the HIV-1nef gene was shown to increase significantly the local frequency of strand transfer (11DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 12Wu W. Blumberg B.M. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 325-332Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In these instances the stalling of reverse transcription is regarded as the limiting step for strand transfer. This idea has, however, recently been challenged by increasing evidence demonstrating that pausing during reverse transcription and strand transfer are not necessarily coupled (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar, 15Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In parallel, studies carried out on RNA templates containing hairpin regions have suggested that such structures could favor template switching by RTs (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Berkhout B. Vastenhouw N.L. Klasens B.I. Huthoff H. RNA. 2001; 7: 1097-1114Crossref PubMed Scopus (88) Google Scholar,17Balakrishnan M. Fay P.J. Bambara R.A. J. Biol. Chem. 2001; 276: 36482-36492Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In these cases it was proposed that the hairpin structures enhance the probability of strand transfer by mediating an interaction between donor and acceptor RNA that increases their spatial proximity (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Berkhout B. Vastenhouw N.L. Klasens B.I. Huthoff H. RNA. 2001; 7: 1097-1114Crossref PubMed Scopus (88) Google Scholar,17Balakrishnan M. Fay P.J. Bambara R.A. J. Biol. Chem. 2001; 276: 36482-36492Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Extensive random searches for the occurrence of recombination hot spots during in vitro reverse transcription by HIV-1 RT had revealed the correlation between the location of these hot spots and the presence of predicted hairpin regions in the RNA template (14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar, 18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar). Based on this observation, it was proposed that template switching proceeds through a two-step mechanism: docking of the acceptor RNA onto the nascent DNA and displacement of the donor RNA by the acceptor RNA (14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). The latter step would be guided by the folding of the RNA. A recent study on the primer binding site of the equine infectious anemia virus has shown that the hairpin present in that region induces a strong pause of reverse transcription that increases the efficiency of the docking step (15Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, in vitro studies on the effect on strand transfer of the nucleocapsid protein (NC), a major co-factor of the reverse transcription complex (19Welker R. Hohenberg H. Tessmer U. Huckhagel C. Krausslich H.G. J. Virol. 2000; 74: 1168-1177Crossref PubMed Scopus (168) Google Scholar), have suggested a mechanism of recombination governed by the structures of the RNA rather than by pausing of reverse transcription (reviewed in Refs. 3Negroni M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google Scholar and 20Negroni M. Buc H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 151-155Crossref PubMed Scopus (41) Google Scholar). Indeed, although NC enhances strand transfer in vitro (reviewed in Ref. 3Negroni M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google Scholar) it does not lead to a parallel increase of pausing during reverse transcription, as predicted for a mechanism of template switching governed by pausing of DNA synthesis (14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). Because the NC is a RNA chaperone (21Clodi E. Semrad K. Schroeder R. EMBO J. 1999; 18: 3776-3782Crossref PubMed Scopus (71) Google Scholar, 22Williams M.C. Rouzina I. Wenner J.R. Gorelick R.J. Musier-Forsyth K. Bloomfield V.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6121-6126Crossref PubMed Scopus (155) Google Scholar), it was suggested that the enhancement of strand transfer observed in its presence was because of its ability to modulate the structures of the RNA templates (14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). Among the several recombinant HIV-1 strains isolated to date, a well defined case is constituted by chimerical genomes between subgroups A and either C or D, generated by recombination on the region coding for the constant portion C2 of the envelope glycoprotein gp120 (23Robertson D.L. Sharp P.M. McCutchan F.E. Hahn B.H. Nature. 1995; 374: 124-126Crossref PubMed Scopus (518) Google Scholar). Recombination on this segment of genome allows reshuffling of the portions of gp120 coding for the variable regions V1 and V2, relative to regions V3 through V5. The spatial arrangement of regions V2 and V3 with respect to the constant regions of the protein has been shown to be critical for allowing the virus to escape neutralizing antibodies raised by the immune system of the host (24Ye Y. Si Z.H. Moore J.P. Sodroski J. J. Virol. 2000; 74: 11955-11962Crossref PubMed Scopus (59) Google Scholar). In a previous report we used several RNA sequences issued from the HIV-1 genome to investigate the mechanism of template switching by HIV-1 reverse transcriptasein vitro. We observed that the genomic sequence coding for the C2 region constituted, indeed, the most important hot spot we found during that work (18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar). Interestingly, a subsequent study on recombination during infection of cells in culture with different HIV-1 subtypes has also shown the occurrence of frequent recombination in the same region (25Quinones-Mateu M.E. Gao Y. Ball S.C. Marozsan A.J. Abraha A. Arts E.J. J. Virol. 2002; 76: 9600-9613Crossref PubMed Scopus (46) Google Scholar). In our previous study, the portion of 200 nt that constituted the hot spot within the C2 domain was called “Eb” and was initially included in a model template where it was surrounded by the sequences that flank it on the viral genome (Fig. 1 A,RNA E2). It was subsequently observed that by changing the surrounding sequences (Fig. 1, RNA G1Eb) the frequency of strand transfer on Eb was decreased by a 4- to 5-fold factor (18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar). We referred to this effect as “context effect.” In this work, we took advantage of the context effect to investigate the role of the RNA structure in the transfer process and to address the question of the mechanism responsible for copy choice by HIV-1 RT. To determine their folding, the various RNA templates were labeled at their 3′-end as follows. A 21-mer oligonucleotide with a sequence complementary to the 3′-end of the RNA to label was annealed, at a molar ratio of 4:1 (oligo:RNA) and in a buffer containing 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, by heating the mixture for 30 s at 95 °C followed by a slow cooling to 30 °C. The oligonucleotide carries at its 5′-end a non-hybridizing tail of six nucleotides constituted by the sequence 5′-CTTTTT-3′. The annealed RNA was then incubated for 20 min at 37 °C in a final volume of 55 μl in a buffer containing 7 mm dithiothreitol, 20 mm Tris-HCl, pH 7.5, 12.5 mm MgCl2, 20 mm NaCl, 40 units of RNasin (Promega), 5 units of Sequenase (United States Biochemicals), and 0.05 mCi of [α32P]dideoxyATP (Amersham Biosciences). Labeled RNAs were purified on 7% polyacrylamide gel and eluted by passive diffusion at 4 °C in a buffer containing 10 mm magnesium acetate, 500 mm ammonium acetate, 0.1% SDS, and 1 mm EDTA. The eluted RNA was extracted with phenol-chloroform and precipitated in ethanol; the dried pellet was conserved under ethanol at −20 °C. For determination of the structure of the RNA, 8 pmol of the labeled RNA were heated in the reverse transcription buffer (50 mm Tris-HCl, pH 7.8, 75 mm KCl, 7 mm MgCl2) at 65 °C for 5 min, slowly cooled to 40 °C, and transferred on ice. The RNA was then treated with either T1 (0.3 and 0.15 units) or T2 (0.04 and 0.02 units) RNases for 5 min at 37 °C. T1 and T2 RNases cleave single-stranded RNA molecules with preference for guanine residues for T1 and adenine residues for T2 (26Ehresmann C. Baudin F. Mougel M. Romby P. Ebel J.P. Ehresmann B. Nucleic Acids Res. 1987; 15: 9109-9128Crossref PubMed Scopus (661) Google Scholar). The reaction was stopped by phenol-chloroform extraction followed by ethanol precipitation. The products were analyzed by autoradiography after electrophoresis on 7% polyacrylamide gels (see Fig. 1). Quantification was performed using phosphorimaging apparatus (Molecular Dynamics). The positions of enzymatic cleavage were identified by reference to a ladder generated by extensive T1 digestion of the same RNA molecule. The residues identified as single-stranded in four independent experiments were introduced as constraints in the structure prediction analysis by the m-fold program (27Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1728) Google Scholar). The various constructs used for RNA synthesis were generated following standard cloning procedures (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Each construct was systematically sequenced before its use in RNA synthesis. RNA synthesis was performed as previously described (29Negroni M. Ricchetti M. Nouvel P. Buc H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6971-6975Crossref PubMed Scopus (37) Google Scholar). Reverse transcription of the donor RNA was carried out in the presence of the acceptor RNA (at a final concentration of 100 nm each, unless otherwise stated) after annealing an oligonucleotide specifically onto the donor template (Fig. 2 A). Annealing was performed at a molar ratio of primer to donor RNA of 10:1 in 50 mm Tris-HCl (pH 7.8), 75 mm KCl, 7 mm MgCl2 at 65 °C for 5 min followed by a slow cooling to 40 °C. Dithiothreitol (1 mm final concentration), the four dNTPs (1 mm each), and RNasin (100 units; Promega) were added after incubation for 5 min on ice. For the experiments with NC (55 amino acids, synthesized as described in Ref. 30De Rocquigny H. Gabus C. Vincent A. Fournie-Zaluski M.C. Roques B. Darlix J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6472-6476Crossref PubMed Scopus (279) Google Scholar), the protein was added at this step at a ratio of 1 molecule of NC/8 nt of total RNA and incubated for 10 min at 37 °C. Reverse transcription was started by the addition of HIV-1 RT at a final concentration of 400 nmand carried out for 90 min. The reaction was stopped by extraction with phenol-chloroform. The samples containing NC were treated, before phenol-chloroform extraction, for 1 h at 56 °C with proteinase K (8 mg/ml), 0.4% (w/v) SDS, and 50 mm EDTA (pH 8.0). The phenol-chloroform-extracted samples were then submitted to RNase treatment. Purification of the reverse transcription product and synthesis of the second DNA strand were performed as previously described (14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). BamHI and PstI digestion, ligation, and Escherichia coli transformation were also carried out as previously described (18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar) and as shown in Fig. 2. For the experiments where the concentration of acceptor template was varied, the procedure of reverse transcription of a fixed amount (100 nm) of donor RNA was identical to the one described above, but the concentration of acceptor included in the assay was varied as detailed in Fig. 5. In all cases HIV-1 RT was used at a concentration of 400 nm.Figure 5Effect of the concentration of the acceptor RNA on copy choice and forced copy choice. A, copy choice; B, forced copy choice. Only the viral portion (“vir” in Fig. 2) of the model templates used in these assays is shown in each panel. A detailed description of these templates is given in Ref. 18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar. The recombination rates refer to recombination occurring on the regions shown in gray on the donor RNAs (the size of which is given in the table). Error bars were calculated as (b 1/2/b), where b is the number of blue colonies, and r is the recombination rate. The standard sample is the one indicated in Fig. 2 for the forced copy choice conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The recombination frequency in the various intervals of the model templates was calculated as follows. In the case of recombination between dE2 and aE2 RNAs, as an example, a NcoI restriction site was present at the boundary between Ea and Eb on the donor RNA, and anApaLI site marked the transition between Eb and Ec on the acceptor RNA (Fig. 3 A, column I). All recombinant molecules NcoI+/ApaLI+were considered to issue from template switching within Eb. We defineF as the overall frequency of recombination observed in the experiment, as given in Fig. 2, left panel. If bis the number of blue colonies whose restriction pattern has been analyzed and c is the number of recombinant coloniesNcoI+/ApaLI+, the frequency of recombination within Eb (f) is given byF(c/b). Recombination rates per nt were calculated by dividing the frequency of recombination within a given region by its size in nt (in the example given here, Eb is 200 nt long, and the recombination rate is therefore given by f/200). Reverse transcription was primed using a 5′-terminal-labeled deoxyoligonucleotide and carried out under the same buffer and RT conditions as above. The templates used in these assays consisted in truncated versions of dG1Eb or dE2, devoid of the sequences coding for the reporter gene and therefore including only the viral sequences. Similarly when the experiments were performed in the presence of the acceptor RNAs, modified versions of aG1Eb and of aE2 RNAs were used, constituted only by the viral sequence. The complex between HIV-1 RT and the primer/template was pre-formed by incubation for 10 min in the same reaction buffer as described above, devoid of dNTPs and MgCl2. The reaction was started by the addition of dNTPs and MgCl2 and stopped at various time intervals by addition of EDTA to a final concentration of 15 mm. All samples were ethanol-precipitated before electrophoresis on 8% (w/v) polyacrylamide gel containing 8 m urea in a loading buffer containing formamide at a final concentration of 22.5%. The intensity of each band was estimated by phosphorimaging as described above. To study the potential correlation between the frequency of recombination on the Eb region and its structure, we determined its folding in solution when it is included either in E2 RNA (condition under which Eb is a recombination hot spot) or in G1Eb RNA (where Eb was not a recombination hot spot). In these two cases we call this region Eb* and Eb°, respectively. The determination of the folding of the RNAs was performed under the same buffer and temperature conditions as the recombination assay (see “Materials and Methods”). These two RNAs were labeled at their 3′-end, subjected to enzymatic probing (Fig.1, panels B and C), and their secondary structures were assessed by including the constraints revealed by these probing assays into the m-fold program (27Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1728) Google Scholar). The most significant difference found between the folding of Eb* and Eb° was in the 3′-terminal region of the sequences. In this part, we identified a stem-loop motif, called here SL (Fig. 1 D), present exclusively in the case of Eb*. As shown in Fig. 1, the SL hairpin is constituted at its 3′-end by a portion of the region Ea, the sequence downstream Eb* in E2 RNA. When Eb is part of G1Eb RNA (Eb°), Ea is replaced by the region G1a and the formation of the SL hairpin is no longer possible (Fig. 1, panels B and C,bottom). No other stable hairpins were found within Eb°. The influence of these structural differences in the strand transfer reaction was then investigated by using a recombination assay previously described (18Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar) and outlined in Fig. 2, left panel. Strand transfer involves two types of RNA templates, the donor and the acceptor. The correlation between the folding of the Eb region and the frequency of strand transfer observed during its reverse transcription was exploited to assess the role played by each of these templates. The experiments were first performed on naked RNA templates. The rationale of the experiment is outlined in Fig.3 A. In the two cases outlined in Fig. 3 A, columns I and IV, the donor and the acceptor RNAs share complete sequence homology on the viral sequence, apart from the presence of the restriction sites indicated in the figure. We refer to these conditions as “symmetric,” because the folding of the region Eb is the same on the donor and the acceptor RNAs (either Eb* on both or Eb° on both). In contrast, under the conditions depicted in columns II and III (“asymmetric conditions”) Eb adopts a different folding between the donor and the acceptor RNAs. Furthermore, under the asymmetric conditions the region Eb constitutes the only region of homology between the two RNAs (in black in the figure). The frequency of template switching within Eb was determined by restriction analysis of the recombinant products, as detailed under “Materials and Methods.” When Eb was in the Eb* conformation on the acceptor RNA, strikingly close frequencies of recombination were observed on the Eb interval, independent of which donor RNA was used (Fig. 3 A,columns I and II, naked RNAs). Conversely, the frequency of strand transfer on Eb was similar when it was in the Eb° conformation on the acceptor RNA, independent from the use of dE2 or of dG1Eb as donor RNA (Fig. 3 A, columns III and IV). Therefore, it was evident that the conformation of Eb on the acceptor RNA determined the frequency of template switching. We then tested whether these conclusions reached on experiments on naked RNAs could apply to recombination occurring in the presence of the NC protein. Also, in this case the frequency of recombination on Eb was found to depend on the folding of the acceptor RNA (Fig. 3 A, RNA· NC complex). To document the role of the SL hairpin in recombination taking place within Eb, we decided to distinguish between the events of strand transfer occurring within SL itself from those taking place outside the hairpin in the Eb interval. We employed aE2 as the acceptor RNA and either dE2 or dG1Eb as donor templates, the conditions under which the highest frequency of transfer was observed on Eb (Fig. 3 A, columns I andII). For these experiments, a point mutation generating anEcoRI site was introduced on both types of donor RNAs, dE2 and dG1Eb, creating dE2-eco and dG1Eb-eco RNAs, respectively (Fig.3 B). This EcoRI site is located immediately 5′ with respect to the base of the SL hairpin and allows mapping of strand transfer within Eb. As shown in Fig. 3 B (naked RNA), the use of either dE2-eco or dG1Eb-eco as donor RNAs yielded a recombination rate higher within the hairpin itself than in the downstream portion. Furthermore the rates of recombination were very close when dE2-eco or dG1Eb-eco was used (15.1 and 14.0 × 10−4 per nt), confirming that the type of donor RNA used does not influence the distribution of the positions of strand transfer within Eb. We then evaluated whether the same conclusions can be drawn from experiments performed in the presence of the NC protein by performing the recombination assays on RNA·NC complexes. Also, in this case strand transfer occurred at high rates on SL, regardless of the type of donor template employed. The observation that the type of donor RNA used does not modify the frequency of template switching strongly suggests that pausing of reverse transcription on the donor RNA is not the trigger for strand transfer. It cannot be ruled out, though, that the pausing pattern on the donor templates is modified when reverse transcription is performed in the presence of aG1Eb or aE2 as acceptor templates. To investigate this point we first carried out a labeled primer extension analysis on dG1Eb and dE2 RNAs (Fig.4 A, lanes 1–4, and B, lanes 1–5). Despite the different folding of the SL region on these RNAs, a prominent pause site was found in both cases at the same position, corresponding to a stretch of four uridine residues. To check whether the presence of an acceptor RNA could induce a change in the pausing pattern on the donor template, reverse transcription of dG1Eb (Fig. 4 A) or of dE2 (Fig. 4 B) was then performed in the presence of either aG1Eb or aE2. As indicated at the bottom of Fig. 4, these conditions reproduce those employed for the recombination assays depicted in Fig.3 A. In no case could a significant change in the pausing pattern be detected. The 4- to 5-fold difference in the frequency of recombination observed, depending on the use of E2 or G1Eb as acceptor RNA (Fig. 3 A), was therefore not associated with an increased stalling of the reverse transcription. To better evaluate the effect of strong arrests of DNA synthesis on template switching, we have developed an experimental system that reproduces the situation described in the forced copy choice model (Fig. 2, right panel). In this system reverse transcription was performed in parallel under two different experimental conditions, referred to in Fig. 2 as “strand transfer” and “standard” samples. In the strand transfer sample, a donor template (“FCC donor RNA,” for forced copy choice) is reverse-transcribed in the presence of an acceptor RNA. The FCC donor RNA is truncated within the region of homology with the acceptor RNA. This system allows strand transfer to occur either from internal positions of the region of homology or at the very 5′-end of the donor template. The reverse transcription products are then treated as for the copy choice experiments and cloned in E. coli (see the Fig. 2 legend and “Materials and Methods" @default.
• W2080498601 created "2016-06-24" @default.
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• W2080498601 date "2003-05-01" @default.
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• W2080498601 title "Evidence for a Mechanism of Recombination during Reverse Transcription Dependent on the Structure of the Acceptor RNA" @default.
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• W2080498601 doi "https://doi.org/10.1074/jbc.m212306200" @default.
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