Nonpolar Thymine Isosteres in the Ty3 Polypurine Tract DNA Template Modulate Processing and Provide a Model for Its Recognition by Ty3 Reverse Transcriptase
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m302374200
ISSN1083-351X
AutoresDaniela Lener, Mamuka Kvaratskhelia, Stuart F.J. Le Grice,
Tópico(s)HIV/AIDS drug development and treatment
ResumoDespite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (–)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T → F substitution and enhanced ribonuclease H (RNase H) activity ∼12–13 bp downstream. In the most pronounced case, dual T → F substitution at PPT positions –1/–2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (–)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT ∼12–13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T → F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5′ vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper. Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (–)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T → F substitution and enhanced ribonuclease H (RNase H) activity ∼12–13 bp downstream. In the most pronounced case, dual T → F substitution at PPT positions –1/–2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (–)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT ∼12–13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T → F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5′ vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper. Although reverse transcriptase (RT) 1The abbreviations used are: RT, reverse transcriptase; RNase H, ribonuclease H; PPT, polypurine tract; HIV, human immunodeficiency virus; F, 2,4-difluoro-5-methylbenzene; PPT-R, 5′-end-labeled PPT-containing RNA extended by 10 ribonucleotides; PPT-D, 5′-end-labeled PPT-containing RNA extended by 10 deoxyribonucleotides; WT, wild type; nt, nucleotide(s).1The abbreviations used are: RT, reverse transcriptase; RNase H, ribonuclease H; PPT, polypurine tract; HIV, human immunodeficiency virus; F, 2,4-difluoro-5-methylbenzene; PPT-R, 5′-end-labeled PPT-containing RNA extended by 10 ribonucleotides; PPT-D, 5′-end-labeled PPT-containing RNA extended by 10 deoxyribonucleotides; WT, wild type; nt, nucleotide(s).-associated ribonuclease H (RNase H) activity degrades RNA of the RNA/DNA replication intermediate with little sequence specificity, it must precisely remove the tRNA and polypurine tract (PPT) primers of (–)-strand (1Taylor J.M. Illmensee R. J. Virol. 1975; 16: 553-558Crossref PubMed Google Scholar) and (+)-strand DNA synthesis (2Sorge J. Hughes S.H. J. Virol. 1982; 43: 482-488Crossref PubMed Google Scholar), respectively, to generate sequences at the 5′ and 3′ termini of the double-stranded DNA recognized by the integration machinery (3Omer C.A. Faras A.J. Cell. 1982; 30: 797-805Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 4Rattray A.J. Champoux J.J. J. Mol. Biol. 1989; 208: 445-456Crossref PubMed Scopus (65) Google Scholar, 5Pullen K.A. Ishimoto L.K. Champoux J.J. J. Virol. 1992; 66: 367-373Crossref PubMed Google Scholar, 6Smith J.S. Roth M.J. J. Biol. Chem. 1992; 267: 15071-15079Abstract Full Text PDF PubMed Google Scholar, 7Pullen K.A. Rattray A.J. Champoux J.J. J. Biol. Chem. 1993; 268: 6221-6227Abstract Full Text PDF PubMed Google Scholar, 8Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Since the PPT is most likely embedded in a considerably larger RNA/DNA hybrid, precise hydrolysis at the PPT-U3 junction observed in vitro (9Rausch J.W. Le Grice S.F. J. Biol. Chem. 1997; 272: 8602-8610Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) suggests unique structural features may participate by correctly positioning this junction in the RNase H catalytic center. Our recent chemical footprinting of HIV-1 PPT-containing RNA/DNA hybrids (10Kvaratskhelia M. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 16689-16696Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and a comparison with the crystal structure of HIV-1 RT bound to a related duplex (11Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (358) Google Scholar) support this notion. Nucleic acid in the RT-RNA/DNA co-crystal is distorted 8–14 bp upstream of the PPT-U3 junction, comprising weakly paired, unpaired, and mispaired bases (Fig. 1A). Subsequent chemical footprinting studies (10Kvaratskhelia M. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 16689-16696Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) revealed that template thymines of this region and thymine +1 (i.e. immediately 3′ to the PPT) deviate from standard Watson-Crick base pairing in the absence of the retroviral enzyme. The finding that these naturally occurring HIV-1 PPT distortions are 10–14 bp apart was particularly intriguing, since this approximates the distance between the thumb subdomain and RNase H catalytic center of the heterodimer-associated p66 subunit (11Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (358) Google Scholar, 12Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1346) Google Scholar, 13Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1114) Google Scholar, 14Kohlstaedt L.A. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9652-9656Crossref PubMed Scopus (63) Google Scholar). Therefore, it is possible that the A-tract-induced HIV-1 PPT distortion plays a role in sequestering RT via an interaction with structural elements at the base of the p66 thumb, thus positioning the RNase H catalytic center over the PPT-U3 junction for correct processing. To examine whether this hypothesis may account for the precision of PPT processing in related long terminal repeat-containing elements, we focused here on the Saccharomyces cerevisiae retrotransposon Ty3. Differences in the primary structure of the HIV-1 and Ty3 RTs (the former is a p66/p51 heterodimer, whereas Ty3 RT is a 55-kDa monomer) as well as in the sequence of their PPTs make the comparison between these two systems very interesting. Our preliminary analysis of Ty3 RT has indicated that its DNA polymerase and RNase H catalytic centers are separated by ∼21 bp (15Rausch J.W. Grice M.K. Henrietta M. Nymark M. Miller J.T. Le Grice S.F. J. Biol. Chem. 2000; 275: 13879-13887Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) rather than the 17 bp observed in HIV-1 RT, suggesting a different spatial arrangement of thumb subdomain and RNase H catalytic center. Moreover, the Ty3 PPT differs in both size (12 bp) and sequence (Fig. 1B) from the HIV-1 counterpart. Whereas Ty3 RT processes its PPT with the appropriate precision in vitro (15Rausch J.W. Grice M.K. Henrietta M. Nymark M. Miller J.T. Le Grice S.F. J. Biol. Chem. 2000; 275: 13879-13887Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 16Lener D. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 26486-26495Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), we show here that it fails to remove (+)-DNA and imprecisely processes (+)-RNA 3′ to the HIV-1 PPT, suggesting co-evolution of enzyme and substrate. RNA/DNA hybrids, whose (–)-DNA template was both individually and dually substituted with 2,4-difluoro-5-methylbenzene (F) for thymine, were used here to investigate structural features of the Ty3 PPT, mediating its recognition and processing. 2,4-Difluoro-5-methylbenzene is isosteric with thymine but has severely reduced hydrogen bonding capacity (17Guckian K.M. Krugh T.R. Kool E.T. Nat. Struct. Biol. 1998; 5: 954-959Crossref PubMed Scopus (129) Google Scholar). Thus, F is a particularly useful tool to study the role of hydrogen bonding and base structure and has been extensively used to evaluate the fidelity of DNA synthesis (18Moran S. Ren R.X. Kool E.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10506-10511Crossref PubMed Scopus (294) Google Scholar, 19Barsky D. Kool E.T. Colvin M.E. J. Biomol. Struct. Dyn. 1999; 16: 1119-1134Crossref PubMed Scopus (52) Google Scholar, 20Morales J.C. Kool E.T. Biochemistry. 2000; 39: 2626-2632Crossref PubMed Scopus (43) Google Scholar, 21Dzantiev L. Alekseyev Y.O. Morales J.C. Kool E.T. Romano L.J. Biochemistry. 2001; 40: 3215-3221Crossref PubMed Scopus (45) Google Scholar). However, to date there have been no studies on the recognition of F-substituted RNA/DNA hybrids. In the present study, T → F substitutions were designed to introduce flexibility and, possibly, structural changes at different positions of Ty3 PPT-containing RNA/DNA hybrids, without changing the sequence of the primer. We show that subtle alterations to the structure of the Ty3 PPT (+)-RNA/(–)-DNA hybrid reposition the RNase H domain, inducing a novel but highly specific cleavage within the U3 region, 12 nt downstream from the site of F insertion. This suggests that correct processing of the (+)-strand primer may proceed through interaction of a structural subdomain of Ty3 RT with sequences immediately 5′ to the PPT and –12 bp from the PPT-U3 junction. Materials—Ty3 RT was expressed and purified as described (16Lener D. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 26486-26495Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Unsubstituted DNA oligonucleotides were obtained from Integrated DNA Technologies (Gaithersburg, MD). 2,4-Difluoro-5-methylbenzene was purchased as the phosphoramidite from Glen Research. The DNA oligonucleotides containing single and pairwise F substitutions were synthesized using the Expedite 8909 automated synthesizer (PerkinElmer Life Sciences). RNA oligonucleotides were purchased from Dharmacon (Boulder, CO). All other reagents were of the highest purity and purchased from Sigma. PPT Selection—To evaluate HIV-1 PPT selection a 55-nt, (–)-strand DNA template (corresponding to nucleotides 9048–9103 of the HIV-1HXB2 genome) was hybridized to a 30-nt, 5′-end-labeled PPT-containing RNA extended by 10 ribonucleotides (HIV-1/PPT-R) or deoxyribonucleotides (HIV-1/PPT-D) beyond the authentic RNase H cleavage site (Fig. 2A). The primer/template mixture was annealed by heating to 90 °C and slow cooling in 10 mm Tris/HCl (pH 7.6), 25 mm NaCl. A reaction mixture containing 50 nm template-primer was prepared in 10 mm Tris/HCl (pH 7.8), 9 mm MgCl2, 80 mm NaCl, 5 mm dithiothreitol. Hydrolysis was initiated by the addition of enzyme to a final concentration of 150 nm in a volume of 80 μl. The reaction mixture was incubated at 37 °C. Ten-μl aliquots were removed at times indicated and mixed with an equal volume of 89 mm Tris borate, pH 8.3, 2 mm EDTA, and 95% (v/v) formamide containing 0.1% (w/v) bromphenol blue and xylene cyanol. Polymerization products were resolved by high voltage denaturing 15% polyacrylamide gel electrophoresis and visualized by phosphor imaging. Quantification was performed using Quantity One software (Bio-Rad). For Ty3 PPT selection, a 46-nt, (–)-strand, DNA template (corresponding to nucleotides 4780–4809 of the Ty3 genome) was hybridized to a 29-nt PPT-containing RNA; the 13 nt 3′ to the authentic cleavage site were either ribonucleotide (Ty3 PPT-R) or deoxyribonucleotide (Ty3 PPT-D; Fig. 1B). The mixture was annealed as described above, and a reaction mixture containing 50 nm template-primer was prepared in 25 mm Tris/HCl (pH 7.8), 9 mm MgCl2, 80 mm NaCl, 5 mm dithiothreitol. Hydrolysis was initiated by the addition of RT to a final concentration of 150 nm in an 80-μl volume. The reaction mixture was incubated at 30 °C. Ten-μl aliquots were removed at the times indicated and processed as above. PPT Selection with 2,4-Difluoro-5-methylbenzene-substituted Substrates—Ty3 PPT-R RNA primer (Fig. 3A) was hybridized to 46-nt, (–)-strand DNA templates harboring single or dual F substitutions, as indicated. Conditions for template-primer annealing, PPT processing, and sample evaluation were as described above. For all experiments evaluating PPT selection, the hydrolysis products were processed as described under "PPT Selection." Modification of RNA/DNA Heteroduplexes with KMnO 4 —KMnO4 sensitivity of Ty3 PPT templates harboring a single +2, or dual –1/–2, –5/–7, or –9/–11 T → F substitution was evaluated by a modification of the protocol of Kvaratskhelia et al. (10Kvaratskhelia M. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 16689-16696Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). RNA/DNA hybrids were incubated at room temperature for 5 min in 20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 100 μm MgCl2. The total reaction volume was 20 μl. Reactions were initiated by adding 2 μl of freshly prepared 25 mm KMnO4 solution and terminated after 30 s with 2 μl of 14 m β-mercaptoethanol. After ethanol precipitation, samples were treated with 100 μl of 1 m piperidine for 30 min at 90 °C. Piperidine was removed by vacuum desiccation. Nucleic acids were washed three times with 50 μl of water and vacuum-dried after each resuspension. Samples were finally resuspended in 89 mm Tris borate, pH 8.3, 2 mm EDTA, and 95% formamide containing 0.1% bromphenol blue and xylene cyanol and analyzed by electrophoresis through 15% denaturing polyacrylamide gels. Modification products were visualized by phosphor imaging. Circular Dichroism Spectra and Thermal Melting Profiles—Equimolar amounts (∼25 μm) of RNA primer were hybridized to +2, –1/–2, –5/–7, and –9/–11 F-substituted 46-nt, (–)-strand PPT-containing DNA templates by heating to 90 °C and slow cooling in degassed 10 mm Na2HPO4/NaH2PO4, pH 7.0, 80 mm NaCl. Circular dichroism spectra were recorded at 30 °C with an AVIV 202 spectrophotometer using a 1-mm path length cuvette. Correction for each spectrum was against the respective buffer-only spectrum. Nucleic acid duplexes were scanned from 190 to 300 nm. For measurement of melting temperatures (T m), 10 μg/ml solutions of the same substrates were analyzed in a Beckman DU 640 spectrophotometer. E 260 was measured at 0.2 °C intervals from 30 to 80 °C. The T m of each hybrid was calculated by the "first derivative" method described by the manufacturer. Altered Processing of the HIV-1 PPT by Ty3 RT—A clear difference between the PPTs of Ty3 and more extensively studied retroviruses is the presence of contiguous (rA:dT) and (rG: dC) tracts in the latter, which might provide a structural basis for recognition. This difference prompted us to investigate the manner in which Ty3 RT processes its cognate PPT and that of HIV-1, for which a considerable body of literature is available (5Pullen K.A. Ishimoto L.K. Champoux J.J. J. Virol. 1992; 66: 367-373Crossref PubMed Google Scholar, 7Pullen K.A. Rattray A.J. Champoux J.J. J. Biol. Chem. 1993; 268: 6221-6227Abstract Full Text PDF PubMed Google Scholar, 8Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 9Rausch J.W. Le Grice S.F. J. Biol. Chem. 1997; 272: 8602-8610Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Wohrl B.M. Moelling K. Biochemistry. 1990; 29: 10141-10147Crossref PubMed Scopus (112) Google Scholar, 23Powell M.D. Beard W.A. Bebenek K. Howard K.J. Le Grice S.F. Darden T.A. Kunkel T.A. Wilson S.H. Levin J.G. J. Biol. Chem. 1999; 274: 19885-19893Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 24Rausch J.W. Lener D. Miller J.T. Julias J.G. Hughes S.H. Le Grice S.F. Biochemistry. 2002; 41: 4856-4865Crossref PubMed Scopus (66) Google Scholar). Fig. 1C indicated that the Ty3 PPT is accurately released from (+) U3 RNA or DNA by Ty3 RT. We next evaluated HIV-1 PPTs extended at the 3′ terminus by either (+)-RNA or (+)-DNA (HIV-1 PPT-R or HIV-1 PPT-D, respectively. Fig. 2A). The hydrolysis pattern obtained with HIV-1 RT on a duplex extended with (+)-RNA (Fig. 2B , i) is similar to the one that we (9Rausch J.W. Le Grice S.F. J. Biol. Chem. 1997; 272: 8602-8610Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and others (7Pullen K.A. Rattray A.J. Champoux J.J. J. Biol. Chem. 1993; 268: 6221-6227Abstract Full Text PDF PubMed Google Scholar, 8Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 22Wohrl B.M. Moelling K. Biochemistry. 1990; 29: 10141-10147Crossref PubMed Scopus (112) Google Scholar) have reported, namely preferential cleavage at the PPT-U3 RNA junction and minor cleavage on either side (we define positions –1 and +1 as the bases on each side of the processing site). However, Ty3 RT cleaved this HIV-1 substrate with significantly altered specificity. Hydrolysis occurred at three positions of the adjacent RNA/DNA hybrid, centered around +2, whereas the correct site was virtually uncleaved (Fig. 2B , ii). Because RNase H cleaves RNA at the PPT-(+)-DNA junction, we examined hydrolysis of an HIV-1 PPT extended by (+)-DNA at its 3′ terminus. In this case, we can only observe cleavage at the PPT-U3 DNA junction, if specific cleavage occurs, or within the PPT itself. The data of Fig. 2B , iii, again show minimal hydrolysis at the PPT-U3 DNA junction. In Fig. 2C, dNTPs were included to examine hydrolysis of the HIV-1 PPT-(+) DNA duplex in the context of DNA synthesis. The results indicated that, despite efficient polymerization from the HIV-1 PPT-D substrate, the removal of U3 DNA was again impaired, even after a 1-h incubation with Ty3 RT, eliminating the possibility that structural features of the HIV-1 substrate prevented binding of Ty3 RT. Following DNA synthesis, the 3′-OH of the extended primer is located 20 bp from the PPT-U3 junction, which would be ideally situated for Ty3 RNase H-mediated hydrolysis (Ty3 RNase H cleaves RNA substrates around 11 and 21 nt from the extremity that directs binding (15Rausch J.W. Grice M.K. Henrietta M. Nymark M. Miller J.T. Le Grice S.F. J. Biol. Chem. 2000; 275: 13879-13887Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)). Similarly, the 5′-end of the HIV-1 PPT-R does not direct hydrolysis, since the same cleavage pattern was observed using a substrate extended by 15 nt at the 5′-end of the PPT. Therefore, altered processing of HIV-1-PPT-R (Fig. 2B , ii) and lack of cleavage of HIV-1-PPT-D by Ty3 RT (Fig. 2B , iii) must reflect recognition of a structural feature assumed by the HIV-1 PPT. In a similar experiment, Ty3 PPT variants were completely and nonspecifically hydrolyzed by HIV-1 RT (data not shown). Dual T → F Substitutions of the Ty3 PPT DNA Template Modulate Cleavage Specificity—The results of Fig. 2 suggest that structural features of the Ty3 PPT may contribute to the specificity of processing. Therefore, we used the base analog 2,4-difluoro-5-methylbenzene (F; Fig. 3A), which is isosteric with thymine but fails to hydrogen-bond with adenine (25Kool E.T. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 1-22Crossref PubMed Scopus (402) Google Scholar). F was substituted for several thymines of the DNA template complementary to the PPT (3′-C-T-C-T-C-T-C-T-C-C-T-T-5′). Such a strategy subtly alters the stability of the PPT-containing heteroduplex, at the site of substitution, and allowed us to determine the impact on both the structure of the duplex and cleavage specificity. Initially, a series of doubly F-substituted Ty3 PPT RNA/DNA hybrids (Fig. 3A) was examined to determine whether localized destabilization of the nucleic acid duplex affected either the kinetics or specificity of processing. Indeed, adjacent F insertion at template positions –1/–2 had a profound effect, redirecting the RNase H catalytic center primarily over position +10/+11 of the non-PPT RNA/DNA hybrid (Fig. 3C , ii). This repositioning of Ty3 RT was also observed with substrates containing dual –5/–7 (Fig. 3C , iii) or –9/–11 substitutions (Fig. 3C , iv), which enhance cleavage at positions +6 and +3 of the RNA/DNA hybrid, respectively. Although less dramatic than the effect observed by a –1/–2 substitution, phosphor imaging and quantification (Fig. 4D) indicated that F-induced +6 and +3 cleavage is equivalent to or exceeds that at the PPT-U3 junction (Fig. 4C , i). Therefore, cleavage at the PPT-U3 junction is affected differently by adjacent or interrupted T → F substitutions. Adjacent substitutions create a more pronounced local destabilization that will sequester the majority of the enzyme at the new recognition site. Alternatively, the distortion induced by the presence of two adjacent T → F substitutions might render the PPT-U3 junction uncleavable. Notably, the combined data of Fig. 4, B and C, also show a constant spatial correlation of 12–13 bp between the site of F insertion and that of enhanced RNase H activity. Characterization of F-substituted Ty3 PPT RNA/DNA Hybrids—Three independent experiments were performed to evaluate if F insertion affected the Ty3 PPT structure (Fig. 4). Since the lack of hydrogen bonding has been correlated with a substantial drop in the T m of shorter nucleic acid duplexes (18Moran S. Ren R.X. Kool E.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10506-10511Crossref PubMed Scopus (294) Google Scholar, 20Morales J.C. Kool E.T. Biochemistry. 2000; 39: 2626-2632Crossref PubMed Scopus (43) Google Scholar), we determined the melting temperature of the F-substituted RNA/DNA hybrids. Wild type Ty3 RNA/DNA hybrid had a T m of 69 °C (Fig. 4A). A single T → F substitution at position +2 (i.e. outside the PPT-containing duplex) reduced this to 65.6 °C, whereas that of substrates harboring dual substitutions varied from 63.0 to 60.5 °C. Thus, whereas T → F substitutions had the expected consequences on decreasing duplex stability, the T m of all RNA/DNA hybrids was considerably higher than the temperature at which PPT processing was evaluated (30 °C). Fig. 4B compares the CD spectrum of each doubly substituted RNA/DNA hybrid to the wild type. The spectra were in good agreement with published data on polypurine-containing RNA/DNA hybrids, which assume an intermediate configuration between A-like and B-like (8Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 26Hung S.H. Yu Q. Gray D.M. Ratliff R.L. Nucleic Acids Res. 1994; 22: 4326-4334Crossref PubMed Scopus (73) Google Scholar, 27Ratmeyer L. Vinayak R. Zhong Y.Y. Zon G. Wilson W.D. Biochemistry. 1994; 33: 5298-5304Crossref PubMed Scopus (166) Google Scholar). Although minor differences were noted in the peak and trough heights at 277 and 210 nm, respectively, mutant substrates differed minimally from the wild type PPT. Finally, in Fig. 4C, we examined the sensitivity of template thymines to chemical modification by KMnO4 following T → F substitution. Previously, we successfully applied this strategy to the HIV-1 PPT, illustrating that template thymines +1 and –10 to –15 adopted a distorted structure (10Kvaratskhelia M. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 16689-16696Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In contrast, very little KMnO4 sensitivity is observed in the wild type Ty3 PPT (lane C), suggesting the absence of preexisting structural perturbations. However, the possibility that these might be induced following enzyme binding could not be excluded. Furthermore, the single +2 F (Fig. 4C , lane 1) or dual –1/–2, –5/–7, and –9/–11 T → F substitutions (Fig. 4C , lanes 2–4, respectively) were accommodated without altering the structure of neighboring A:T base pairs. Since F is insensitive to KMnO4 oxidation, this prohibited any direct evaluation on the structure of the dF:rA pair. The combined data of Fig. 4 therefore provide a strong argument that T → F substitution within or adjacent to the Ty3 PPT is not accompanied by global changes in structure but rather a subtle and localized alteration in hydrogen bonding. Single T → F Substitutions of the Ty3 PPT RNA/DNA Hybrid—To conduct a more detailed analysis of Ty3 PPT architecture, a second series of RNA/DNA hybrids was prepared containing single T → F substitutions from positions –1to –11 of the (–) DNA template (Fig. 5A). In each case, processing at the PPT-U3 RNA junction was preserved (Fig. 5B) but was accompanied by an alteration in specificity that again directed cleavage ∼12 bp downstream. Introducing F as template nucleotide –1 enhanced cleavage at position +10, and to a lesser extent at position +11 (Fig. 5, B and C , ii). This substitution also decreased cleavage at the PPT-U3 RNA junction and increased cleavage at position +2 and +3 (Fig. 5, B and C, compare i with ii). A –2 substitution likewise resulted in significantly increased cleavage at position +10 without affecting the physiological cleavage site. These results suggest that Ty3 RT partitions between the correct recognition site and a second artificially induced as a consequence of F insertion. A –1 F substitution also promoted increased cleavage at several positions downstream from the junction. (Fig. 5B , ii). The extent of +10 cleavage in the case of both monosubstituted substrates exceeded that at the authentic junction, and was comparable with what was observed with a dual –1/–2 substitution. Introducing T → F substitutions at the 5′-end of the (–)-DNA template (positions –9 and –11) had a similar effect, inducing RNase H activity ∼12–13 bp from the site of insertion (Fig. 5B , iv and v). Although –5 and –7 T → F substitutions yielded a similar result, the alteration in cleavage specificity was not as pronounced (Fig. 5C , iv and v). These data provide additional support that T → F substitution of the PPT (–) DNA template induces a subtle alteration in duplex architecture and promotes the interaction with a structural component of Ty3 RT 12–13 bp from the RNase H active site. Moreover, the gradual increase in RNase H activity on the non-PPT RNA/DNA hybrid as the position of F substitution approaches the 3′-end of the (–)-DNA template suggests that regions immediately upstream of the PPT may work in concert with the F-induced destabilization. Altering Sequences 5′ of the Ty3 PPT Affects Processing— Taken together, the T → F substitution data of Figs. 3 and 5 imply that this non-hydrogen-bonding thymine isostere introduces flexibility and possibly localized structural changes in the RNA/DNA hybrid. This may serve as a determinant for Ty3 RT binding that directs RNase H cleavage ∼12–13 bp from the site of substitution. However, it is important to consider these observations with respect to the features of the wild type PPT-containing duplex that sequester Ty3 RT and result in correct cleavage at the PPT-U3 junction. Since the Ty3 PPT/(–) DNA hybrid is 12 bp (Fig. 1B), our data suggest that initial sequestration of the retrotransposon polymerase is mediated by sequences adjacent to the 5′-end of the polypurine tract. This possibility was tested by introducing two alterations immediately 5′ of the Ty3 PPT, changing the sequence from 5′-rC-rC-rC-rU-3′ to either 5′-rC-rU-rC-rU-3′ (mutant C → U) or 5′-rU-rU-rU-rU-3′ (mutant 3C → 3U). The latter sequence closely resembles the U-rich region 5′ of the HIV-1 PPT, which has been shown to play a role in its utilization (31Bacharach E. Gonsky J. Lim D. Goff S.P. J. Virol. 2000; 74: 4755-4764Crossref PubMed Scopus (29) Google Scholar, 32Ilyinskii P.O. Desrosiers R.C. EMBO J. 1998; 17: 3766-3774Crossref PubMed Scopus (48) Google Scholar). To control for potential destabilization of the duplex resulting from C → U substitutions, each RNA primer was extended by an additional 7 nucleotides at its 5′ terminus (Fig. 6A). As a consequence, the 5′ flanking region of the PPT served as an additional substrate for Ty3 RNase H, resulting in precise cleavage at both the 5′ and 3′ PPT junctions (Fig. 6B , i). This was expected, because our previous data on HIV-1 and Ty3 PPT selection indicated that they are ac
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