Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (–)-DNA Template and (+)-RNA Primer
2005; Elsevier BV; Volume: 280; Issue: 20 Linguagem: Inglês
10.1074/jbc.m411228200
ISSN1083-351X
AutoresHye Young Yi-Brunozzi, Stuart F.J. Le Grice,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoA variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (–)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO4 footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (–)-DNA template between positions –15 and –13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (–11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile –1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis. A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (–)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO4 footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (–)-DNA template between positions –15 and –13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (–11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile –1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis. In retroviruses and long terminal repeat (LTR) 1The abbreviations used are: LTR, long terminal repeat; DAb, abasic deoxyriboside linkage; HIV-1, human immunodeficiency virus, type 1; PPT, polypurine tract; RAb, abasic riboside linkage; RNase H, ribonuclease H; RT, reverse transcriptase; U3, unique 3′ sequence; nt, nucleotide. -containing retrotransposons, second or (+)-strand synthesis requires (a) specific cleavage between the 3′ end of the (+)-strand, polypurine tract (PPT), and the 3′ unique sequence (U3) of the LTR; (b) DNA-dependent DNA synthesis from the newly created primer; and (c) removal of the PPT primer from nascent (+)-DNA (1Rausch J.W. Le Grice S.F. Int. J. Biochem. Cell Biol. 2004; 36: 1752-1766Crossref PubMed Scopus (87) Google Scholar) (see Fig. 1A). Each of these steps can be accurately recapitulated in vitro when PPT sequences are embedded within a larger RNA/DNA hybrid (2Pullen K.A. Rattray A.J. Champoux J.J. J. Biol. Chem. 1993; 268: 6221-6227Abstract Full Text PDF PubMed Google Scholar, 3Rausch J.W. Le Grice S.F. J. Biol. Chem. 1997; 272: 8602-8610Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 4Schultz S.J. Zhang M. Kelleher C.D. Champoux J.J. J. Biol. Chem. 2000; 275: 32299-32309Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 5Wilhelm M. Uzun O. Mules E.H. Gabriel A. Wilhelm F.X. J. Biol. Chem. 2001; 276: 47695-47701Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 6Dash C. Yi-Brunozzi H.Y. Le Grice S.F. J. Biol. Chem. 2004; 279: 37095-37102Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), i.e. where the nucleic acid termini cannot influence enzyme orientation, implicating structural features of the PPT in both resistance to internal reverse transcriptase-associated ribonuclease H (RT/RNaseH) cleavage and specific processing at its 3′ terminus from (+)-RNA and (+)-DNA. Early NMR studies, using a short fragment of this hybrid, identified a 15° bend at the HIV-1 PPT/U3 junction (7Fedoroff O.Y. Ge Y. Reid B.R. J. Mol. Biol. 1997; 269: 225-239Crossref PubMed Scopus (62) Google Scholar), which may contribute to the accuracy of cleavage. Subsequent crystallographic studies with HIV-1 reverse transcriptase (RT) bound to a PPT-containing hybrid indicated a pattern of weakened base pairing centered ∼13 bp upstream of the PPT/U3 junction (8Sarafianos 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 (360) Google Scholar), a notion supported by chemical footprinting of the duplex in the absence of RT (9Kvaratskhelia 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). Interestingly, the distance between the PPT/U3 junction and the upstream region of weakened base pairing is close to the spatial separation between the p66 thumb subdomain and RNase H catalytic center of HIV-1 RT (8Sarafianos 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 (360) Google Scholar, 10Jacobo-Molina A. Clark Jr., A.D. Williams R.L. Nanni R.G. Clark P. Ferris A.L. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10895-10899Crossref PubMed Scopus (68) Google Scholar, 11Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar). These combined observations suggested that an induced fit between the HIV-1 RT thumb subdomain and the upstream portion of the PPT might position the RNase H catalytic center over the PPT/U3 junction. As a consequence, this would render intervening regions inaccessible and RNase H-resistant. Implicating regions upstream of the PPT/U3 junction in enzyme positioning (9Kvaratskhelia 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, 12Rausch J.W. Qu J. Yi-Brunozzi H.Y. Kool E.T. Le Grice S.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11279-11284Crossref PubMed Scopus (28) Google Scholar) is supported by our studies of PPT recognition in the LTR-containing retrotransposon Ty3. Although the Ty3 PPT lacks the hallmark rA·dT and rC·dG homopolymeric stretches characteristic of retroviral (+)-strand primers (13Rattray A.J. Champoux J.J. J. Mol. Biol. 1989; 208: 445-456Crossref PubMed Scopus (65) Google Scholar), altering nucleic acid geometry 10–12 bp upstream of the PPT/U3 junction also affects the precision of PPT cleavage (14Lener D. Kvaratskhelia M. Le Grice S.F. J. Biol. Chem. 2003; 278: 26526-26532Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 2C. Dash, D. Lener, and S. Le Grice, unpublished observations. One approach to studying PPT cleavage specificity is by introducing structural changes in a manner preserving sequence and spatial context. As an example, we substituted the thymine analog 2,4-difluoro-5-methylbenzene and cytosine analog 2-fluoro-4-methylbenzene throughout the HIV-1 PPT (–)-DNA template to remove hydrogen bonding and locally increase flexibility (12Rausch J.W. Qu J. Yi-Brunozzi H.Y. Kool E.T. Le Grice S.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11279-11284Crossref PubMed Scopus (28) Google Scholar, 15Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (337) Google Scholar). This approach indicated that the RNase H catalytic center could be relocated ∼4bp3′ of the site of analog insertion, indirectly implicating the "RNase H primer grip" of the p66 subunit (8Sarafianos 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 (360) Google Scholar) in positioning the scissile bond in the RNase H-active center. A similar approach with the Ty3 PPT relocated its RNase H catalytic center ∼12 bp downstream of the site of analog insertion (14Lener D. Kvaratskhelia M. Le Grice S.F. J. Biol. Chem. 2003; 278: 26526-26532Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), suggesting that alternative structural elements of the retrotransposon enzyme may participate in PPT recognition. The converse strategy, i.e. decreasing local flexibility, was evaluated by introducing locked nucleic acid analogs (16Petersen M. Nielsen C.B. Nielsen K.E. Jensen G.A. Bondensgaard K. Singh S.K. Rajwanshi V.K. Koshkin A.A. Dahl B.M. Wengel J. Jacobsen J.P. J. Mol. Recognit. 2000; 13: 44-53Crossref PubMed Scopus (184) Google Scholar, 17Nielsen K.E. Singh S.K. Wengel J. Jacobsen J.P. Bioconjugate Chem. 2000; 11: 228-238Crossref PubMed Scopus (98) Google Scholar, 18Petersen M. Bondensgaard K. Wengel J. Jacobsen J.P. J. Am. Chem. Soc. 2002; 124: 5974-5982Crossref PubMed Scopus (231) Google Scholar) into the HIV-1 PPT (–)-DNA template, indicating that regions at both the 5′ and 3′ end of the RNA/DNA hybrid are critical for correct enzyme processing. This study (6Dash C. Yi-Brunozzi H.Y. Le Grice S.F. J. Biol. Chem. 2004; 279: 37095-37102Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) also supports a finding of Schultz et al. (19Schultz S.J. Zhang M. Champoux J.J. J. Virol. 2003; 77: 5275-5285Crossref PubMed Scopus (21) Google Scholar), suggesting two PPT cleavage modes, namely the PPT/U3 junction and ∼5 bp into the U3 region, the latter of which might contribute to efficient (+)-strand synthesis. Related studies evaluating the interaction of protein subdomains with nucleic acid exiting the DNA polymerase catalytic center (20Latham G.J. Lloyd R.S. J. Biol. Chem. 1994; 269: 28527-28530Abstract Full Text PDF PubMed Google Scholar, 21Forgacs E. Latham G. Beard W.A. Prasad R. Bebenek K. Kunkel T.A. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1997; 272: 8525-8530Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 22Latham G.J. Forgacs E. Beard W.A. Prasad R. Bebenek K. Kunkel T.A. Wilson S.H. Lloyd R.S. J. Biol. Chem. 2000; 275: 15025-15033Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) illustrate the value of nucleoside analogs in dissecting protein-nucleic acid interactions. The "unzipped" portion of the HIV-1 PPT reported by Sarafianos et al. (8Sarafianos 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 (360) Google Scholar) was of particular interest to us, because template base –11 and primer base –13 (defining position –1 as the base pair 5′ to the PPT/U3 junction, Fig. 1B) are unpaired, thereby enhancing the flexibility of this region. At the same time, we and others have noted that template base +1 of the HIV-1 and murine leukemia virus PPT is surprisingly tolerant to substitution, accepting substitution with non-hydrogen-bonding pyrimidine isosteres and base mismatches with minimal effect on the accuracy and overall rate of hydrolysis (2Pullen K.A. Rattray A.J. Champoux J.J. J. Biol. Chem. 1993; 268: 6221-6227Abstract Full Text PDF PubMed Google Scholar, 9Kvaratskhelia 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, 13Rattray A.J. Champoux J.J. J. Mol. Biol. 1989; 208: 445-456Crossref PubMed Scopus (65) Google Scholar). The latter observations might indicate that, as the RNA/DNA hybrid enters the RNase H catalytic center, the DNA strand is displaced, allowing "docking" of the scissile phosphodiester bond in the active site, a model consistent with the role proposed for the p66 RNase H primer grip (8Sarafianos 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 (360) Google Scholar). A detailed understanding of how duplex geometry influences the accuracy of PPT cleavage is important in understanding this critical step in the reverse transcription cycle. In this communication, we examined the effect of introducing abasic lesions (23Lhomme J. Constant J.F. Demeunynck M. Biopolymers. 1999; 52: 65-83Crossref PubMed Scopus (188) Google Scholar) into the unzipped region of both the HIV-1 (–)-DNA template and (+)-RNA primer between positions –15 and –11, as well as around the PPT/U3 cleavage junction. Although there is little information on abasic lesions within RNA/DNA hybrids, structural studies with duplex DNA suggest that local elimination of the base does not affect the sugar-phosphate backbone (24Goljer I. Kumar S. Bolton P.H. J. Biol. Chem. 1995; 270: 22980-22987Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 25Coppel Y. Berthet N. Coulombeau C. Garcia J. Lhomme J. Biochemistry. 1997; 36: 4817-4830Crossref PubMed Scopus (83) Google Scholar) but, in general, increases flexibility (25Coppel Y. Berthet N. Coulombeau C. Garcia J. Lhomme J. Biochemistry. 1997; 36: 4817-4830Crossref PubMed Scopus (83) Google Scholar). An abasic site can also affect whether its unpaired complement assumes an intraor extrahelical configuration. Collectively, our data suggest positions –15, –14, and –13 of the HIV-1 PPT (–)-DNA template are important modulators of cleavage specificity, either through directly contacting the thumb subdomain or imparting local flexibility to nucleic acid in its vicinity. The previously reported (8Sarafianos 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 (360) Google Scholar) unpaired template base (–11C) could also be removed without affecting PPT processing. In contrast, abasic lesions in the (+)-RNA primer between positions –15 and –11 are tolerated with very little alteration in cleavage specificity. With respect to structural requirements at the PPT/U3 junction, we show here that, although eliminating primer bases 5′ and 3′ of the scissile bond destroys cleavage, removing their complement on the (–)-DNA template is only partially inhibitory, indicating that, consistent with studies on human RNase H1 (26Lima W.F. Nichols J.G. Wu H. Prakash T.P. Migawa M.T. Wyrzykiewicz T.K. Bhat B. Crooke S.T. J. Biol. Chem. 2004; 279: 36317-36326Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), a DNA base at the scissile bond is not essential for hydrolysis. Oligonucleotide and Enzyme Preparation—Substituted 40-nt oligodeoxynucleotides and 30-nt oligoribonucleotides were synthesized at a 1 μmol scale on a PE Biosystems Expedite 8909 oligonucleotide synthesizer by standard phosphoramidite chemistry and deprotected according to the manufacturer's specifications. r-Spacer (abasic RNA lesions) and d-Spacer (abasic DNA lesions) phosphoramidites were purchased from Glen Research, Sterling, VA. Stepwise coupling yields for incorporation of each analog was >98%, determined by trityl cation monitoring. Oligonucleotides were purified by preparative polyacrylamide gel electrophoresis and quantified spectrophotometrically (260 nm), assuming a molar extinction coefficient equal to the sum of the constituent bases. A wild type 30-nt RNA containing the HIV-1 3′ PPT and flanking sequences at its 5′ and 3′ termini was purchased from Dharmacon Research (Boulder, CO). DNA/RNA hybrids were prepared by heating equimolar amounts of RNA and DNA to 95 °C in 10 mm Tris-HCl, pH 7.8, 25 mm NaCl for 5 mins, followed by slow cooling to 4 °C. The samples were stored at –20 °C. p66/p51 HIV-1 RT was purified according to published procedures (27Le Grice S.F. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-144Crossref PubMed Scopus (120) Google Scholar). Thermal Melting Profiles—Equimolar amounts of 30-nt RNA primer and 40-nt, (–)-DNA templates were annealed by heating to 90 °C and slow cooling in degassed 10 mm Na2HPO4/NaH2PO4, pH 7.0, 80 mm NaCl. Non-denaturing gel electrophoresis was used to determine that complete hybridization had been achieved. For measurement of melting temperatures (Tm), 10 μg/ml solutions of each substrate were analyzed in a Beckman DU 640 spectrophotometer. E260 was measured at 0.2 °C intervals from 30 to 85 °C. The Tm of each hybrid was calculated by the "first derivative" method described by the manufacturer. KMnO4 Footprinting of PPT Variants—RNA/DNA hybrids containing abasic lesions in the template or primer 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 μlof14 m β-mercaptoethanol. After ethanol precipitation and dessication, the 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. RNase H-mediated PPT Processing—Specific hydrolysis at the PPT 3′ terminus was evaluated as previously described (6Dash C. Yi-Brunozzi H.Y. Le Grice S.F. J. Biol. Chem. 2004; 279: 37095-37102Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) using p66/p51 HIV-1 RT and 30-nt RNA/40-nt DNA hybrids containing abasic lesions. 5′ end labeling of the PPT-containing RNA was performed with T4 polynucleotide kinase and [γ-32P]ATP. PPT cleavage was initiated by adding HIV-1 RT to RNA/DNA hybrids in 10 mm Tris-HCl (pH 8.0), 80 mm NaCl, 5 mm dithiothreitol, and 6 mm MgCl2 at 37 °C, with enzyme and RNA/DNA hybrid present at final concentrations of 50 and 200 nm, respectively. Hydrolysis was terminated at the times indicated in the Fig. 3 legend by adding an equal volume of 95% (v/v) formamide containing 0.1% (w/v) bromphenol blue and xylene cyanol, and the products fractionated by high voltage electrophoresis through denaturing 15% (w/v) polyacrylamide/7 m urea gels, visualized by autoradiography, and quantified following phosphorimaging. Thermal Stability of Abasic PPT Duplexes—A portion of the HIV-1 PPT-containing hybrid used in our studies is illustrated in Fig. 1B, highlighting anomalous base pairing observed in the RT-RNA/DNA co-crystal between positions –15 and –9 (defining –1 as the first base pair 5′ to the scissile phosphate) (8Sarafianos 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 (360) Google Scholar). For the present study, abasic lesions were introduced into the (–)-DNA template and (+)-RNA primer between positions –15 and –11. The second region selected for analysis was the –1/+1 scissile bond corresponding to the PPT/U3 junction, where lesions were introduced on either side of the junction and downstream at position +2. To determine how these substitutions affected duplex stability, the melting temperature (Tm) of each hybrid was determined, the results of which are presented in Table I.Table IMelting temperatures of PPT substrates containing abasic lesions(-)-DNATm(+)-RNATm°C°CWT67.1 ± 0.96WT67.1 ± 0.96-15DAb68.0 ± 1.62-15RAb68.2 ± 0.76-14DAb68.1 ± 0.74-14RAb66.8 ± 1.25-13DAb (mp)69.0 ± 0.12-13RAb (up)65.0 ± 0.95-12DAb (mp)65.9 ± 0.95-12RAb (mp)66.1 ± 1.39-11DAb (up)65.6 ± 0.49-11RAb (mp)65.3 ± 1.04-1DAb56.9 ± 0.95-1RAb54.8 ± 0.99+1DAb58.9 ± 1.67+1RAb57.6 ± 0.59+2DAb56.3 ± 0.64+2RAb57.9 ± 0.12 Open table in a new tab For PPT variants containing substitutions between positions –15 and –11, most melted at temperatures similar to that of the wild type hybrid (67.1 °C), regardless of whether the nucleobase was eliminated from the (–)-DNA template or (+)-RNA primer. The maximum decrease in Tm was observed with duplexes containing the unpaired bases defined by Sarafianos et al. (8Sarafianos 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 (360) Google Scholar) i.e. template base –11 (Tm = 65.6 °C) and primer base –13 (Tm = 65.0 °C). From these results, we concluded that the stability of the unzipped portion of the HIV-1 PPT was not seriously affected by base elimination. In contrast, removing nucleobases around the PPT/U3 cleavage junction was significantly more destabilizing, depressing the Tm by as much as 12.3 °C (–1RAb). The destabilizing effect was evident as far as position +2, suggesting that abasic lesions inserted at the PPT/U3 junction influenced neighboring base pairs, possibly through the alteration of stacking interactions. For a duplex of the length used in our studies (30 bp), a 10 °C reduction in Tm can be likened to introducing three base mismatches. Thus, if duplex geometry at the unsubstituted PPT/U3 junction is altered, as suggested by NMR (7Fedoroff O.Y. Ge Y. Reid B.R. J. Mol. Biol. 1997; 269: 225-239Crossref PubMed Scopus (62) Google Scholar) and chemical footprinting studies (9Kvaratskhelia 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), this appears to be further destabilized as a consequence of nucleobase elimination. Susceptibility of Template Thymines to KMnO4 Oxidation—To assess how targeted nucleobase elimination altered PPT structure, susceptibility of template thymines between positions –15 and +1 to KMnO4 oxidation was investigated (see Fig. 1B). This strategy can determine whether thymines are unpaired (28Farah J.A. Smith G.R. J. Mol. Biol. 1997; 272: 699-715Crossref PubMed Scopus (49) Google Scholar, 29Zou Y. Van Houten B. EMBO J. 1999; 18: 4889-4901Crossref PubMed Scopus (92) Google Scholar) or are structurally distorted but exhibit weak hydrogen bonding (30Reddy Y.V. Rao D.N. J. Mol. Biol. 2000; 298: 597-610Crossref PubMed Scopus (49) Google Scholar). Template thymine reactivity in response to abasic insertions in both strands of the RNA/DNA hybrid was determined. Fig. 2A presents thymine sensitivity when template nucleobases between positions –15 and –12 were removed. Clearly, removing any thymine will eliminate that particular hydrolysis product. Eliminating nucleobases –15, –14, and –13 slightly increased reactivity of adjacent thymines (Fig. 2A, lanes 1–3), which in conjunction with the Tm data of Table I, suggests local alteration in base stacking rather than extensive disruption of the duplex. Eliminating template base –12T had little effect on thymine reactivity at positions –13, –14, and –15 (Fig. 2A, lane 4). In contrast, –12T was highly susceptible to oxidation following removal of the unpaired nucleobase, –11C (Fig. 2A, lane 5), suggesting disruption of the original –12T/–11rG mispair suggested by Sarafianos et al. (8Sarafianos 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 (360) Google Scholar) (Fig. 1B) when the stacking environment of the template is altered. The notion that eliminating base –11C is more destabilizing is supported by the observation that this substitution also affects –10T reactivity. With this exception, template substitutions between position –15 and –12 did not alter KMnO4 sensitivity within the adjacent r(A)4·d(T)4 tract between –10T and –7T. Reactivity of template nucleobase +1T in response to introducing abasic lesions at positions –1, +1, and +2 is shown in Fig. 2B. As expected, no product is evident for substrate +1DAb (Fig. 2B, lane 8). Also, altered migration of the +1 hydrolysis product on substrate +2DAb (Fig. 2B, lane 9) is accounted for by the fact that the DNA template was 5′ end-labeled, thus the cleavage product lacks one base. Substitutions –1DAb and +2DAb resulted in enhanced +1T reactivity (Fig. 2B, lanes 7 and 9, respectively). Table I indicates that the Tm for substrates containing lesions at these positions was reduced by ∼10 °C, which is equivalent to loss of hydrogen bonding over three base pairs. The data of Fig. 2B and Table I thus indicate that abasic template lesions are more destabilizing when positioned at the PPT/U3 junction. Fig. 2, C and D, indicate template thymine reactivity in response to removing nucleobases from the (+)-RNA primer. In general, substitutions –15RAb to –11RAb increased KMnO4 sensitivity between bases –15T and –12T, the effect being more pronounced at thymines opposite the site of nucleobase removal (Fig. 2C, lanes 1–5). In an analogous manner to its template counterpart, primer substitution –11RAb increased –12T and –10T reactivity (Fig. 2C, lane 5), suggesting local disruption of the RNA/DNA hybrid. As might be expected, removing primer nucleobase +1 enhanced KMnO4 sensitivity of template thymine +1 (Fig. 2D, lane 8). Enhanced +1T reactivity was also evident following removal of primer nucleobase –1 (Fig. 2D, lane 7). Finally, +1T reactivity of substrate +2RAb was similar to that of the unsubstituted duplex (Fig. 2D, lanes 9 and 6, respectively), suggesting that stacking of template nucleobases at the scissile bond was unaffected by removing primer nucleobase +2. Removing Template Nucleobases between Positions –15 and –11 Affects PPT Cleavage—Cleavage at the PPT/U3 junction in response to eliminating template nucleobases between positions –15 and –11 is shown qualitatively in Fig. 3A and quantitatively in Fig. 3B (for the former, lane d of each hydrolysis profile was selected). Using a 5′ end-labeled RNA primer, the wild type RNA/DNA hybrid (Fig. 3, A and B, panels i) was hydrolyzed predominantly at the –1/+1 junction and, to a lesser extent, between positions –2 and +6. No template substitution gave rise to aberrant cleavage within the PPT itself (data not shown). Removing template nucleobase –15 induced relaxed cleavage specificity, with the consequence that specific cleavage at the PPT/U3 junction was reduced (Fig. 3, A and B, panels ii). This effect was even more pronounced with substrate –14DAb, where the PPT/U3 junction was the least-preferred cleavage site (Fig. 3, A and B, panels iii). Although substrate –13DAb was hydrolyzed with improved specificity at the –1/+1 junction, positions +5 and +6 were still the favored sites (Fig. 3, A and B, panels iv). The data of Fig. 3, A and B, panels ii–iv, suggest alternative scenarios, namely that (a) specific contacts between the DNA strand between positions –15 and –13 and a structural motif of HIV-1 RT have been disturbed or (b) enhanced duplex flexibility upon nucleobase removal is incompatible with its trajectory between the catalytic centers of HIV-1 RT. Each of these possibilities will be discussed later. In contrast, we observed enhanced specificity for cleavage at the PPT/U3 junction with substrates –12DAb and –11DAb (Fig. 3, A and B, panels v and vi, respectively), which are mispaired and unpaired, respectively, in the HIV-1 RT/PPT structure described by Sarafianos et al. (8Sarafianos 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 (360) Google Scholar). Interestingly, a –11DAb substitution increased reactivity of template nucleobase –12T to KMnO4 oxidation (Fig. 2A, lane 5) to a level equal to or exceeding that of any template thymine rendered unpaired by removal of its primer counterpart, suggesting that –12T is selectively unpaired when template nucleobase –11 is removed. In all structures of HIV-1 RT containing nucleic acid (8Sarafianos 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 (360) Google Scholar, 10Jacobo-Molina A. Clark Jr., A.D. Williams R.L. Nanni R.G. Clark P. Ferris A.L. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10895-10899Crossref PubMed Scopus (68) Google Scholar, 11Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar), the duplex undergoes an A- to B-form transition downstream of the DNA polymerase catalytic center, an event accompanied by a 40° bend centered ∼11 bp from the RNase H catalytic center. Locating the RNase H catalytic center over the PPT/U3 junction, as depicted in the model of Fig. 1B, would center this bend around position –11. Thus, local weakening of duplex architecture via targeted nucleobase removal (i.e. substrates –12DAb and –11DAb
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