Requirements for DNA Unpairing during Displacement Synthesis by HIV-1 Reverse Transcriptase
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m409134200
ISSN1083-351X
AutoresJamie Winshell, Benjamin A. Paulson, Ben Buelow, James J. Champoux,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoDNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO4 oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein. DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO4 oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein. HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; DTT, dithiothreitol; WT, wild type. reverse transcriptase is a multifunctional enzyme with a polymerase domain that catalyzes both RNA-dependent and DNA-dependent DNA synthesis and an RNase H domain that catalyzes degradation of RNA when it is hybridized to DNA (for reviews, see Refs. 1Varmus H. Brown P. Berg D. Howe M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 53-108Google Scholar and 2Arts E. Le Grice S. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 339-393Crossref PubMed Scopus (77) Google Scholar). Viral reverse transcription is initiated from a specific tRNA primer that anneals near the 5′-end of the plus-sense RNA genome at the primer binding site. Extension of the tRNA primer followed by the first strand transfer allows the completion of the minus DNA strand and generates the substrate for the RNase H cleavage that creates the plus-strand primer at the polypurine tract. Removal of the tRNA allows a second strand transfer that occurs via complementary primer binding site regions at the 3′-ends of both plus and minus strands. Completion of both plus and minus strands to produce the long terminal repeat-flanked linear product required for integration is believed to occur through a circular intermediate in which reverse transcriptase must carry out strand displacement synthesis through a stretch of DNA ∼600 bp in length (3Gilboa E. Mitra S.W. Goff S. Baltimore D. Cell. 1979; 18: 93-100Abstract Full Text PDF PubMed Scopus (421) Google Scholar, 4Whiting S.H. Champoux J.J. J. Virol. 1994; 68: 4747-4758Crossref PubMed Google Scholar). Reverse transcriptases have been shown to be capable of displacing either a DNA or an RNA non-template strand ahead of the primer terminus during polymerase chain elongation in vitro (4Whiting S.H. Champoux J.J. J. Virol. 1994; 68: 4747-4758Crossref PubMed Google Scholar, 5Huber H.E. McCoy J.M. Seehra J.S. Richardson C.C. J. 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Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1763) Google Scholar, 17Jacobo-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. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1122) Google Scholar, 18Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (303) Google Scholar, 19Sarafianos 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) and for conformational changes in reverse transcriptase induced by dNTP binding (20Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar). Kinetic analyses have supported the concept of an ordered polymerization mechanism with a conformational change accompanying primer/template binding, followed by an additional conformational change associated with dNTP binding that leads to a productive complex (21Patel P.H. Jacobo-Molina A. Ding J. Tantillo C. Clark Jr., A.D. Raag R. Nanni R.G. Hughes S.H. Arnold E. Biochemistry. 1995; 34: 5351-5363Crossref PubMed Scopus (177) Google Scholar, 22Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (468) Google Scholar, 23Lanchy J.M. Ehresmann C. Le Grice S.F. Ehresmann B. Marquet R. EMBO J. 1996; 15: 7178-7187Crossref PubMed Scopus (109) Google Scholar, 24Lanchy J.M. Keith G. Le Grice S.F. Ehresmann B. Ehresmann C. Marquet R. J. Biol. Chem. 1998; 273: 24425-24432Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Wohrl B.M. Krebs R. Goody R.S. Restle T. J. Mol. Biol. 1999; 292: 333-344Crossref PubMed Scopus (72) Google Scholar). Importantly, this latter conformational change that results in the formation of the ternary complex involving the enzyme, primer/template, and dNTP shifts the fingers domain closer to the palm to more tightly wrap the enzyme around the DNA (20Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar). Furthermore, the structure of this ternary complex reveals that the three unpaired template residues ahead of the primer terminus are folded back away from the DNA axis and interact with the fingers domain. A cocrystal structure showing DNA bound to an N-terminal fragment of Moloney murine leukemia virus reverse transcriptase similarly implicates residues in the fingers sub-domain in template binding (26Najmudin S. Cote M.L. Sun D. Yohannan S. Montano S.P. Gu J. Georgiadis M.M. J. Mol. Biol. 2000; 296: 613-632Crossref PubMed Scopus (47) Google Scholar). Recently, amino acid changes at position 61 within the fingers domain of HIV-1 reverse transcriptase have revealed a role for this residue in both template binding and displacement synthesis (27Fisher T.S. Darden T. Prasad V.R. J. Mol. Biol. 2003; 325: 443-459Crossref PubMed Scopus (37) Google Scholar). Whereas changing Phe61 to tyrosine or leucine increased the efficiency of displacement synthesis with some loss in processivity, a change to tryptophan at this position was shown to significantly reduce the displacement capability of the polymerase without affecting processivity. Model building based on the crystal structure of the enzyme with a bound dNTP (20Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar) suggests that an interaction between Phe61 and the template base two nucleotides in front of the primer terminus (referred to as the +2 position) is involved in destabilizing the helix at this position and thereby promoting displacement synthesis. We have shown previously that reverse transcriptases are capable of DNA displacement synthesis through several hundred base pairs at a rate ∼4-fold slower than that of non-displacement synthesis (4Whiting S.H. Champoux J.J. J. Virol. 1994; 68: 4747-4758Crossref PubMed Google Scholar, 6Whiting S.H. Champoux J.J. J. Mol. Biol. 1998; 278: 559-577Crossref PubMed Scopus (41) Google Scholar, 9Kelleher C.D. Champoux J.J. J. Biol. Chem. 1998; 273: 9976-9986Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Moreover, based on the similar effects of temperature on non-displacement and displacement synthesis, it seems likely that displacement synthesis occurs by an active mechanism as opposed to a strictly passive process dependent on breathing of the helix in advance of the primer terminus (6Whiting S.H. Champoux J.J. J. Mol. Biol. 1998; 278: 559-577Crossref PubMed Scopus (41) Google Scholar). However, a strict helicase-like mechanism (28Lohman T.M. J. Biol. Chem. 1993; 268: 2269-2272Abstract Full Text PDF PubMed Google Scholar) would appear to be ruled out by the finding that the polymerase is unable to melt the DNA helix in the presence of either ATP or dNTPs under conditions where extension is prevented (4Whiting S.H. Champoux J.J. J. Virol. 1994; 68: 4747-4758Crossref PubMed Google Scholar). Thus, strand separation ahead of the advancing polymerase in the displacement mode could either be facilitated directly by protein-nucleic acid interactions and/or coupled to the translocation of the polymerase with each nucleotide added. Consistent with either of these possibilities is the observation based on DNase I footprinting results that the enzyme contacts the DNA 7 and 9 nucleotides downstream of the primer terminus in the template and non-template strands, respectively (29Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F. Biochemistry. 1995; 34: 5343-5356Crossref PubMed Scopus (80) Google Scholar, 30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). Further support for an active mechanism involving direct contacts between the enzyme and the duplex region ahead of the primer terminus comes from our use of KMnO4 sensitivity to show that the two base pairs in front of the polymerase are unpaired in a temperature-independent fashion in model displacement constructs (30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). Here we describe additional studies using KMnO4 oxidation as a probe for unpaired thymine residues to investigate the nature of the protein-DNA interactions responsible for unpairing the duplex in front of the primer terminus during displacement synthesis. We show that the unpairing reaction requires the cognate dNTP, but is independent of the length of the already-displaced DNA flap. Furthermore, the length of the unpaired region is not influenced by the base composition of the DNA beyond the +2 position. Finally, we show that replacing Phe61 with tryptophan, which results in a diminished capacity to carry out displacement synthesis (27Fisher T.S. Darden T. Prasad V.R. J. Mol. Biol. 2003; 325: 443-459Crossref PubMed Scopus (37) Google Scholar), results in a reduced melting of the DNA at the +1 and +2 positions. Enzymes, Reagents, and Buffers—Heterodimeric recombinant HIV-1 reverse transcriptase (14–24 units/μl, ∼22 units/μg) was purchased from Worthington Biochemical Corp. T4 polynucleotide kinase (10,000 units/ml) and bovine serum albumin were purchased from New England Biolabs, Inc. Denaturing polyacrylamide gels (8.3 m urea) were made with Sequagel Sequencing System reagents purchased from National Diagnostics. The 5% non-denaturing polyacrylamide gels (29:1 acrylamide:bis) were prepared in a high ionic strength buffer containing 0.16 m glycine, 25 mm Tris-HCl, pH 8.5. SDS-PAGE gels were prepared using Protogel reagents purchased from National Diagnostics. [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences Research Products, and dNTPs and ddNTPs were from Amersham Biosciences. KMnO4 and piperidine were purchased from Sigma, and 2-mercaptoethanol was purchased from Aldrich. Standard reverse transcriptase reaction buffer (1× RT buffer) was 50 mm Tris-HCl, pH 8.3, 50 mm KCl, 6 mm MgCl2. Reverse transcriptase dilution buffer (RT dilution buffer) was 20% (v/v) glycerol, 20 mm Tris-HCl, pH 8.0, 1 mg/ml bovine serum albumin, 2 mm DTT. HZ stop solution was 0.3 m sodium acetate, pH 7.0, 1 mm EDTA, 25 μg/ml salmon sperm DNA. 6× SDS sample buffer was 350 mm Tris-HCl, pH 6.8, 36% glycerol, 10% SDS, 600 mm DTT, and 0.12% bromphenol blue. Escherichia coli strains containing plasmids coding for the WT and the F61W mutant forms of p66 and a His-tagged form of p51 were kindly provided by Dr. V. Prasad (27Fisher T.S. Darden T. Prasad V.R. J. Mol. Biol. 2003; 325: 443-459Crossref PubMed Scopus (37) Google Scholar). To purify the heterodimeric HIV-1 reverse transcriptase, bacteria expressing p66 or p51 were separately grown to mid-log phase, and after induction and further growth, the bacterial cell pellets were collected and mixed. After lysis of the cells, the mixtures were incubated overnight at 4 °C prior to purification of the heterodimeric proteins as described by Fisher et al. (27Fisher T.S. Darden T. Prasad V.R. J. Mol. Biol. 2003; 325: 443-459Crossref PubMed Scopus (37) Google Scholar). The specific activities of the purified WT and F61W reverse transcriptases were determined by the poly(rA):oligo(dT) assay as described previously (31Hou E.W. Prasad R. Beard W.A. Wilson S.H. Protein Expr. Purif. 2004; 34: 75-86Crossref PubMed Scopus (30) Google Scholar) using WT HIV-1 reverse transcriptase from Worthington Biochemical Corp. (21.6 units/μg) as an activity standard. One unit of activity incorporates 1 nmol of labeled dTMP in 20 min at 37°C using poly(rA): oligo(dT) as the template primer. The specific activities of the purified WT and F61W reverse transcriptases were 9.1 and 5.2 units/μg, respectively. Oligonucleotides and Preparation of Displacement Constructs— Mlv13dideoxy was purchased from Sigma Genosys, and non-temp3moreTs and temp3moreTs were purchased from Operon. All other oligonucleotides (mlv13primer, non-temp3, temp3, non-temp4, temp4, non-temp3T2, temp3T2, non-temp3-gap2, non-temp3-gap1, non-temp3-OH1, non-temp3-OH3 and non-temp3-OH6) were purchased from Invitrogen. All oligonucleotides were purified by denaturing PAGE. The names, lengths, and sequences of the DNA oligonucleotides are as follows: mlv13 primer (30-mer, 5′-CAGGGGTCTCCCGATCCC-GGACGAGCCCCC-3′), mlv13dideoxy (30-mer, 5′-CAGGGGTCTCCCG-ATCCCGGACGAGCCCC(ddC)-3′), non-temp3 (41-mer, 5′-CCAGTAA-TGTGTACGTGAGTCGTGCTTGACTGGATCCCGAG-3′), temp3 (59-mer, 5′-CGGGATCCAGTCAAGCACGACTCACGTGGGGGCTCGTCC-GGGATCGGGAGACCCCTGGA-3′), non-temp4 (40-mer, 5′-CCAGTAA-TGTGTTGCACTCAGCACGAACTGACCTAGGCAG-3′), temp4 (58-mer, 5′-GCCTAGGTCAGTTCGTGCTGAGTGCAGGGGGCTCGTCCG-GGATCGGGAGACCCCTGGA-3′), non-temp3T2 (41-mer, 5′-CCAGTA-ATGTGTGTCAGAGTCGTGCTTGACTGGATCCCGAG-3′), temp3T2 (59-mer, 5′-CGGGATCCAGTCAAGCACGACTCTGACGGGGGCTCGT-CCGGGATCGGGAGACCCCTGGA-3′), non-temp3moreTs (41-mer, 5′-CCAGTAATGTGTACGTGTTTCGTGCTTGACTGGATCCCGAG-3′), temp3moreTs (59-mer, 5′-CGGGATCCAGTCAAGCACGAAACACGTG-GGGGCTCGTCCGGGATCGGGAGACCCCTGGA-3′), non-temp3-gap2 (27-mer, 5′-GTGAGTCGTGCTTGACTGGATCCCGAG-3′), non-temp3-gap1 (28-mer, 5′-CGTGAGTCGTGCTTGACTGGATCCCGAG-3′), non-temp3-OH1 (30-mer, 5′-TACGTGAGTCGTGCTTGACTGGATCCCGA-G-3′), non-temp3-OH3 (32-mer, 5′-TGTACGTGAGTCGTGCTTGACTG-GATCCCGAG-3′), and non-temp3-OH6 (35-mer, 5′-ATGTGTACGTGA-GTCGTGCTTGACTGGATCCCGAG-3′). Where indicated, 10 pmol of the oligonucleotides were 5′-end-labeled with equimolar [γ-32P]ATP and 10 units of polynucleotide kinase for 45 min at 37 °C in the reaction buffer supplied by the manufacturer. The reactions were terminated by chelating the Mg2+ with EDTA and heating the reaction to 65 °C for 10 min. DNA displacement constructs were prepared as follows: 5′-32P-end-labeled non-template/template/primer DNAs were annealed in a 1:2:3 molar ratio in 1.5× RT buffer using a PerkinElmer Life Sciences thermocycler programmed as follows: 1 min at 95 °C, 10 min ramp to 70 °C, hold 30 min at 70 °C, 10 min ramp to 33 °C, hold 30 min at 33 °C, cool and hold at 4 °C. DTT was then added to a final reaction concentration of 5 mm. Annealing for primer extension assays was performed as described above except that the molar ratio was 1:2:3 for the 5′-32P-end-labeled primer/template/non-template DNAs. To confirm that the labeled oligonucleotide was completely annealed and that no single-strands persisted that could contribute to the KMnO4 cleavage pattern, the complexes were routinely analyzed by electrophoresis through non-denaturing polyacrylamide gels at 4 °C, as described previously (30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). Thermostability calculations employing the thermodynamic parameters of Sugimoto et al. (32Sugimoto N. Nakano S. Yoneyama M. Honda K. Nucleic Acids Res. 1996; 24: 4501-4505Crossref PubMed Scopus (389) Google Scholar) indicated that the region of non-template strand downstream from the primer terminus would remain stably annealed at 37 °C even when the reverse transcriptase had extended to the 3′-most position required by the experiments. Use of KMnO4 as a Probe for Unpaired Nucleotides in Front of the Primer Terminus on Displacement Templates—Displacement reactions (final volume, 28 μl) containing 10 nm 5′-32P-end-labeled non-template strand annealed as described above were preincubated with HIV-1 reverse transcriptase (40 nm final concentration) in 1× RT buffer containing 5 mm DTT at 37 °C for 1 min. Control reactions were incubated with enzyme in the absence of dNTPs. Displacement synthesis was initiated by the addition of dNTPs and/or ddNTPs (final concentration, 200 μm each) as indicated in figure legends. Constructs employing dideoxy-terminated primer were initiated in an identical manner. After a 20-min incubation at 37 °C, a 10-μl portion of each reaction was removed and combined with 2.5 μl of 50% glycerol. These samples were held on ice for ∼10 min before being electrophoresed through a 5% non-denaturing polyacrylamide gel at 4 °C as previously described (30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar) to confirm that all of the labeled DNA was bound to the enzyme (not shown). To the remaining 18 μl of each reaction was added 2 μl of freshly made 200 mm KMnO4 (final concentration, 20 mm) followed by an incubation at room temperature for 2.5 min. The oxidation reaction was quenched with 2 μl of 14.4 m 2-mercaptoethanol. 180 μl of HZ stop solution was added, and the samples were ethanol-precipitated twice, resuspended in 100 μl of 1 m piperidine and incubated at 90 °C for 30 min. To decrease background smearing in the gel caused by residual piperidine, samples were lyophilized to dryness after resuspension in 50 μl of H2O, three times. The final dried samples were resuspended in 10 μl of 98% formamide, 10 mm EDTA containing a trace amount of xylene cyanol. Before loading on a denaturing 20% polyacrylamide gel, samples were heated at 65 °C for 1 min. Gels were dried and exposed to a PhosphorImager screen and analyzed using ImageQuant software. The same sensitivity and background parameters were used for an entire gel image. Each lane on a gel was analyzed separately, calculating the percentage of the total signal in the lane represented by each detectable band. Comparisons were made between lanes based on these calculations. Time Course Extension Assays with Non-displacement and Displacement Constructs—The procedures and DNA oligonucleotides for carrying out extension assays have been described previously (6Whiting S.H. Champoux J.J. J. Mol. Biol. 1998; 278: 559-577Crossref PubMed Scopus (41) Google Scholar). Briefly a single-stranded template DNA from recombinant M13 phagemid pB-SMOLTR(+) DNA was isolated and linearized. For the displacement synthesis assays, oligonucleotide MLVU5I was annealed to the template and extended to the 5′-end of the template using Sequenase. For both non-displacement and displacement synthesis, extension was initiated from 5′-32P-end-labeled oligonucleotide MLVPBSII under standard reaction conditions (20-μl final volume) using 5 units of either WT or F61W reverse transcriptase. Control reactions with T4 DNA polymerase, which is capable of only very limited displacement synthesis, were used to verify the structure of the displacement template. Extension products were analyzed by electrophoresis in a 6% denaturing polyacrylamide gel alongside size markers prepared as described previously (6Whiting S.H. Champoux J.J. J. Mol. Biol. 1998; 278: 559-577Crossref PubMed Scopus (41) Google Scholar). Gels were analyzed using a PhosphorImager and ImageQuant software. Displacement Constructs—The displacement constructs used in this study are shown in Fig. 1. The basic displacement construct (I) is formed by annealing a 30-mer primer (mlv13primer), a 59-mer template strand (temp3), and a 41-mer non-template strand (non-temp3). The non-template strand contains a single-stranded flap (12 bases) at its 5′-end to simulate a displacement intermediate, but the sequence of the flap region is different from the sequence at the 3′-end of the primer to prevent branch migration. Each end of the construct has a two-base 3′ overhang to prevent recognition of the 3′-ends as primer termini by reverse transcriptase. This construct was designed so that incorporation of different chain-terminating dideoxynucleotides would arrest movement of reverse transcriptase and position the primer terminus at a fixed distance from thymine residues in the downstream non-template strand. Because unpaired thymine bases are sensitive to KMnO4 oxidation, this experimental design allowed us to test for DNA melting by the polymerase at positions downstream of the primer terminus. In control primer-extension experiments with 5′-end-labeled mlv13primer, synthesis was found to proceed to the end of the template in the presence of all four dNTPs, whereas extension reactions carried out in the presence of chain-terminating ddNTPs produced fragments with the predicted lengths (data not shown) (30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). Variations on this basic construct were designed to examine the influence of synthesis, flap length, and sequence context on melting of the non-template strand downstream from the primer terminus. The mlv13dideoxy oligonucleotide used in constructs II and III was synthesized with a 2′,3′-dideoxynucleotide at its 3′-end to prevent extension while allowing for the formation of a ternary complex (reverse transcriptase, primer/template/non-template, and dNTP) and was confirmed to be unextendable by a standard primer extension assay (data not shown). Construct IV was designed to examine the effects of sequence context on the extent of melting by including a stretch of three consecutive thymines in the region downstream from the primer terminus. Construct V (see Fig. 4) was identical to the basic displacement construct except for variations in the length of the single-stranded non-template strand flaps. Effect of Next Correct Nucleotide on DNA Melting at +1 and +2 Positions—KMnO4 oxidation of thymine residues in regions of single-stranded DNA, followed by alkaline cleavage of the modified residues generates 5′-phosphate-terminated oligonucleotide fragments (33Hayatsu H. Ukita T. Biochem. Biophys. Res. Commun. 1967; 29: 556-561Crossref PubMed Scopus (117) Google Scholar, 34Hänsler U. Rokita S.E. J. Am. Chem. Soc. 1993; 115: 8554-8557Crossref Scopus (14) Google Scholar, 35Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar, 36Ramaiah D. Koch T. Orum H. Schuster G.B. Nucleic Acids Res. 1998; 26: 3940-3943Crossref PubMed Scopus (16) Google Scholar). Using model displacement constructs built from oligonucleotides containing thymine residues at various distances from the primer terminus in both the template and non-template strands, we previously used this property of unpaired thymines to show that the +1 and +2 base pairs are melted by reverse transcriptase (30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). However, these assays were carried out in the presence of three dNTPs plus a ddNTP, so it is likely that the nucleotide binding pocket was occupied by the next correct dNTP in every case. Here we systematically investigate whether the next correct dNTP is required for the observed melting and by extension whether the melting is dependent on the protein conformational change that accompanies dNTP binding (20Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar). HIV-1 reverse transcriptase was incubated with displacement construct I containing a 5′-32P-end-labeled non-template strand under conditions of saturating enzyme (data not shown, see Ref. 30Winshell J. Champoux J.J. J. Mol. Biol. 2001; 306: 931-943Crossref PubMed Scopus (15) Google Scholar). Incubation in the presence of ddGTP plus dATP and dCTP arrested synthesis at the point where thymine 16 in the non-template strand is at position +1, whereas extension in the presence of ddCTP and dATP placed the same thymine residue at position +2. The cleavage patterns after KMnO4 oxidation a
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