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• W1966873031 abstract "The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through −21 and primer nucleotides −1 through −24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe2+ fails to replace Mg2+ in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase. The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through −21 and primer nucleotides −1 through −24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe2+ fails to replace Mg2+ in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase. phosphate-buffered saline feline immunodeficiency virus reverse transcriptase human immunodeficiency virus isopropyl-1-thio-β-d-galactopyranoside polypurine tract long terminal repeat nucleotide(s) base pair(s) integrase equine infectious anemia virus Moloney murine leukemia virus Following infection, retroviruses initiate their DNA synthesis program from a host-derived tRNA hybridized to a specific region at the 5′ end of their (+) strand RNA genome, designated the primer binding site or PBS.1 However, tRNA use is somewhat heterogeneous, i.e. while avian viruses exploit tRNATrp, Moloney murine leukemia virus uses tRNAPro and d-type and human spumaretroviruses tRNALys1,2 (1.Leis J. Aiyar A. Cobrinik D. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 33-47Google Scholar). In the case of HIV and related lentiviruses of simian, feline, and equine origin, tRNALys3 is selected as the replication primer. Early experimentation suggested complementarity between the PBS and sequences at the 3′ terminus of the replication primer as the sole specificity determinant during initiation of (−) strand synthesis (2.Taylor J.M. Illmensee R. J. Virol. 1975; 16: 553-558Crossref PubMed Google Scholar). However, extensive analyses with Rous sarcoma virus (3.Cobrinik D. Aiyar A Ge Z. Katzman M. Huang H. Leis J. J. Virol. 1991; 65: 3864-3872Crossref PubMed Google Scholar, 4.Aiyar A. Ge Z. Leis J. J. Virol. 1994; 68: 611-618Crossref PubMed Google Scholar, 5.Miller J.T. Ge Z. Morris S. Das K. Leis J. J. Virol. 1997; 71: 7648-7655Crossref PubMed Google Scholar) and HIV-1 (6.Wakefield J.K. Wolf A.G. Morrow C.D. J. Virol. 1995; 69: 6021-6029Crossref PubMed Google Scholar, 7.Li Y. Zhang Z. Kang S.-M. Buescher J.L. Morrow C.D. Virology. 1997; 238: 273-282Crossref PubMed Scopus (9) Google Scholar, 8.Zhang Z. Kang S.-M. Li Y. Morrow C.D. RNA. 1998; 4: 394-406Crossref PubMed Scopus (244) Google Scholar, 9.Liang C. Li X. Rong L. Inouye P. Quan Y. Kleiman L. Wainberg M.S. J. Virol. 1997; 71: 5750-5757Crossref PubMed Google Scholar, 10.Isel C. Ehresmann C. Keith G. Ehresmann B. Marquet R. J. Mol. Biol. 1995; 247: 236-250Crossref PubMed Scopus (229) Google Scholar, 11.Isel C. Lanchy J.-M. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. EMBO J. 1996; 15: 917-924Crossref PubMed Scopus (177) Google Scholar, 12.Arts E.J. Ghosh M. Ehresmann B. Le Grice S.F.J. J. Biol. Chem. 1996; 271: 9054-9061Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13.Arts E.J. Stetor S. Li X. Rausch J.W. Howard K.J. Ehresmann B. North T.W. Goody R.S. Wainberg M.A. Le Grice S.F.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10063-10068Crossref PubMed Scopus (68) Google Scholar, 14.Lanchy J.M. Keith G. Le Grice S.F.J. Ehresmann B. Ehresmann C. Marquet R. J. Biol. Chem. 1998; 273: 24425-24432Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) have provided a convincing argument that additional intermolecular base pairing between the replication primer and sequences of the viral genome 5′ to the PBS play a major role in controlling initiation. In the latter case, chemical footprinting data (11.Isel C. Lanchy J.-M. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. EMBO J. 1996; 15: 917-924Crossref PubMed Scopus (177) Google Scholar, 15.Isel C. Westhof E. Massire C. Le Grice S.F.J. Ehresmann B. Ehresmann C Marquet R. EMBO J. 1999; 18: 1036-1046Crossref Scopus (104) Google Scholar) and kinetic analysis (12.Arts E.J. Ghosh M. Ehresmann B. Le Grice S.F.J. J. Biol. Chem. 1996; 271: 9054-9061Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 14.Lanchy J.M. Keith G. Le Grice S.F.J. Ehresmann B. Ehresmann C. Marquet R. J. Biol. Chem. 1998; 273: 24425-24432Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) indicate a two-step initiation program. The first of these is characterized by slow addition of the first 5 dNTPs, during which DNA synthesis is highly distributive; subsequently, the replication machinery moves into a rapid and processive elongation mode. Avian viruses display a similar control mechanism, although the intermolecular interactions underlying this are subtly different,i.e. while the anticodon loop of tRNALys,3 is in intimate contact with U5-IR loop bases of the HIV genome, this occurs between the TΨC arm of tRNATrp and U5-IR stem bases in Rous sarcoma virus (3.Cobrinik D. Aiyar A Ge Z. Katzman M. Huang H. Leis J. J. Virol. 1991; 65: 3864-3872Crossref PubMed Google Scholar, 4.Aiyar A. Ge Z. Leis J. J. Virol. 1994; 68: 611-618Crossref PubMed Google Scholar, 5.Miller J.T. Ge Z. Morris S. Das K. Leis J. J. Virol. 1997; 71: 7648-7655Crossref PubMed Google Scholar). Although restricted to an intracellular life cycle in the absence of an envelope gene, LTR-containing retrotransposons of the budding yeastSaccharomyces cerevisiae, representatives of which include Ty1 and Ty3, share many features of the reverse transcription cycle with their retroviral counterparts (16.Boeke J. Stoye J.P. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 343-432Google Scholar). Both are LTR-containing elements requiring a host-derived tRNA primer, in this case tRNAiMet, to initiate (−) strand synthesis. In contrast, a distinguishing feature of these retrotransposons is the limited complementarity between PBS sequences at the 5′ end of the genome and the tRNA primer, which in Ty3 is reduced from 18 to 8 nt. However, Keeney et al. (17.Keeney J.B. Champan K.B. Lauermann V. Voytas D.F. Astrom S.U. von Pawel-Rammingen U. Byström A. Boeke J.D. Mol. Cell. Biol. 1995; 15: 217-226Crossref PubMed Scopus (55) Google Scholar) demonstrated that features of the TΨC arm are critical to transposition, and more recently Gabus et al. (18.Gabus C. Ficheux D. Rau M. Kieth G. Sandmeyer S. Darlix J.L. EMBO J. 1998; 17: 4873-4880Crossref PubMed Scopus (58) Google Scholar) provided experimental evidence that Ty3 compensates for this by exploiting a bipartite PBS. According to this model, a region with extensive complementarity (12 nucleotides) to the TΨC arm of the tRNA primer is located at the 3′ end of the genome. Although speculative, these authors have also suggested an initiation complex of two genomic RNAs could be stabilized through a short autocomplementary sequence in tRNAiMet, which induces dimerization. A similar scenario prevails with Ty1, where reduced complementarity to the 3′ end of the tRNA primer (10 nt) is compensated by extended interactions with the D arm (19.Friant S. Heyman T. Wilhelm M.L. Wilhelm F.X. Nucleic Acids Res. 1996; 24: 441-449Crossref PubMed Google Scholar). This notion of co-operativity between distal cis-acting sequences on the genome may be not be unique to retrotransposons. Brule et al. (20.Brulé F. Bec G. Keith G. Le Grice S.F.J. Roques B.P. Ehresmann B. Ehresmann C. Marquet R. Nucleic Acids Res. 2000; 28: 634-640Crossref PubMed Scopus (32) Google Scholar) have found that (−) strand transfer in HIV can benefit from complementary sequences in the tRNA anticodon stem and bases in the U3 region at the 3′ end of the genome. A better understanding of cis-acting sequences cooperating in (−) strand DNA synthesis in retrotransposons would therefore be beneficial. As in retroviruses, (+) strand synthesis in retrotransposons initiates from an RNase H-resistant, purine-rich sequence immediately adjacent to the U3 region at the 3′ end of the genome and designated the polypurine tract or PPT. This sequence must be (i) selected from the (+) RNA/(−) DNA replication intermediate, (ii) extended at its 3′ terminus into (+) strand DNA, and (iii) excised from the nascent (+) strand to generate the appropriate 5′ LTR sequences for recognition by the integration machinery. Since imprecise removal of the PPT from (+) DNA may have consequences for integration, PPT selection and removal must by necessity be a highly accurate process. In this respect, Kirchner and Sandmeyer (21.Kirchner J. Sandmeyer S.B. J. Virol. 1996; 70: 4737-4747Crossref PubMed Google Scholar) and Wilhelm et al. (22.Wilhelm M. Heyman T. Friant S. Wilhelm F.X. Nucleic Acids Res. 1997; 25: 2161-2166Crossref PubMed Scopus (9) Google Scholar) indicated that several ribonucleotides at the 3′ terminus of the Ty3 and Ty1 PPT could serve as (+) strand initiation sites. These studies have relied exclusively on analysis of DNA isolated from virus-like particles since, until recently, purified Ty3 RT and a reconstituted system recapitulating in vivo events have been unavailable. The goal of the present study was to prepare recombinant Ty3 RT and analyze both the nucleoprotein complexes and enzymatic activities (DNA polymerase and RNase H) mediating these events. DNase I footprinting of binary polymerization complexes indicates an organization unlike that demonstrated for several retroviral enzymes (23.Wohrl B.M. Georgiadis M. Telesnitsky A. Hendrickson W. Le Grice S.F.J. Science. 1995; 267: 96-99Crossref PubMed Scopus (53) Google Scholar, 24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar, 25.Rausch J.R. Wohrl B.M. Le Grice S.F.J. J. Mol. Biol. 1996; 257: 500-511Crossref PubMed Scopus (17) Google Scholar). A system of “PPT scanning” was also exploited to evaluate the precision with which Ty3 (+) strand synthesis is initiated. Surprisingly, this system indicated that the specificity of primer selection and removal was dependent on the nature of the PPT-containing RNA primer. Finally, alignment of amino acid sequences from the RNase H domains of several LTR-containing retrotransposons and plant caulimoviruses suggests an alternative distribution of catalytic residues. The 55-kDa RT open reading frame was amplified from the Ty3POL3 gene (26.Kirchner J. Sandmeyer S.B. J. Virol. 1993; 67: 19-28Crossref PubMed Google Scholar) by the polymerase chain reaction as aBamHI/HindIII fragment and inserted between the equivalent sites of plasmid p6HRT (27.Le Grice S.F.J. Grüninger-Leitch F. Eur. J. Biochem. 1990; 178: 307-314Crossref Scopus (299) Google Scholar). This procedure generated plasmid p6HTy3RT, which allows IPTG-inducible expression of a polyhistidine extended enzyme. RT was purified from logarithmically grown and IPTG-induced cultures by a combination of metal chelate (nickel-nitrilotriacetic acid-Sepharose) and ion exchange chromatography (S-Sepharose). Purified enzyme was demonstrated to be free of contaminating nucleases and stored at −20 °C in a 50% glycerol-containing buffer (28.Le Grice S.F.J. Cameron C.E. Benkovic S.J. Campbell J.L. DNA Replication: Methods in Enzymology. Academic Press, New York1995: 130-147Google Scholar) at a concentration of 0.25 mg/ml. Under these conditions, we observed minimal loss of DNA polymerase or RNase H activity over several months. For comparative purposes, the p66/p51 form of either FIV or HIV-1 RT was included in several experiments. Methods for preparation and purification of these enzymes have been provided elsewhere (28.Le Grice S.F.J. Cameron C.E. Benkovic S.J. Campbell J.L. DNA Replication: Methods in Enzymology. Academic Press, New York1995: 130-147Google Scholar). Immunological analysis of Ty3 RT expressed in Escherichia coli was performed using rabbit polyclonal antibodies against the purified protein. The molecular weight and quaternary structure of Ty3 RT was evaluated by size exclusion chromatography using a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) connected to a DuoFlow (Bio-Rad) chromatography system. For Calibration purposes, 50–250 μg of several proteins of known molecular weight were applied to the column in a buffer of 50 mm Tris HCl (pH 7.0), 25 mm NaCl, 1 mm EDTA at a flow rate of 0.4 ml/min. These include human IgG (150,000 Da), HIV RT p66/p51 (117,000 Da), bovine serum albumin (67,000 Da), HIV RT p51 (52,000 Da), β-lactoglobulin (35,000 Da), and cytochrome c (12,000 Da). 62 μg of Ty3 RT was likewise applied. Elution of proteins was detected spectrophotemetrically (E 280), and migration times plotted against log molecular weight to create a molecular weight standard curve. The best fit dependence of mass on migration time was determined using the logarithmic curve-fitting function of Delta Graph graphing software (Design Sciences, Inc.). DNA-dependent DNA polymerase activity was evaluated on a 71-nt template hybridized to a 5′ end-labeled 36-nt primer, the former of which contains a short stem-loop in the single stranded template (29.Wohrl B.M. Howard K.J. Jacques P.S. Le Grice S.F.J. J. Biol. Chem. 1994; 269: 8541-8548Abstract Full Text PDF PubMed Google Scholar). Twenty nm template-primer (annealed by incubation at 95 °C in 10 mm Tris/HCl, pH 7.5, 25 mm MgCl2 and slow cooling to room temperature) was incubated with 40 nm RT on ice for 5 min, in a buffer comprising 10 mm Tris/HCl, pH 7.5, 10 mmMgCl2, 50 mm KCl, and 5 mmdithiothreitol. DNA synthesis was initiated at 30 °C by addition of dATP, dGTP, dCTP, and TTP to a final concentration of 100 μm. Aliquots were removed at times indicated in the text and mixed with an equal volume of 7 m urea containing 0.1% bromphenol blue and xylene cyanol. Polymerization products were resolved by high voltage denaturing polyacrylamide gel electrophoresis and evaluated by autoradiography. RNase H activity was initially evaluated on a 5′ end-labeled 90-nt RNA template (prepared by in vitro transcription) hybridized to the 36-nt DNA primer used to evaluate polymerase function (30.Ghosh M. Cameron C.E. Hughes S.H. Benkovic S.J. Le Grice S.F.J. J. Biol. Chem. 1995; 270: 7068-7076Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 10 nm enzyme was incubated with 20 nmtemplate-primer in a buffer containing 10 mm Tris/HCl, pH 7.5, 50 mm KCl, 5 mm dithiothreitol. Hydrolysis was initiated by addition of MgCl2 to a final concentration of 10 mm and allowed to continue at 30 °C. Aliquots were again removed at times indicated in the text and processed as described above. In a minor modification to this technique, RNase H activity was also examined on the same substrate whose 3′ terminus was end-labeled with [32P]Cp and RNA ligase (New England Biolabs) under conditions recommended by the manufacturer. DNase I footprinting (24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar) was conducted on the 71-nt template/36-nt primer described above and whose template or primer was end-labeled with γ-32P and polynucleotide kinase. End-labeling followed protocols specified by the manufacturer (Roche Molecular Biochemicals). 50 mm end-labeled template-primer was incubated with 85 nm Ty3 RT in 10 mm Tris/HCl, pH 8.0, 6 mm MgCl2, 80 mm NaCl for 10 min at room temperature. Two units of DNase I were added, and digestion allowed to proceed for 30 s. Hydrolysis was terminated by addition of an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). Nucleic acids in the aqueous phase were recovered by ethanol precipitation; dried; resuspended in a solution of 8 m urea, 0.1% bromphenol blue, and 0.1% xylene cyanol; and fractionated by high voltage denaturing polyacrylamide gel electrophoresis. Hydrolysis products were visualized by autoradiography. S1 footprinting (24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar) required modification of the DNase I protection protocol. Following preparation of protein/nucleic acid complexes, the sample was supplemented with 40 units of S1 (Roche Molecular Biochemicals) in a concentrated S1 buffer, such that the final composition of the reaction mixture was 33 mm sodium acetate, pH 4.5, 50 mm NaCl, and 30 μm ZnSO4. Following 30 s of S1 treatment, hydrolysis was terminated and nucleic acids processed as described above. Under these conditions, the replication complex remains stable over the digestion period. Control S1 digests of extended substrates in the absence of RT were also prepared. Experiments evaluating Ty3 PPT utilization required a combination of both DNA polymerase and RNase H activities (31.Rausch J.W. Le Grice S.F.J. J. Biol. Chem. 1997; 272: 8602-8610Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A 65-nt, chemically synthesized (−) strand DNA template (Integrated DNA Technologies) containing the PPT complement was hybridized to synthetic (+) strand RNA primers (Dharmacon Research) spanning the PPT by heating to 90 °C and slow cooling in 10 mm Tris/HCl, pH 7.5, 25 mm MgCl2. The final concentration of all template-primer combinations following hybridization was 20 μm. These substrates were incubated at room temperature for 45 min with Ty3 RT in buffer containing (final concentration) 10 mmTris/HCl, pH 8.0; 80 mm NaCl; 6 mmMgCl2; 5 mm dithiothreitol; 1 μmtemplate-primer; 340 nm RT; 100 μm each dATP, dGTP, dCTP, and TTP; 85 nm [α-32P]dATP. After 45 min, the reactions were terminated by heating to 90 °C for 2 min, after which unincorporated radioactivity was removed by spin-column Sephadex G25 gel filtration (Amersham Pharmacia Biotech). The eluate was divided into equal portions to visualize nascent (+) DNA containing or lacking the RNA primer. One portion was treated with 0.3 volumes of 1 n NaOH at 65 °C to hydrolyze all RNA primers, then neutralized by adding an equivalent volume of 1n HCl. Nucleic acids were precipitated with ethanol; precipitated; dried; and resuspended in 7 m urea, 0.1% bromphenol blue, and 0.1% xylene cyanol. The remaining portion (i.e. containing RNA primers) was precipitated as described above and resuspended in the same gel loading buffer. DNA synthesis products were fractionated by high voltage denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Replacement of Mg2+ in the RNase H domain with Fe2+ and hydroxyl radical-mediated cleavage of duplex DNA followed the protocol of Goette et al. (32.Gotte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Substrate was the 71-nt template/36-nt primer used to evaluate DNA-dependent DNA polymerase activity, the template of which was 5′ end-labeled with [γ-32P]ATP and polynucleotide kinase according to standard protocols. Enzyme (1 μm) and template primer (50 nm) were incubated 5 min at room temperature in a buffer of 80 mmHEPES, pH 8.0, 50 mm NaCl. The following reactants were subsequently pipetted onto the wall of the reaction tube: 1 μl of 50 mm dithiothreitol, 1 μl of freshly prepared H2O2, 2 μl of 2 mmFe(NH4)2(SO4)2·6H2O. Reaction vessels were carefully closed and centrifuged to initiate of Fe2+-mediated hydroxyl radical cleavage. After 5 min, the reaction was terminated by adding 40 μl of stop solution (0.1m thiourea, 10.0 mm EDTA, 0.6 mNaOAc, pH 6.2), and 1 μl of glycogen. Nucleic acids were precipitated with ethanol, collected by centrifugation, dried, and resuspended in urea-based gel loading buffer. Hydrolysis products were fractionated by high voltage denaturing gel electrophoresis and visualized by autoradiography. Although most lentiviral RTs studied to date exhibit a dimeric structure of asymmetrically organized subunits, the purified MLV enzyme is a monomer. Although surprising, the possibility of a monomeric RT organization is supported by recent data with recombinant enzyme from bovine leukemia virus (33.Perach M. Hizi A. Virology. 1999; 259: 176-189Crossref PubMed Scopus (29) Google Scholar), which was shown by rate sedimentation analysis to migrate as a monomer in both the absence and presence of duplex DNA. Following expression and purification of Ty3 RT (Fig.1, A and B), its quaternary structure was evaluated by size exclusion chromatography. As indicated in Fig. 1 B, the Ty3 enzyme migrated slightly faster than the monomeric polyhistidine-tagged p51 subunit of HIV-1 RT (mass 52 kDa) but behind bovine serum albumin (mass 67 kDa), which is consistent with a monomeric organization. However, these results do not rule out the possibility of other RT forms are required during Ty3 replication. The ability of Ty3 RT to support processive DNA synthesis in the absence of accessory factors such as the nucleocapsid protein was initially assessed. At the same time, we also wished to determine the extent to which processivity might be influenced by temperature, since yeast strains harboring Ty elements are maintained at 30 °C and RT activity in VLPs is temperature-sensitive (34.Hansen L.J. Chalker D.L. Orlinsky K.J. Sandmeyer S.B. J. Virol. 1992; 66: 1414-1424Crossref PubMed Google Scholar). DNA synthesis was evaluated on a 71-nt DNA template/36-nt DNA primer used to characterize many of the retroviral RTs in our collection (24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar). A fortuitous feature of this substrate is the intramolecular duplex adopted by the single-stranded template immediately ahead of the primer 3′ terminus (Fig. 2 A). This structure has been exploited to evaluate the processivity of wild type and mutant variants of HIV-1 and EIAV RT. As an example, Wöhrl et al. demonstrated that the p51 subunit of EIAV RT efficiently initiates DNA synthesis on this substrate, but fails to polymerize into the hairpin (29.Wohrl B.M. Howard K.J. Jacques P.S. Le Grice S.F.J. J. Biol. Chem. 1994; 269: 8541-8548Abstract Full Text PDF PubMed Google Scholar). A similar phenotype was obtained with HIV-1 enzymes harboring mutations within the p66 primer grip motif (35.Ghosh M. Jacques P.S. Rodgers D. Ottmann M. Darlix J.-L. Le Grice S.F.J. Biochemistry. 1996; 35: 8553-8562Crossref PubMed Scopus (93) Google Scholar). Thus, as a preliminary characterization, the response of the Ty3 enzyme to this structure was investigated, the results of which are presented in Fig.2 (B and C). DNA polymerase activity of Ty3 RT was affected by both the template hairpin and temperature at which the assay was performed (Fig.2 B). At 37 °C, i.e. where the HIV-1 enzyme was most active, DNA-dependent DNA synthesis catalyzed by Ty3 RT stopped predominantly between positions P + 10 and P + 15, which define the base of the template hairpin (Fig. 2 A). Since DNase I and S1 footprinting experiments have verified the presence of the stem-loop (24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar), it appears that Ty3 RT inefficiently resolves this structure at 37 °C. Lowering the incubation temperature to 30 °C conferred on Ty3 RT the capacity to polymerize through the hairpin, although the overall level of polymerase activity was lower than that obtained with the HIV-1 enzyme. In Fig. 2 C, a time course of DNA-dependent DNA synthesis was performed with both the HIV-1 and Ty3 enzymes at 30 °C. Although it is again clear that Ty3 RT is less active than its HIV-1 counterpart, pausing between template nucleotides +10 and +15 is only observed with the latter, suggesting the Ty3 enzyme may have a more robust strand displacement activity. Enzymatic footprinting of HIV (24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar), EIAV (25.Rausch J.R. Wohrl B.M. Le Grice S.F.J. J. Mol. Biol. 1996; 257: 500-511Crossref PubMed Scopus (17) Google Scholar), and MLV replication complexes (23.Wohrl B.M. Georgiadis M. Telesnitsky A. Hendrickson W. Le Grice S.F.J. Science. 1995; 267: 96-99Crossref PubMed Scopus (53) Google Scholar) indicates that the retroviral polymerase is in close contact with DNA from template nucleotide +7 to −24/−27 of the template-primer duplex. Since the size of the Ty3 enzyme is considerably different from those we have previously evaluated, it was of interest to determine if this resulted in an altered enzymatic footprint on the same template-primer duplex. A complete picture of the nucleoprotein complex can only be achieved by independent evaluation of resistance to the nucleases S1 and DNase I, which hydrolyze single-stranded and double-stranded DNA, respectively (Fig. 3 A). For comparison, replication complexes containing p66/p51 HIV-1 RT were evaluated in parallel. The results of S1 probing are illustrated in Fig. 3 B. Since the single-stranded template of our substrate assumes an intramolecular base paired structure (Fig. 3 A), only template nucleotides between positions +1 and +10 are revealed in Fig. 3 B(hydrolysis products in the immediate vicinity of the 5′ terminus lie outside the resolving capacity on the gel). Incubation of template-primer with the heterodimeric HIV-1 enzyme results in protection of template nucleotides between positions +1 and +7 from hydrolysis, which is in keeping with our previous findings (23.Wohrl B.M. Georgiadis M. Telesnitsky A. Hendrickson W. Le Grice S.F.J. Science. 1995; 267: 96-99Crossref PubMed Scopus (53) Google Scholar, 24.Wohrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar). A similar S1 hydrolysis profile was obtained with the Ty3 enzyme, suggesting that the finger subdomains of each polymerase make equivalent contact with nucleotides ahead of the DNA polymerase catalytic center. In contrast, the manner in which the HIV-1 and Ty3 enzymes contact the template-primer duplex is significantly different (Fig. 3 C). The protection pattern derived from the HIV-1 enzyme extends as far as template nucleotide −22, within which positions −19/−20 remain nuclease-accessible. In the presence of Ty3 RT, the protection pattern extends to position −24, while template nucleotides between positions −16 and −19 remain nuclease-accessible. A similar pattern emerges when contact to primer nucleotides of the template-primer duplex is investigated (Fig. 3 D). In this case, HIV-1 RT protects primer nucleotides between positions −1 and −25, within which positions −19/−20 remain accessible. With the Ty3 enzyme, the protection pattern also extends as far as primer nucleotide −25, but within this footprint positions −16 to −18 are rendered nuclease-susceptible. Combining the Ty3 RT-derived template and primer hydrolysis profiles suggests duplex DNA between positions −16 and −19 remains freely accessible to DNase I. Such data may indicate that the N-terminal DNA polymerase and C-terminal RNase H domains of Ty3 RT form independent domains separated by a small linker, as has been proposed for the murine enzyme (36.Telesnitsky A. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1276-1280Crossref PubMed Scopus (87) Google Scholar). Alternatively, an interaction of Ty3 RT with the template-primer duplex may alter its structure sufficiently to render it locally hypersensitive to DNase I digestion. Currently available crystallographic (37.Kohlstaedt L.A. Wang J. Friedman M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1749) Google Scholar, 38.Jacobo-Molina A. Ding J. Nanni R.G. Clark A.D. Lu X. Tantillo C. Wi" @default.
• W1966873031 created "2016-06-24" @default.
• W1966873031 creator A5003900273 @default.
• W1966873031 creator A5038719903 @default.
• W1966873031 creator A5039741438 @default.
• W1966873031 creator A5044685547 @default.
• W1966873031 creator A5084314666 @default.
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• W1966873031 date "2000-05-01" @default.
• W1966873031 modified "2023-09-29" @default.
• W1966873031 title "Interaction of p55 Reverse Transcriptase from theSaccharomyces cerevisiae Retrotransposon Ty3 with Conformationally Distinct Nucleic Acid Duplexes" @default.
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