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J. Biol. Chem., Vol. 277, Issue 29, 26486-26495, July 19, 2002

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Mutating Conserved Residues in the Ribonuclease H Domain of Ty3 Reverse Transcriptase Affects Specialized Cleavage Events^*

Daniela Lener, Scott R. Budihas, and Stuart F. J. Le Grice

From the Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702

Received for publication, January 16, 2002, and in revised form, March 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg²⁺- and Mn²⁺-dependent RNase H function. Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg²⁺, whereas in the presence of Mn²⁺ these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA·DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although many steps in the life cycle of Saccaromyces cerevisiae long terminal repeat (LTR)¹-containing retrotransposons are readily amenable to genetic analysis, there have been limited biochemical studies on enzymes supporting these events. With respect to reverse transcriptase (RT)-mediated conversion of the (+) strand RNA genome into integration-competent, double-stranded proviral DNA, several notable differences from their retroviral counterparts have been documented. Examples include (a) the use of a noncontiguous primer binding site to initiate () strand DNA synthesis in Ty3 (1); (b) long range interactions between D-loop nucleotides of the cognate tRNA primer (tRNA^iMet) and the RNA genome controlling initiation of () strand DNA synthesis (2); (c) initiation of () strand DNA synthesis from an internal region of the tRNA primer in Ty5 (3); (d) an alternate model of tRNA primer inheritance in Ty1 (4, 5); and (e) divergence in both the length and sequence of their (+) strand, polypurine tract (PPT) primers (6, 7). In an initial step toward dissecting these complex events at the molecular level, we reported the purification of recombinant p55 Ty3 RT and preliminary characterization of its DNA polymerase and ribonuclease H (RNase H) activities (8). More recently, an active form of Ty1 RT has also been described by Wilhelm and co-workers (9, 10). Although the Ty3 enzyme would recapitulate precise selection, extension, and excision of its (+) strand PPT primer, we were unsuccessful in replacing Mg²⁺ in the Ty3 RNase H domain with Fe²⁺ to support hydroxyl radical-mediated cleavage of duplex DNA, a feature common to the RTs of human and feline immunodeficiency viruses (8, 11). Such a result suggested that the mode of metal ion coordination in the Ty3 RNase H domain might differ from the extensively characterized retroviral enzymes.

Sequence alignment shows that a conserved histidine and a cluster of four carboxylate residues constitute the -D-E-D-H-D- motif common to the catalytic site of both retroviral and prokaryotic RNases H (12-14). In the proposed general acid-base model of catalysis (15, 16) Asp-134 of Escherichia coli RNase H positions the attacking water molecule to donate a proton to His-124, Glu-48 anchors the water molecule acting as a general acid, and Asp-10 and Asp-70 coordinate divalent metal in the active site. Biochemical studies with HIV-1 and E. coli RNases H have indicated that only the first three carboxylate residues are critical for catalysis. Replacing HIV-1 His-539 and E. coli His-124 with Asn, Asp, or Ala only reduces RNase H activity (17, 18), and a similar effect accompanies replacement of HIV-1 Asp-549 and E. coli Asp-134 with Asn (18-20). These findings suggest that the -H-D- component of the -D-E-D-H-D- motif can assist catalysis but are dispensable. In view of the sequence similarities between retroviral and retrotransposon RNases H, a surprising observation was the presence of tyrosine (Tyr-459) in the Ty3 RNase H domain at a position generally occupied by histidine. A contribution of His-124 of E. coli RNase H to catalysis has been proposed (15, 21, 22), but it was not immediately clear how this function might be fulfilled by tyrosine in the retrotransposon enzyme without invoking its activation by a nearby acidic residue. However, a compilation of RNase H sequences from the gypsy group of retrotransposons (8) suggested a conserved histidine immediately adjacent to one of the catalytic carboxylate residues (Asp-426) might be implicated in catalysis, leading to our proposal of a -D-E-DH-Y-D- motif (Fig. 1A). Interestingly however, a recent phylogenetic compilation of RNase H sequences from LTR- and non-LTR-containing elements suggests that retrotransposon enzymes may lack the flexible "His loop" of retroviral and bacterial enzymes (13) (Fig. 1B), which may result in decreased RNase H activity. The same paper also indicates that although the DNA polymerase domains Ty3/gypsy elements and retroviruses are closely related, their RNase H domains display much greater divergence. Such findings open the possibility that the retrotransposon RNase H domain could function in the absence of a "His loop," and that Y459 of Ty3 RT need not be directly involved in catalysis. Thus, a more detailed biochemical analysis of the evolutionarily conserved residues of the Ty3 RNase H domain is clearly warranted.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   A, comparison of HIV-1 catalytic residues with Ty3 RNase H conserved amino acids. Amino acid substitutions introduced into the Ty3 enzyme are indicated. B, spatial distribution of conserved residues constituting the active center the HIV-1 and Ty3 RT RNase H. A partial superimposition of the peptide backbones is illustrated. HIV structural elements and amino acid numbering are in yellow, and those of Ty3 are in red. C, purity of Ty3 enzymes. 1 µg of total protein was fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M, molecular mass markers (in kDa). WT, wild type.

In this paper, we investigated the role of several residues in the catalytic site of Ty3 RT-RNase H using purified, recombinant enzymes, in addition to monitoring transposition of the Ty3 element containing these mutations. Of the conserved carboxylate residues, RNase H activity is eliminated by replacement of Asp-358, Glu-401, and Asp-426 with their amidated counterpart, whereas the equivalent replacement of Asp-469 only partially reduced activity. Reduced activity was also noted for mutants H427A and Y459A. In contrast to our biochemical observations, all mutations prevented Ty3 transposition. Further evaluation of mutants H427A, Y459A, and D469N indicated that they failed to support two specific events in Ty3 transposition, namely DNA strand transfer and processing of the (+) strand PPT primer. Taken together, our data suggest that although the Asp-358/Glu-401/Asp-426 triad constitutes the biologically relevant metal binding site and is indispensable for RNase H function, His-427, Tyr-459, and Asp-469 may be more important in position the substrate for specific cleavage events. Differences in RNase H activity between Ty3 and HIV-1 RT were also observed when Mn²⁺ was substituted for Mg²⁺ as the divalent cation, suggesting differences in the mode of metal ion coordination between retrotransposon and retroviral enzymes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Culture Conditions-- E. coli and S. cerevisiae strains were cultured and transformed by standard methods. S. cerevisiae yTM443 (23) (MATa trp1-H3 ura3-52 his3 200 ade2-101 lys2-1 leu1-12 can1-100 Ty3 bar::hisG Gal3⁺), a derivative of yVB110 containing no endogenous copies of Ty3 (24), was used for transposition assays, and protein analyses. E. coli CJ236 (New England Biolabs) (F' cat ( = pCJ105; M13^sCm^r)/dut ung-1 thi-1 relA1 spoT1 mcrA) was used for production of single-stranded DNA for site-directed mutagenesis (25).

Modeling the Ty3 RNase H Domain-- The C-terminal Ty3 RNase H domain (residues 341-476) was modeled using the RNase H domain of p66 HIV-1 RT (PDB 1HYS, residues 427-553) as the reference protein. The Ty3 structure was generated using the Modeler function within the Homology module of InsightII (Accelrys). The conserved carboxylates Asp-358, Glu-401, Asp-426, and Asp-469 were aligned manually to the equivalent RT residues (Asp-443, Glu-478, Asp-498, and Asp-549, respectively). Optimization was set to high, and all other options remained at the defaults.

Site-directed Mutagenesis-- Point mutations in the RNase H domain of Ty3 RT expressed on plasmid p6HTy3RT (8) were introduced by one of two PCR strategies. For mutants E401Q, D426N, and H427A, 3'- and 5'-primers homologous to the mutation site, each containing the desired mutated codon, were used in separate PCRs (26), paired with the appropriate primer for either an upstream or downstream restriction site (5'-Ty3 RT SalI and 3'-Ty3 RT HindIII, respectively). The PCR products generated overlap at the ends containing the mutation site; these products were then used as templates to amplify the entire mutated construct with outside primers. Mutants D358N, Y459A, and D469N were generated using the appropriate 5'- and 3'-primers containing the desired mutated codon. The resulting fragments were cleaved and subcloned into p6HTy3RT. The final constructs were sequenced completely in the region derived by PCR amplification. These same mutations were introduced into plasmid pEGTy3-1 (24). 3'-Primers homologous to the mutation site were used for site-directed mutagenesis as described by Kunkel (25).

Expression and Purification of Ty3 RT Mutants-- Ty3 RT variants were purified from logarithmically grown and isopropyl-1--D-thiogalactopyranoside-induced E. coli cultures by a combination of metal chelate (nickel-nitrilotriacetic acid-Sepharose, Qiagen) and size exclusion chromatography (Superdex 200, Amersham Biosciences). Purified enzymes were free of contaminating nucleases and stored at 20 °C in a 50% glycerol-containing buffer (50 mM NaH₂PO₄/Na₂HPO₄, pH 7.8, 0.7 M NaCl). Under these conditions we observed minimal loss of activity over several months.

DNA Polymerase Activity-- RNA-dependent DNA polymerase activity was evaluated on a 138-nucleotide RNA template (prepared by in vitro transcription), corresponding to nucleotides 4851-4977 of Ty3 genome plus 12 additional nucleotides, hybridized to a ³²P-5'-end-labeled 20-nucleotide DNA primer (Integrated DNA Technologies). Template-primer was annealed by incubation at 95 °C in 10 mM Tris/HCl, pH 7.8, 2.5 mM MgCl₂ and slow cooling to room temperature. A reaction mixture containing 50 nM template-primer and 250 µM dNTPs was prepared in a buffer comprising 10 mM Tris/HCl, pH 7.8, 9 mM MgCl₂, 80 mM NaCl, and 5 mM dithiothreitol. DNA synthesis was initiated at 30 °C by the addition of wild type or mutant RT to a final concentration of 50 nM in a final reaction volume of 10 µl. Aliquots were removed after 5 min and mixed with an equal volume of 89 mM Tris borate, pH 8.3, 2 mM EDTA, and 7 M urea containing 0.1% bromphenol blue and xylene cyanol. Polymerization products were resolved by high voltage denaturing PAGE and evaluated by autoradiography.

RNase H Activity-- RNase H activity was initially evaluated concomitant with polymerization, using the substrate indicated above but relocating radiolabel to the 5'-terminus of the RNA template. A reaction mixture containing 50 nM template-primer and 250 µM dNTPs was prepared in a buffer of 10 mM Tris/HCl, pH 7.8, 9 mM MgCl₂, 80 mM NaCl, and 5 mM dithiothreitol. Hydrolysis and polymerization were initiated by adding wild type or mutant RT to a final concentration of 250 nM in a 10-µl reaction and allowed to continue at 30 °C for 20 min. Hydrolysis was terminated as above. Products were resolved by high voltage denaturing electrophoresis and evaluated by autoradiography. In the absence of polymerization, RNase H activity was evaluated on a 5'-end-labeled 40-nucleotide RNA template (Dharmacon Research) annealed to a 30-nucleotide DNA primer (Integrated DNA Technologies). A reaction mixture containing 50 nM template-primer was prepared in a buffer of 10 mM Tris/HCl, pH 7.8, 80 mM NaCl, 5 mM dithiothreitol, 9 mM MgCl₂ or 1 mM MnCl₂ was used for Mg²⁺- or Mn²⁺-dependent hydrolysis, respectively. Hydrolysis was initiated by the addition of enzyme to a final concentration of 50 nM in a final volume of 60 µl. 10-µl aliquots were removed at the times indicated and processed as above.

DNA Strand Transfer-- Strand transfer reactions were performed using a 40 nucleotide donor RNA template annealed to a ³²P-5'-end-labeled 20-nucleotide DNA primer (Integrated DNA Technology) and a 40-nucleotide acceptor RNA (Dharmacon Research). Donor and acceptor templates were designed to share 20 nucleotides of homology at their 5'- and 3'-termini, respectively. Polymerization and successful strand transfer produced a 60-nucleotide cDNA product. A reaction mixture containing 50 nM donor RNA template-DNA primer, 250 nM acceptor RNA template, and 250 µM dNTPs was prepared in a buffer of 10 mM Tris/HCl, pH 7.8, 9 mM MgCl₂, 80 mM NaCl, 5 mM dithiothreitol. Polymerization was initiated by the addition of Ty3 RT to a final concentration of 250 mM in a final volume of 60 µl. Ten-µl aliquots were removed at the times indicated in the text and processed as above. The same system was adapted to evaluate RNase H activity during DNA synthesis, using a ³²P-5'-end-labeled 40-nucleotide donor RNA annealed to the 20-nucleotide DNA primer.

PPT Selection-- To evaluate Ty3 PPT selection a 65-nucleotide () strand DNA template (corresponding to nucleotides 4848-4913 of the Ty3 genome) containing the PPT complement was hybridized to a variety of 5'-end-labeled (+) strand RNA primers spanning the PPT by heating to 90 °C and slow cooling in 10 mM Tris/HCl, pH 7.5, 2.5 mM MgCl₂. A reaction mixture containing 50 nM template-primer was prepared in a buffer of 10 mM Tris/HCl, pH 7.8, 9 mM MgCl_2, 80 mM NaCl, 5 mM dithiothreitol. Hydrolysis was initiated by the addition of RT to a final concentration of 50 mM in a 10-µl volume and allowed to continue at 30 °C for 20 min. Reactions were stopped and hydrolysis products resolved as above.

Transposition Assays-- Qualitative plasmid-based suppressor target assays were performed as described previously (27). The assay is based on expression of Ty3 under control of the GAL1-10 promoter on a URA3-marked donor plasmid (pEGTy3-1) and subsequent integration of the replicated Ty3 into a HIS3-marked target plasmid (pCH2bo19V) (28). The target plasmid contains two divergent tRNA genes. One of these tRNA genes acts to recruit Ty3 to the target site. The other is a transcriptionally inactive ochre suppressor tRNA^Tyr gene (sup2-o), which is activated by Ty3 integration into the target site. Transposition is scored by suppression of the ochre nonsense mutations, ade2-101 lys2-1, in yeast strain yTM443. Suppression in cells that have undergone transposition results in papillations on synthetic complete medium containing glucose (SD) and lacking adenine and lysine. yTM443 cells were transformed with pEGTy3-1, carrying wild type or mutant Ty3, and the target plasmid pCH2bo19V and plated onto SD medium lacking uracil and histidine. Three independent colonies from each transformation were patched onto SD URA His. Plates were incubated at 30 °C for 24 h, and the cells were replica plated to SD medium Ade Lys and to synthetic complete medium containing galactose (SG) lacking uracil and histidine to induce Ty3 transposition. After 48 h at 30 °C on SG medium, the patches were replica plated onto SD medium Ade Lys and incubated at 30 °C for 6 days. Transposition was scored as papillations on SD medium Ade Lys.

Whole Cell Extraction-- Cultures (10 ml) of yTM443 cells transformed with pEGTy3-1, carrying wild type or mutant Ty3, were grown in SG medium to an absorbance of ~1.0 at 600 nm and the cells collected by centrifugation. Whole cell extracts were prepared essentially as described previously (27). Briefly, pelleted cells were resuspended in 1.2 ml of whole cell extract buffer (0.1 mM EDTA, 25 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 10% glycerol) containing 1 µg/ml leupeptin and pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Cells were lysed by vortexing in presence of glass beads; the lysate was clarified by centrifugation, and protein concentration was determined using the micro BCA assay kit (Pierce).

Virus-like Particle (VLP) Preparation-- 1-liter cultures of yTM443 cells transformed with pEGTy3 derivatives were grown to late log phase in SG medium to induce Ty3 expression. A mock VLP preparation was made with nontransformed yTM443 cells. VLPs were partially purified from whole cell extract as described previously (29). Briefly, the cells were harvested, washed in buffer, digested with zymolyase, and lysed by vortexing with glass beads. Whole cell extract was fractionated over a 70, 30, and 20% (5, 5, and 15 ml, respectively) sucrose step gradient by centrifugation in a Surespin 630/36-ml rotor (Sorvall) at 22,000 rpm for 3 h at 4 °C. 4 ml of the 70%/30% interface, where VLPs sediment, was collected and divided in two portions. Each portion was concentrated by centrifugation in a Surespin 630/17-ml rotor (Sorvall) at 24,000 for 1 h at 4 °C, and the pellet was resuspended in 100 µl of buffer containing 9 mM HEPES, pH 7.8, 13.5 mM KCl, 4.5 mM MgCl₂, and 10% glycerol. VLP proteins were used for integrase immunoblot analysis.

Immunological Analysis-- Proteins from whole cell extracts and VLPs were fractionated by SDS-PAGE, transferred to nitrocellulose membranes (Hybond ECL; Amersham Biosciences), and probed with antibody to Ty3 nucleocapsid protein (a generous gift from J. L. Darlix, ENS-INSERM U412, Lyon, France), capsid protein, or integrase (23). Secondary antibodies to rabbit IgG were detected by chemiluminescence, using the ECL system as described by the manufacturer (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preliminary Characterization of Ty3 RNase H Mutants-- The recombinant enzymes were purified, and their purity was estimated by Coomassie Brilliant Blue staining (Fig. 1C). Prior to evaluating Ty3 RNase H function, it was important to confirm that altering conserved residues in this domain did not induce global changes in enzyme structure. Using the approach of Fig. 2A, RNA-dependent DNA polymerase and RNase H activity were monitored concomitantly by locating the ³²P label to the 5'-terminus of the 20-nucleotide primer or 138-nucleotide RNA template, respectively. The DNA polymerase profiles of Fig. 2B, i, obtained after a 5-min incubation, indicate minimal differences between the recombinant enzymes. The exception to this was mutant D358N, which consistently displayed lower activity with extended incubation. Equivalent results were obtained on several different substrates (data not shown). Thus, to a first approximation, mutating conserved residues of the Ty3 RNase H domain had minimal effects on the structure of the DNA polymerase catalytic center.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   RNA-dependent DNA polymerase and RNase H activities of Ty3 RT variants. A, schematic representation of the RNA-dependent DNA polymerase and RNase H assay. The substrate is a 138-nucleotide RNA template annealed to a 20-nucleotide DNA primer. i, in the RNA-dependent DNA polymerase assay, the 20-nucleotide DNA primer is 5'-radiolabeled to allow visualization of DNA synthesis. ii, in the RNase H assay the 138-nucleotide RNA is 5'-radiolabeled and annealed to the 20-nucleotide DNA primer. Ty3 RT RNase H activity, during DNA synthesis, hydrolyzes the radiolabeled RNA. Arrows indicate potential cleavage sites at the 3'- and 5'-end of the RNA. B, i, RNA-dependent DNA polymerase activity catalyzed by Ty3 RT wild type and mutants. Full-length product (cDNA, 138 nucleotides) and the 32P-labeled 20-mer DNA primer are indicated on the right. ii, RNase H hydrolysis profiles. The full-length uncleaved RNA template is indicated on the left. The 21- and 12-nucleotide products (a) represent cleavage by enzymes that have polymerized to the 5'-end of the template. Additional hydrolysis products derived from enzymes stalled on the initial template-primer duplex (b) and during polymerization (c) are indicated. Lane 1, substrate in absence of enzyme; lane 2, wild type Ty3 RT; lane 3, Ty3 RT D358N; lane 4, Ty3 RT E401Q; lane 5, Ty3 RT D426N; lane 6, Ty3 RT H427A; lane 7, Ty3 RT Y459A; lane 8, Ty3 RT D469N.

In a second experiment, relocating radiolabel to the template 5'-terminus allowed RNase H activity to be evaluated during RNA-dependent DNA synthesis. Under such conditions, the primary hydrolysis products generated by wild type Ty3 RT are 21 and 12 nucleotides (region a), which would be the expected products from a template on which the primer had been fully extended. Despite prolonged incubation, these products are absent for mutants D358N, E401Q, and D426N (Fig. 2B, ii, lanes 3-5, respectively). By analogy with a recent model for RNase H-mediated catalysis (16), Asp-358 and Asp-426 would directly coordinate the divalent metal, with Glu-401 positioning the water molecule acting as a general acid. Altering any of these three residues might be expected to eliminate hydrolysis. In contrast, His-427, Tyr-459, and Asp-469 appear dispensable for Ty3 RNase H activity (Fig. 2C, ii, lanes 6-8, respectively). In addition to the 21- and 12-nucleotide hydrolysis products, cleavage at two additional regions of the template is evident. The larger of these products (region b) is only slightly smaller than the intact template, suggesting longer residency of these mutants on the initial template-primer duplex and low level template hydrolysis prior to polymerization. Further downstream, a second region of the RNA template is susceptible to hydrolysis. Because the DNA synthesis profiles of these mutants show no major termination products in this region, this might reflect (a) transient pausing at a region of template secondary structure; (b) resolution of this structure via RNase H-mediated hydrolysis; and (c) continued polymerization. Such a mechanism has in fact been proposed for HIV-1 RT (30-32), and RNA folding programs indicate a stable stem-loop structure in this region (data not shown). Thus, although presenting a more complex hydrolysis profile, the cumulative data with mutants H427A, Y459A, and D469N indicate that they retain significant RNase H activity on a heteropolymeric RNA·DNA hybrid.

Divalent Cation Requirement of Ty3 RT Mutants-- Previous work indicated that the HIV-1 RNase H mutant p66^E478Q/p51 (33) recovered polymerization-dependent RNase H activity in Mn²⁺ on both a random heteropolymeric RNA·DNA hybrid and a second substrate mimicking release of the tRNA primer (33, 34). We therefore determined whether such a phenotype could be reproduced with Ty3 RT and whether this was restricted to the equivalent catalytic residue, Glu-401. For this analysis, a ³²P-labeled 40-nucleotide RNA·30-nucleotide DNA hybrid (Fig. 3A) was employed. In keeping with our recent studies (8) and the data of Fig. 2, the primary Mg²⁺-dependent products with wild type Ty3 RT indicate cleavage at template nucleotides 21 and 13 (Fig. 3B, lane W, and Fig. 3C, i), corresponding to the polymerase-dependent and -independent modes of hydrolysis, respectively, as described by Peliska and Benkovic (35) and Gopalakrishnan et al. (36). However, in contrast with what is typically seen with HIV RT (8, 33), polymerase-independent cleavage appears to predominate over polymerase-dependent cleavage when multiple binding events are permitted. However, in experiments conducted in the presence of heparin (which restricts Ty3 RT to a single binding event), the more established pattern of hydrolysis emerges: polymerase-dependent (21) and polymerase-independent (13) cleavages represent 65 and 35% of the total product, respectively (data not shown). Although speculative, it is possible that when Ty3 RT cleaves at position 21, this is immediately followed by cleavage of the same substrate at position 13 when multiple binding events are permitted. This subsequent cleavage, however, is suppressed in the presence of heparin because rebinding of Ty3 RT cannot occur. Experiments to address this possibility are being considered.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Mg2+ and Mn2+-dependent RNase H activities of Ty3 RT variants. A, schematic representation of the substrate used for analysis. The major Ty3 RT-derived cleavage sites on the 40-nucleotide RNA template are indicated, designating the 1st bp of the RNA·DNA hybrid in the DNA polymerase catalytic center 1. The shaded ellipse indicates the enzymatic footprinting of Ty3 using nucleases S1 and DNase I (8). B, hydrolysis profiles of Ty3 RT mutants D358N (lanes 1 and 4), E401Q (lanes 2 and 5), and D426N (lanes 3 and 6) in Mg2+ and Mn2+. The major Ty3 RT-derived cleavage sites are indicated. C and D, hydrolysis profiles of Ty3 RT mutants H427A, Y459A, and D469N in Mg2+ or Mn2+, respectively. For each enzyme, a time course is presented. Lanes 1-7 represent samples analyzed after 0, 1, 2, 5, 10, 30, and 60 min, respectively. Lanes C, alkaline hydrolysate of RNA template. RNase H activity was evaluated in the absence of DNA synthesis.

As expected, substitution within the Asp-358/Glu-401/Asp-426 triad eliminated Mg²⁺-dependent RNase H activity (Fig. 3B, lanes 1-3), and this was reduced upon replacement of His-427, Tyr-459, and Asp-469 (Fig. 3C, ii-iv). Surprisingly, introducing Mn²⁺ as the divalent cation (Fig. 3, B and D) had several consequences for the latter mutants. First, in contrast to HIV-1 RT, Mn²⁺ failed to stimulate any mutant of the Asp-358/Glu-401/Asp-426 triad (Fig. 3B, lanes 4-6, respectively). Based on data from E. coli (37) and HIV-1 RNase H (38), Mn²⁺ might be expected to occupy two sites in the Ty3 RNase H domain, i.e. at site 1, coordinated via Asp-358/Glu-401/Asp-426 and site 2 via Asp-358/D469. Because both potential sites share Asp-358, amidation of Glu-401 or Asp-426 appears to affect Asp-358 geometry such that the occupancy by either divalent cation is affected. Second, the Mn²⁺-dependent activity of mutants H427A, Y459A, and D469N exceeded that observed in Mg²⁺ (Fig. 3D, ii-iv). Retention of Mn²⁺-dependent activity again suggests that these residues are less critical for catalysis. Third, wild type Ty3 RT exhibited relaxed RNase H specificity in Mn²⁺, hydrolyzing the template at almost every position between nucleotides 24 and 6 (Fig. 3C, lane 2). Although indirect, the lack of Mn²⁺-dependent activity with mutants of the Asp-358/Glu-401/Asp-426 triad (Fig. 3B) ruled out the trivial possibility of E. coli RNase H contamination in our enzyme preparations. Finally, the data of Fig. 3D indicate that in the presence of Mn²⁺, cleavage products accumulate rapidly during the 1st min of the reaction. However, almost no additional cleavage is observed at subsequent time points, and hydrolysis does not reach the level observed in the presence of Mg²⁺ (compare the hydrolysis profile of WT Ty3 RT in Fig. 3D with that of Fig. 3C). This suggests slower turnover, i.e. the enzyme, having cleaved the substrate, fails to dissociate from the product, and as a result, additional cleavage is not observed. Enhanced Mn²⁺-dependent activity, for mutants H427A, Y459A, and D469N, was directed primarily on 13 cleavage, yielding hydrolysis profiles qualitatively similar to that observed with wild type enzyme in Mg²⁺. Stated differently, if the His-427/Tyr-459/Asp-469 triad is implicated in site 2 metal binding, abrogating this event results in catalysis mediated by metal bound exclusively at the biologically relevant site.

RNase H Mutations Are Lethal for Ty3 Transposition-- Although the data of Figs. 2 and 3 indicate retention of RNase H activity with Ty3 RT mutants H427A, Y459A, and D469N, it was unclear whether these levels could support transposition, which requires a combination of nonspecific and highly accurate processing of the replication intermediate. Consequently, all mutations were introduced into the RT domain of pEGTy3-1, a plasmid harboring a replication-competent Ty3 element (39), to be tested in a transposition assay. The assay is based on expression of Ty3 RNA upon induction with galactose and subsequent insertion of its double-stranded DNA genome into the target plasmid pCH2bo19V (28) (Fig. 4A). Integration occurs between two divergent tRNA genes, sup2bo and tRNA^Val (AAC). The sup2bo gene is a transcriptionally inactive ochre suppressor tRNA^Tyr, which is activated by Ty3 integration into the target site and suppresses the ade2-101, lys2-1 ochre nonsense mutations in the yeast host yTM443. Therefore, transposition is scored as papillations on a SD medium lacking adenine and lysine.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Ty3 RNase H mutants fail to support transposition. A, schematic representation of the transposition assay. B, transformants were patched onto selective (His, URA) SD to repress transposition and were grown for 2 days. Cells were then replica plated onto SD medium lacking lysine and adenine (upper panel) and onto selective SG medium to induce transposition. Cells, grown on selective SG medium, were then replica plated onto SD medium lacking lysine and adenine (lower panel). Papillations on SD medium lacking lysine and adenine indicate transposition.

The results of this assay are presented in Fig. 4B. The upper panel (non transposed) shows that prior to induction, none of the constructs used permitted growth on selective medium. In the lower panel (transposed) transcription of Ty3 RNA was induced by galactose, and, in the case of the wild type Ty3 element, papillations were observed. As expected from the analysis of recombinant Ty3 RT, RNase H-inactivating mutations D358N, E401Q, and D426N were lethal for transposition. Surprisingly, the same was true for mutants H427A, Y459A, and D469N. To determine whether the barrier to transposition reflected imprecise maturation of either the Gag3 (capsid protein, nucleocapsid protein) or Pol3 polyproteins (protease, RT, integrase, and RT/integrase), an immunological evaluation of whole cell (Fig. 5A) or VLP proteins (Fig. 5B) was undertaken. Because of the absence of specific antibodies, RT could only be evaluated in the context of the RT/integrase polyprotein. In general, the relative capsid protein and nucleocapsid protein levels in cell extracts (Fig. 5A) and VLPs (data not shown) were not significantly influenced, whereas reduced amounts of integrase and RT/integrase were noted for mutants Y459A and D469N in VLPs. However, in no case was accumulation of an aberrant maturation intermediate evident. Identification of appropriately sized integrase and RT/integrase also excluded the possibility that loss of transposition activity did not reflect frameshifting errors inadvertently introduced into the Ty3 clones with the RNase H point mutations. The inability of mutants retaining RNase H activity to transpose suggests that they fail to provide a "threshold" hydrolysis level required in vivo and that the Mn²⁺-dependent activity observed in vitro is not biologically relevant. Alternatively, although Ty3 RT mutants H427A, Y459A, and D469N could process random, heteropolymeric substrates, the same might not hold true for precise RNase H-mediated events required during replication. This notion was investigated in the next sections.

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Immunological analysis of Ty3 proteins. A, analysis of Ty3 structural proteins from whole cell extract isolated from yTM443 cells overexpressing wild type or RT mutant Ty3. Proteins were detected using a polyclonal rabbit anti-capsid (CA) protein IgG antibody and polyclonal rabbit anti-nucleocapsid (NC) protein IgG antibody. Molecular mass markers (in kDa) are indicated on the left. The positions of the Gag3 precursor protein (38 kDa), mature capsid protein (26 kDa), and nucleocapsid protein (7.9 kDa) are indicated on the right. B, analysis of Ty3 integrase from VLPs isolated from yTM443 cells overexpressing wild type or RT mutant Ty3. The amount of VLP protein was normalized to mature capsid protein. Protein was detected using a polyclonal rabbit anti-integrase (IN) IgG antibody. For both panels, lanes 1-7 represent wild type, D358N, E401Q, D426N, H427A, Y459A, and D469N Ty3 RT, respectively. Molecular mass markers are indicated on the left. The positions of the RT-integrase fusion protein (115 kDa) and mature integrase (61 kDa) are indicated on the right.

RNase H-proficient RT Mutants Fail to Support DNA Strand Transfer-- DNA strand transfer, i.e. relocation of nascent DNA to an acceptor template (35), is a specialized event in Ty3 replication requiring RNase H activity. Although this has been studied in retroviruses (35, 40, 41), model Ty3 systems to investigate the mechanism and its dependence on RNase H function have not been reported. The features of our DNA strand transfer system (Fig. 6A) are similar to those described by Peliska and Benkovic (35), where extension of a 20-nucleotide DNA primer to the 5'-terminus of the donor RNA template yields a 40-nucleotide strand transfer intermediate; transfer to the acceptor template, followed by resumption of DNA synthesis, yields a 60-nucleotide strand transfer product. In this model system, efficient strand transfer requires that polymerization-independent RNase H activity reduces the donor RNA template to a size permitting its dissociation and relocation of nascent DNA onto the acceptor (33, 35, 42, 43).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Strand transfer activity of mutant Ty3 RT. A, schematic representation of the strand transfer system, comprising a 40-nucleotide donor and acceptor RNAs templates sharing 20 nucleotides of homology. Extension of the 20-nucleotide DNA primer (solid arrow) to the 5'-terminus of the donor template (14) generates a 40-nucleotide strand transfer intermediate. Homology between the donor and acceptor templates allows transfer and continued synthesis, resulting in a 60-nucleotide strand transfer product. B, strand transfer activity of Ty3 RT variants. Migration positions of the DNA primer (20 nucleotides), strand transfer intermediate (40 nucleotides), and full-length, 60-nucleotide strand transfer product are indicated. WT, wild type. Lanes 1-7, represent tine points of 0, 1, 2, 5, 10, 30, and 60 min, respectively. C, RNase H cleavage of the 40-nucleotide donor template during polymerization. The full-length (uncleaved) 40-nucleotide RNA and the major Ty3 RT-derived cleavage sites are indicated on the left. Lanes 1-7 represent samples evaluated after 0, 1, 2, 5, 10, 30, and 60 min, respectively.

Fig. 6B summarizes the strand transfer activities of wild type Ty3 RT and mutants retaining RNase H function. In each case we observed efficient DNA synthesis on the donor template to produce the 40-nucleotide strand transfer intermediate. Although a slight degree of pausing was evident in the immediate vicinity of the primer 3'-OH, the activity of these enzymes was comparable, confirming that their DNA polymerase domains were not structurally compromised. However, although strand transfer product accumulates with wild type Ty3 RT, it is barely detectable with mutants H427A, Y459A, and D469N. Fig. 6C follows the same reaction using a 5'-end-labeled donor RNA template rather than radiolabeled primer, which allowed us to monitor RNase H function prior to and concomitant with DNA strand transfer. The accumulation of a 30-nucleotide hydrolysis product early in the time course (Fig. 6B, i) correlates with transient pausing shortly after DNA synthesis is initiated. Thereafter the primary hydrolysis products are 18 nucleotides and shorter, each of which results from an enzyme that has completed DNA synthesis to the 5'-terminus of the donor template. A comparison of the hydrolysis and polymerization products gives insight into the size to which the donor template must be reduced to allow strand transfer. For example, although we observe rapid accumulation of an 18-nucleotide hydrolysis product, strand transfer and continued synthesis are not evident. With time, the 18-nucleotide RNA diminishes and is replaced with fragments of 11 and 10 nucleotide. As these accumulate, there is a parallel rise in strand transfer activity, suggesting that the donor RNA template must be reduced to ~10 nucleotides to permit its displacement and relocation of the growing point onto the acceptor template.

Despite retaining both DNA polymerase and RNase H activity, the three mutant enzymes barely support strand transfer (Fig. 6C, ii-iv). This is particularly significant for mutant Y459A (Fig. 6C, iii) because this enzyme yields appreciable amounts of 18-nucleotide hydrolysis product. Data with this enzyme support our contention that cleaving the donor template 18 nucleotides from its 5'-terminus leaves an RNA fragment stably bound to nascent DNA, denying access to the acceptor template and preventing strand transfer. Low level transfer activity with this mutant correlates well with the rate at which the 11/10-nucleotide hydrolysis product appears. Trace amounts of strand transfer product visible with mutants H427A and D469N (Fig. 6C, ii and iii), despite undetectable 11/10-nucleotide RNase H hydrolysis product might be explained by low level dissociation of the residual 18-nucleotide donor template from nascent DNA over the course of the 1-h reaction.

PPT Selection by Ty3 Mutants-- During Ty3 replication, the PPT primer must be (i) excised from (+) RNA; (ii) extended into (+) DNA; and (iii) excised from (+) DNA to provide a correct 5'-LTR terminus for integration. PPT processing thus represents a second specialized RNase H-mediated event. The experimental protocol for examining PPT utilization is shown in Fig. 7A and follows processing of RNA primers hybridized to different positions of the same () DNA template. The non-PPT primer (Fig. 7A, i) served as a control, whereas the PPT/5'- and PPT/3'-primers (Fig. 7A, ii and iii) flank the 13-nucleotide PPT at its 5'- or 3'-terminus with 11 ribonucleotides, respectively. PPT/5'-3'-primer flanks the PPT with 5 and 6 nucleotides at the 5'- and 3'-termini, respectively. On the non-PPT template, wild type Ty3 RT produces a major 21-nucleotide fragment, suggesting that the DNA polymerase catalytic center is positioned at the template 3'-OH. A second series of hydrolysis products corresponding to cleavage between 11 and 14 is also evident (Fig. 7B, i). Although the latter products are absent with mutants H427A, Y459A, and D469N, each supports hydrolysis at position 21. However, only Y459A RT yields levels of 21 product equivalent to those of the wild type enzyme. A different picture emerges when these mutants process the PPT from adjacent 5'- (+) RNA. Fig. 7B, ii, indicates that removal of this RNA would yield an 11-nucleotide radiolabeled fragment. Although this is achieved by wild type RT, mutant enzymes are virtually inactive. Precise processing of the PPT flanked by 11 ribonucleotides at its 3'-terminus was predicted to yield a 13-nucleotide radiolabeled fragment (Fig. 7A, iii). 13- and 20-nucleotide fragments are produced by wild type Ty3 RT (Fig. 7B, iii), the latter most likely arising from positioning of its polymerase domain over the 5'-terminus of the PPT-containing primer (44-46). Interestingly, although the three mutants hydrolyze the non-PPT portion of the primer, cleavage at the PPT/U3 (+) RNA junction is impaired. Finally, release of the PPT 3'-OH on substrate PPT/5'-3' is predicted to generate an 18-nucleotide radiolabeled fragment. This is the primary product observed with wild type RT (Fig. 7B, iv). Additional fragments of 21/22 nucleotides most likely reflect positioning of the polymerase on the 5'-terminus of the PPT-containing primer and cleavage in a polymerization-dependent fashion. Internal cleavage of the PPT is indicated by the 12/11-nucleotide fragments (Fig. 7B, iv), a feature that was also observed with both the HIV-1 and murine leukemia virus enzymes (47, 48). Again, Ty3 mutants fail to select the PPT 3'-OH of this substrate. Thus, in keeping with data of Fig. 6, the stringency imposed by the conformation of PPT-containing substrates has a significant impact on selection by RNase H mutants.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Selection of the Ty3 polypurine tract. A, schematic representation of the model PPT selection system, comprising a 65-nucleotide DNA template to which one of three RNA primers is hybridized. The non-PPT primer (i) corresponds to the 24-nucleotide RNA sequence immediately upstream from the PPT. The arrows indicate the major Ty3 RT wild type-derived cleavage sites. PPT/5' (ii) and PPT/3' (iii) represent the PPT primer extended at its 5'- and 3'-terminus, respectively, by 11 nucleotides. In iv, the PPT is flanked at its 5'- and 3'-termini by five and six nucleotides, respectively. Arrows indicate the predicted cleavage sites. B, hydrolysis profiles from substrates i-iv. The major Ty3 RT-derived cleavage sites are indicated on the left of each panel. Lanes 1, no enzyme; lanes 2, Ty3 RT wild type; lanes 3, Ty3 RT H427A; lanes 4, Ty3 RT Y459A; lanes 5, Ty3 RT D469N. The junction between the PPT 3'-terminus and U3 RNA sequences is indicated on each panel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The availability of recombinant RT from the S. cerevisiae retrotransposons Ty1 (9, 49, 50) and Ty3 (1, 8) has spurred efforts to define a unifying mechanism of RNase H-mediated hydrolysis for bacterial, retroviral, and retrotransposon enzymes, as well as understand how specific RNase H-dependent events are achieved in the context of nonspecific hydrolysis of the RNA·DNA replication intermediate. In this paper, we describe how altering conserved residues of the Ty3 RNase H domain affects both metal ion dependence and hydrolysis of a variety of biologically relevant substrates. However, before comparing or contrasting the Ty3 RNase H domain with more extensively studied systems, a recent phylogenetic study conducted by Malik and Eickbush (13) is particularly relevant. These authors have documented that although the DNA polymerase domain of retroviral RTs is a sister group to the Ty3/gypsy elements and caulimoviruses, their C-terminal RNase H domains are only distantly related. More importantly, the flexible His-loop of bacterial and retroviral RNases H, the role of which is still unresolved, is absent from LTR-containing retrotransposon enzymes when the conserved carboxylates are aligned (Fig. 1B). Thus, the catalytic mechanism for Ty3 RNase H need not be strictly reconciled with other more extensively studied bacterial and retroviral enzymes, perhaps exemplified by recent hydroxyl radical footprinting efforts (8) and in this report by the activity of wild type and mutant enzymes as a function of divalent cation requirement.

One unequivocal feature of our data is complete loss of Mg²⁺- and Mn²⁺-dependent RNase H activity when any residue of the Asp-358/Glu-401/Asp-426 triad is altered, whereas substitutions in the His-427/Tyr-459/Asp-469 triad are only partially inhibitory. Thus, in keeping with bacterial and retroviral RNases H, we propose that Asp-358/Glu-401/Asp-426 constitute the primary metal binding site of Ty3 RNase H, which presumably is occupied with Mg²⁺ in vivo. Relaxed RNase H specificity and reduced enzyme turnover with wild type Ty3 RT in the presence of Mn²⁺ was surprising but not entirely without precedent. This profile is reminiscent of the activity of EcoRV (51) and TaqI endonucleases (52) when Mn²⁺ replaces Mg²⁺ in the active site. In the presence of Mn²⁺ both endonucleases show increased phosphodiester bond hydrolysis and lower K_m for the substrate, i.e. tighter binding that slows down product release, with the consequence of decreasing enzyme turnover and inducing relaxed specificity. Indeed, for EcoRV, Mn²⁺ was shown to accelerate the chemical reaction and stabilize the enzyme-substrate complex. Under these conditions, extended residency at a noncognate site might be predicted to enhance hydrolysis, thus accounting for relaxed specificity (51). Invoking this argument, the data of Fig. 3D suggest that the relaxed specificity of wild type Ty3 RT in Mn²⁺ reflects enhanced affinity for the substrate, prolonged residency, and accelerated hydrolysis. Moreover, in the presence of Mn²⁺, two divalent metals can occupy the RNase H domain, whereas a third will be coordinated by the carboxylate triad of the DNA polymerase catalytic center. Mn²⁺ occupancy at both catalytic centers may increase the affinity for nucleic acid at both domains. For mutants H427A, Y459A, and D469N, we predict that the biologically relevant metal binding sites of the RNase H (site 1) and DNA polymerase catalytic centers will be occupied by Mn²⁺. Single-metal occupancy at the RNase H domain would favor correct positioning on the substrate, whereas coordination of Mn²⁺ at the polymerase catalytic center increases the K_m to reduce the rate of product release. This scenario would account for a Mn²⁺-dependent hydrolysis profile of mutants His-427/Tyr-459/Asp-469, which is qualitatively similar to wild type enzyme in Mg²⁺. Finally, retention of RNase H activity on several substrates, despite the absence of a flexible RNase H His-loop in Ty3 RT, suggests that Tyr-459, His-427, and possibly Asp-469 may participate in catalysis by positioning the substrate such that the RNA strand of an RNA·DNA hybrid finds the appropriate trajectory into the RNase H catalytic center for hydrolysis.

This study also provides the first in vitro demonstration of strand transfer activity for a retrotransposon RT and the requirement for RNase H activity. A lag between completion of DNA synthesis and appearance of transfer product with wild type Ty3 RT suggests that RNase H-mediated removal of the donor template and relocation of nascent DNA on the acceptor are rate-limiting steps. At this stage, coordination between polymerization-dependent and -independent template hydrolysis is important and most likely involves distinct binding modes. Although the 18-nucleotide product reflects an enzyme whose polymerase and RNase H catalytic centers remain in close contact with the RNA·DNA duplex, hydrolysis at positions 11/10 suggests relocation of the enzyme beyond the duplex terminus such that polymerase domain contacts are largely lost, and the interaction with the RNase H domain becomes more critical. Although Y459A RT catalyzes polymerization-dependent cleavage, critical nucleic acid contacts within the RNase H domain may have been altered or lost, destabilizing the nucleoprotein complex during translocation to the duplex terminus. Dissociation and loss of polymerization-independent hydrolysis would leave the fragmented template stably bound to the growing point, thereby interrupting strand transfer. Although indirect, data with this mutant also indicate that Ty3 RT lacks any helicase activity to induce removal of residual template. The ability of mutants H427A and D469N to cleave the template-primer duplex immediately after the onset of DNA synthesis contrasts with reduced activity when the polymerizing enzymes reach the template 5'-terminus. Because extended contact with the single-stranded portion of the template is possible during initiation, the activity of these mutants suggests that once synthesis to the template 5'-terminus is complete, the resulting nucleoprotein complex is unstable and cannot translocate to catalyze polymerization-independent hydrolysis. These results could explain the in vivo defects we observed for all RNase H mutants. Based on published observations with murine leukemia virus (53-55), HIV-1 (56, 57), and Ty1 (49), we predict that transposition of Ty3 mutants whose RNase H domain is completely (D358N, E401Q, and D426N) or partially inactivated (H427A, Y459A, and D469N) would be interrupted at () strand DNA transfer.

Finally, the inability of otherwise RNase H-proficient Ty3 RT mutants to hydrolyze RNA·DNA hybrids with a more unique structure is best exemplified in PPT selection. Here, although cleavage at the junction with U3 RNA is severely impaired, non-PPT portions of the same template are hydrolyzed efficiently. Although the Ty3 PPT sequence (5'-GAGAGAGAGGAAGA-3') differs significantly from its retroviral counterparts (e.g. 5'-AAAAGAAAAGGGGGG-3' for HIV-1), selective processing by wild type enzyme infers an unusual structure that (a) renders it RNase H-resistant, yet (b) allows precise cleavage at its 3'-terminus to liberate U3 DNA sequences. We recently used chemical footprinting to demonstrate that in the absence of protein, the HIV-1 PPT RNA·() DNA hybrid (58) contains two structural distortions, namely the A:T base pair adjacent to the 3'-terminus and within a distal r(A)₄·d(T)₄ duplex. Although the precise mechanism remains to be elucidated, the interdependence between these unique structures clearly contributes to the selectivity of PPT processing. Preliminary studies suggest that similar structural anomalies may also be a feature of the Ty3 PPT,² although the extent of distortion may be less severe. We therefore suggest that, similar to the RNase H primer grip of HIV-1 RT (59) His-427, Tyr-459, and Asp-469 constitute a subset of residues whose interaction with an RNA·DNA hybrid induces the appropriate trajectory of the RNA template into the RNase H catalytic center for hydrolysis. Although speculative, Tyr-459 of Ty3 RT, a residue conserved in many LTR-containing retrotransposons, may be the counterpart of Tyr-501 of the HIV-1 RNase H primer grip, alteration of which to alanine yields an enzyme exhibiting an abnormal PPT processing phenotype (60). Experiments to understand better the role of this Ty3 residue are currently under way.

In summary, our data show that Asp-358, Glu-401, and Asp-426 constitute the primary Mg²⁺ binding site of the Ty3 RNase H domain and are required for catalysis. A second triad of conserved residues, His-427, Tyr-459, and Asp-469, may interact with substrate to ensure that the RNA template is positioned appropriately for catalysis. Based on phylogenetic (13), biochemical, and modeling data (this work), a catalytic role for Tyr-459 akin to that proposed for His-539 of HIV-1 RT (15) seems highly unlikely. Thus, whether these distantly related RNase H domains follow different catalytic mechanisms remains an open question. Finally, although we propose a single metal binding site in the Ty3 RNase H domain, this does not necessarily mean that the catalytic mechanism is single-metal-catalyzed because a second metal could be introduced by the substrate.

	ACKNOWLEDGEMENTS

We thank J. W. Rausch, G. J. Klarmann, J. T. Miller (NCI-Frederick), and M. K. Bona-Le Grice (SAIC-Frederick) for useful suggestions and critical reading of the manuscript. We also thank S. Sandmeyer (University of California at Irvine) and H. M. Nymark-McMahon (The Salk Institute) for the gift of and help with the in vivo Ty3 system.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, 1050 Boyles St., P. O. Box B, Frederick, MD 21702. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice@ncifcrf.gov.

Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M200496200

² M. Kvaratskhelia and S. F. J. Le Grice, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: LTR, long terminal repeat; HIV-1, human immunodeficiency virus type 1; PPT, polypurine tract; RNase H, ribonuclease H; RT, reverse transcriptase; SD, synthetic complete medium containing glucose; SG, synthetic complete medium containing galactose; VLP, virus-like particle.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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