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Nucleic Acids Research, 2002, Vol. 30, No. 14 3118-3129
© 2002 Oxford University Press

The role of template-primer in protection of reverse transcriptase from thermal inactivation

Gary F. Gerard*, R. Jason Potter, Michael D. Smith, Kim Rosenthal, Gulshan Dhariwal, Jun Lee and Deb. K. Chatterjee

Invitrogen Corporation, 7335 Executive Way, Frederick, MD 21704, USA

*To whom correspondence should be addressed. Tel: +1 240 379 4728; Fax: +1 240 379 4333; Email: gary.gerard{at}invitrogen.com

Received March 28, 2002; Revised and Accepted May 16, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reverse transcriptase (RT) is both an RNA- and DNA-directed DNA polymerase that is used extensively in recombinant DNA technology to synthesize cDNA from mRNA. Retroviral RT is encoded by the pol gene and is expressed as part of a polyprotein precursor that is processed into mature RT by viral-coded protease. The portion of the precursor from which RT is derived includes three structural domains arranged in the following order: NH2-Polymerase-RNase H-Integrase-COOH (1). However, the mature structural form of RT found in the prototypic retroviruses, Moloney murine leukemia virus (M-MLV), human immunodeficiency virus (HIV) and avian sarcoma and leukosis virus (ASLV) varies both with respect to the number of structural domains and subunits present. The RT from M-MLV is a monomeric polypeptide of ~80 kDa that contains the polymerase and RNase H domains (2,3). M-MLV RT has been cloned and over expressed in Escherichia coli (4,5) and its RNase H-minus forms (6) have been used extensively as tools to synthesize cDNA (7,8). HIV RT is a heterodimer with a larger 66-kDa subunit having both the polymerase and RNase H domains, and a smaller 51-kDa subunit that is identical except it lacks the RNase H domain (9–11). The polymerase activity of the HIV RT heterodimer resides only in the larger subunit (12,13). Recombinant HIV RT has been studied extensively (14), but has not been used as a tool to copy mRNA because of its relatively high error rate (15). ASLV RT [includes avian myeloblastosis virus (AMV) and Rous sarcoma virus (RSV) RT] is also heterodimeric consisting of a larger 95-kDa subunit (ß) and a smaller 63-kDa subunit ({alpha}) (16). ASLV RT ß contains all three domains, N-terminal polymerase, RNase H and C-terminal integrase, while {alpha} contains only the polymerase and RNase H domains, the integrase domain having been deleted by proteolysis (17–19). In distinction to HIV RT, the subunits of the ASLV RT heterodimer are folded in such a way that its DNA polymerase and RNase H activities reside only on the smaller {alpha} subunit (20,21). In spite of this, the integrase domain of the larger ß subunit retains enzymatic activity (19). Native, virion-derived AMV RT has also been used widely to copy mRNA (22), but until recently (23) recombinant forms of the enzyme were not available, because avian RT expressed in E.coli tends to be insoluble (24).

In addition to polymerase activity, RT possesses RNase H activity that degrades the RNA in an RNA/DNA hybrid. The presence of this degradative activity that is essential to the enzyme’s function in vivo is detrimental to the synthesis of cDNA from mRNA in vitro (25). The RNase H domain of RT can be mutated to reduce or eliminate RNase H activity while maintaining mRNA-directed DNA polymerase activity (6,26). Removal of RT RNase H activity improves the efficiency of cDNA synthesis from mRNA catalyzed by RT (6). A second significant drawback to copying mRNA with retroviral RT is its tendency to pause during cDNA synthesis resulting in the generation of truncated products (27,28). This pausing is due in part to the secondary structure of RNA (27,29). Performing cDNA synthesis at reaction temperatures that begin to melt the secondary structure of mRNA (>55°C) helps to alleviate this problem (30). Several laboratories have demonstrated that AMV RT can be used to copy mRNA at higher temperatures than M-MLV RT (31,32). In addition, mutations in the RNase H active site of M-MLV RT that eliminate catalytic activity appear to enhance thermal stability of RT polymerase by an unknown mechanism (33). We have compared the biochemical properties of the DNA polymerase of RNase H-plus (H+) and RNase H-minus (H–) forms of recombinant M-MLV and AMV RT in order to understand the causes of these differences in apparent thermal stability. We have found that RT interaction with the template-primer rather than intrinsic protein structural stability plays the most critical role in creating these differences. The practical implications of these differences are examined also.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Buffers
Binding buffer was composed of 50 mM Tris–HCl pH 8.4, 75 mM KCl, 1 mM DTT and 7.5 mM MgCl2. cDNA synthesis reaction buffer was identical except the DTT concentration was 10 mM. DNA denaturing buffer contained 96% (v/v) formamide, 20 mM EDTA, pH 8.0, 3 mg/ml bromphenol blue and 3 mg/ml xylene cyanol.

Materials
Native, virion-derived AMV RT was purchased from Life Sciences, Inc. SuperScript II RT (H– M-MLV RT), recombinant H+ M-MLV RT and recombinant H– and H+ RSV RT were from Invitrogen.

RNA and DNA
Synthetic RNAs (cRNAs), 1.4, 2.4, 4.4, 7.5 and 9.5 kb in length, containing a poly(A) tail at the 3' end were obtained from Invitrogen. (rA)20 and (dT)20 were from Invitrogen and (rA)630 was purchased from Miles. p(dT)12–18, p(dT)25–30, (dT)263 and (dA)246 were purchased from Amersham Pharmacia. Chloramphenicol acetyl transferase (CAT) cRNA (~900 nt), RSV cRNA (~1450 nt; derived from the pol gene of RSV, nt 3620–5009; 34) and MAP-4 cRNA (5.2 kb) were synthesized by T7 RNA polymerase run-off transcription from linearized plasmid DNAs (35). The sequence at the 3' end of these cRNAs if run-off transcription was complete is 5'-(N)n-(A)40-UUAAAGUAUAUUACCA-3'. The cRNAs were selected on oligo(dT)-cellulose to ensure the presence of a poly(A) tail. The 5' end of CAT cRNA was dephosphorylated with alkaline phosphatase. A DNA 24mer complementary to CAT cRNA that annealed between nucleotides 679 and 692 with its 5' end 146 nt distant from the first base at the 5' end of the CAT cRNA poly(A) tail was from Invitrogen.

CAT cDNA was synthesized from CAT cRNA in a reaction mixture (100 µl) containing cDNA synthesis reaction buffer, 500 µM each of dCTP, dATP, dTTP and [3H]dGTP (36 c.p.m./pmol), 145 pmol poly(A)-tailed CAT cRNA, 145 pmol (dT)20 and 3750 U SuperScript II. After incubation at 37°C for 15 min and 50°C for 15 min, EDTA was added to 50 mM and the reaction product was extracted with phenol/chloroform/isoamyl alcohol and ethanol precipitated. After removal of RNA by treatment with 0.3 N KOH (37°C for 16 h) and ethanol precipitation, 43 pmol of full-length single-stranded CAT cDNA was recovered. An RNA 24mer complementary to CAT cDNA that annealed between nt 679 and 692 with its 3' end 146 nt distant from the first base at the 3' end of the CAT cDNA oligo(dT) tail was purchased from Dharmacon Research.

Cloning and expression of AMV RT
AMV viral RNA was prepared (36) from purified AMV obtained from Life Sciences. The AMV pol gene cDNA was prepared from the viral RNA using the SuperScript cDNA Synthesis and Plasmid Cloning System (Invitrogen) following the manufacturer’s instructions. The cDNA was cloned into plasmid pSPORT1 (Invitrogen) between the SalI and NotI sites. Using a combination of restriction site cloning and polymerase chain reaction amplification, the gene encoding the {alpha} subunit of AMV RT was cloned into pFastBac Dual (Invitrogen) from the AMV pol gene in pSPORT1. The gene was cloned downstream of the p10 promoter while introducing a start codon adjacent and 5' to nt 2796 and a stop codon adjacent and 3' to nt 4511 (37). The gene encoding the ß subunit of AMV RT was cloned into the vector containing the {alpha} gene. The ß gene was cloned downstream of the polyhedrin promoter while introducing a start codon adjacent and 5' to nt 2796 and a coding sequence for a His6 tag followed by a 3' stop codon downstream of nt 5369 (37).

Site-directed mutagenesis (38) was used to change the aspartic acid residue to alanine at amino acid position 450 of AMV RT (37) in both the {alpha} and ß subunits. This resulted in AMV {alpha}ß RT, designated H– RT, that lacked detectable RNase H activity.

Recombinant baculovirus was formed by transforming pFastBac Dual DNA with AMV RT genes into a DH10Bac E.coli host (Invitrogen) bearing a low copy plasmid (bacmid) with the baculovirus genome. This resulted in transposition of the RT genes and expression control sequences into the bacmid. For expression of AMV RT, Sf 21 cells were infected with virus at a multiplicity of infection of 2. After 72 h at 27°C, cells were harvested by centrifugation for 5 min at 2500 g, and cell pellets were stored at –80°C.

Purification and characterization of AMV RTs
Recombinant AMV RTs were purified from frozen infected insect cells by sequential chromatography on columns of nickle-charged chelating Sepharose (Amersham Pharmacia), AF-heparin-650 M (TosoHaas) and Mono S (Amersham Pharmacia). The enzyme was stored in 0.2 M potassium phosphate pH 7.1, 0.05% (v/v) Triton X-100, 50% (v/v) glycerol, 0.01 mM EDTA and 1 mM DTT at –80°C. As judged by SDS–PAGE, the AMV RTs were >95% homogeneous and contained equimolar amounts of {alpha} and ß. They contained no detectable contaminating RNase, DNA endonuclease or DNA exonuclease. The RNA-directed DNA polymerase specific activities assayed with (rA)630•p(dT)12–18 (39) of H+ and H– recombinant AMV RT were the same (57 500 U/mg) and were similar to that of native AMV RT (46 250 U/mg).

DNA polymerase assays
RT DNA polymerase unit activity was assayed with (rA)630•p(dT)12–18 (39). One unit of DNA polymerase activity is the amount of RT that incorporates 1 nmol of deoxynucleoside triphosphate into acid-insoluble product at 37°C in 10 min.

cDNA synthesis from cRNA catalyzed by H– AMV RT was carried out in reaction mixtures (20 µl) containing cDNA synthesis reaction buffer, 1 mM each of dATP, dTTP, dGTP and [{alpha}-32P]dCTP (250 c.p.m./pmol), 35 U RNase inhibitor, 60 nM in ends (1–3 µg) cRNA, 500 nM in ends (0.1 µg) p(dT)25–30 and 80 nM (15 U) RT. Incubation was at 45–58°C for 30–60 min. When H+ AMV RT was used, 4 mM sodium pyrophosphate (22,40) was added and the MgCl2 concentration was adjusted to 12 mM. An aliquot of the reaction mixture was precipitated with TCA to determine total yield of cDNA synthesized, and the remaining cDNA product was size fractionated on an alkaline 1.2% agarose gel (41). Reaction mixtures for H– M-MLV RT were identical except MgCl2 was 3 mM, dNTPs were each 0.5 mM and RT was at 50–400 nM (25–200 U). Reaction mixtures for H+ M-MLV RT also contained 50 µg/ml actinomycin D.

To establish monovalent and divalent metal ion reaction optima, initial reaction rates were determined under conditions of limiting RT concentration during a 10 min incubation at 45°C. Reaction mixtures (20 µl) contained 3 µg (11 pmol) CAT cRNA, 0.5 µg p(dT)25–30, 0.6 pmol RT, 50 mM Tris– HCl, pH 8.4, 10 mM DTT, 1 mM each of dATP, dTTP, dCTP and [3H]dGTP (40 c.p.m./pmol), 35 U RNase inhibitor, and KCl and MgCl2, varied in concentration one at a time.

To establish RT cDNA synthetic catalytic activity at elevated temperatures (>45°C), reactions were carried out in 0.5-ml tubes in a thermocycler. To ensure reactions were initiated at the desired temperature, RT was added to pre-heated reaction tubes that were not removed from the thermocycler wells during mixing.

Measurement of KD by filter binding
A nitrocellulose filter-binding assay (42,43) was used to determine the nucleic acid binding constants (KD) of RTs for various nucleic acids. Polynucleotides were labeled at the 5' end with [{gamma}-32P]ATP and T4 polynucleotide kinase. Oligonucleotides were annealed to complementary polynucleotides in a buffer containing 10 mM Tris–HCl pH 7.5, and 80 mM KCl at 65°C for 5 min followed by room temperature for 15 min. The molar ratios were (dA)246•(dT)20 1:2, CAT cRNA•(dT)20 1:20, CAT cRNA•DNA 24mer 1:10 and CAT cDNA•RNA 24mer 1:10. Reaction mixtures (100 µl) containing binding buffer, 0.003 or 0.03 nM labeled polynucleotide, and 0.01 to 10 nM avian RT were incubated at 23°C for 5 min. After incubation, the mixture was filtered through a nitrocellulose filter (Millipore, HA 0.45 µm) soaked in binding buffer, which was then washed with binding buffer. For M-MLV RTs, reaction mixtures contained binding buffer with the MgCl2 concentration reduced to 3 mM, 0.03 nM labeled polynucleotide and 2–200 nM RT. The KD is equal to that enzyme concentration at which 50% of the labeled polynucleotide is bound. For this method of analysis to be valid, the polynucleotide concentration in the reaction must be substantially below KD, so that the total enzyme concentration approximates the concentration of free unbound enzyme.

Half-life determination
Mixtures (20 µl) incubated in 0.5-ml tubes in a thermocycler at 50°C contained cDNA synthesis reaction buffer and 40–80 nM RT. With murine RTs, 0.1% (v/v) Triton X-100 was also present. In some cases 200 nM CAT cRNA or 1600 nM (rA)20 and 5400 nM p(dT)12–18 were added. Incubation was stopped by placing tubes in ice. An aliquot (5 µl) was assayed for residual activity with (rA)630•p(dT)12–18.

Reaction temperature optimum determination
RTs (1.5 U in 50-µl reaction mixture) were incubated under unit assay reaction conditions with (rA)630•(dT)30 (39) as template-primer for 5 min at temperatures ranging from 37 to 68°C. The amount of acid-insoluble DNA product synthesized at each temperature was determined.

Measurement of template breakdown catalyzed by RT RNase H
To assess the impact of RT RNase H early in a reaction on the structure of poly(A)-tailed mRNA annealed to p(dT)12–18, CAT cRNA was labeled at the 3' end with [{alpha}-32P]ddATP (Amersham Pharmacia) and yeast poly(A) polymerase (USB) following the manufacturer’s protocol. Reaction mixtures (2.5 µl) containing cDNA synthesis reaction buffer, 1.2 pmol CAT [32P]cRNA (24 000 c.p.m./pmol), 27 pmol p(dT)12–18 and 0.42 pmol avian RT were incubated for various times at 37 or 55°C. In some cases, 1 mM each of dTTP, dATP, dCTP and dGTP was present in reaction mixtures. Escherichia coli RNase H (1 U) was used as a positive control. Incubations were terminated by the addition of 2.5 µl of DNA denaturing buffer. After heating at 65°C for 5 min, the RNA was fractionated by electrophoresis in a 20% polyacrylamide gel containing 7 M urea.

Processivity measurement
To assess the processivity of ASLV RTs, cDNA synthesis was carried out in the presence of a heparin trap for RSV cRNA annealed to a DNA 20mer (Invitrogen) labeled at the 5' end with 32P. The DNA primer was annealed as already described to the RNA with its 3' end 1390 nt from the 5' end of the RNA. Reaction mixtures (2.5 µl) contained 50 mM Tris–HCl pH 8.4, 75 mM KCl, 10 mM DTT, 0.1 pmol RSV cRNA, 0.1 pmol DNA primer and 0.2 pmol ASLV RT and were pre-incubated at 45°C for 2 min. Synthesis was initiated by the addition of a solution (2.5 µl) containing Tris–HCl, KCl and DTT at the same concentrations, plus 15 mM MgCl2, 2 mM each of dATP, dTTP, dGTP and dCTP and 200 mg/ml heparin. Synthesis was terminated after incubation at 45°C for 0.5 or 2 min by the addition of 5 µl of DNA denaturing buffer. A control reaction (4 µl) to test the effectiveness of the trap contained all the components listed and was incubated at 45°C for 2 min after the addition of 1 µl (0.2 pmol) of cloned H+ AMV RT to initiate the reaction. Reaction mixtures (4 µl) to assess the length of the cDNA product synthesized in the absence of trap contained all the components listed except heparin. They were incubated at 45°C for 0.5 or 2 min after addition of 1 µl (0.2 pmol) of ASLV RT. Reaction mixtures for M-MLV RT were carried out in an identical manner with the following exceptions. Incubations were at 37°C; the MgCl2 and dNTP concentrations added to initiate synthesis were reduced to 6 and 1 mM, respectively; and 0.5 pmol of M-MLV RT was used. Samples were heated at 65°C for 5 min and the DNAs were fractionated by electrophoresis in a 6% polyacrylamide gel containing 7 M urea.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reaction optima of recombinant AMV RT
Since recombinant forms of AMV RT have not been available for study until recently, we established reaction optima of recombinant AMV RTs and compared them with those of the native enzyme. Monovalent and divalent metal ion optima, as well as pH optima, were determined for the RNA-directed DNA polymerase activity of native AMV, cloned H+ AMV and cloned H– AMV RT utilizing CAT cRNA primed with p(dT)25–30 (Materials and Methods). The optima for all RTs tested were identical: 75 mM KCl, 7.5 mM MgCl2 and pH 8.4 in Tris–HCl buffer in the presence of 1 mM dNTPs (data not shown). Substituting Na+ or NH4+ for K+ ions did not alter the optima, nor did replacing Cl– with CH3COO– salts. Because of the similarity of native and recombinant AMV RT, we focused subsequent experiments on the recombinant avian enzymes assayed under these optimal conditions.

In the process of establishing reaction optima, we also determined the reaction temperature optima of H– and H+ AMV RT. The optima of H– and H+ M-MLV RT were determined for comparison. The optima of both avian and murine RTs were much higher than expected, ranging from 43 to 54°C (Fig. 1). Strikingly, H– AMV RT still maintained near maximum relative activity even at 58°C. The optimum of each H– RT was 4°C higher than that of its respective H+ counterpart. The optimum of each avian RT was 7°C higher than that of the respective murine RT (Fig. 1). We explored the causes of these differences, and examined their practical implications.



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  		Figure 1. Reaction temperature optima of RTs. The relative RNA-directed DNA polymerase activities of H+ M-MLV RT (inverted triangle), H– M-MLV RT (triangle), H+ AMV RT (open circle) and H– AMV RT (filled circle) were measured (Materials and Methods) at the temperatures indicated. The optima (middle of the temperature range at which the relative activity was >0.9) are indicated by broken vertical lines.

 
Half-lives of RTs at elevated temperature
We chose to examine RT half-lives at 50°C, the maximum temperature at which AMV RT has been used to synthesize cDNA. Table 1 shows that the half-lives at 50°C of H+ and H– forms of AMV RT in the absence of template-primer were short and were similar. The same was true for H+ and H– M-MLV RT. These results indicate removal of RT RNase H activity by altering the amino acids at the active site does not change the intrinsic thermal stability of RT. Table 1 also shows that the presence of cRNA•p(dT)12–18 at 50°C increases the half-lives of most RTs substantially. The presence of such an increase supports the hypothesis that binding by RT to template-primer imparts protection from thermal inactivation. We assume that in the bound state RT is more resistant to heat inactivation than when free in solution. The increased thermal protection reflected in the increased half-life in the presence of template-primer was much greater for H– RTs. In the case of H– AMV RT, a 70-fold increase was observed. The greater increases in half-lives of H– over H+ RTs in the presence of template-primer are consistent with the optima differences in Figure 1 and could be explained by several alternatives. The first is that an increase in binding affinity for template-primer was created by the mutation introduced at the RNase H active site. This possibility seems unlikely as elimination of catalytic activity at a RT active site would be expected to leave unaltered or reduce template-primer binding. A second alternative is that RNase H activity plays a role in decreasing the thermal stability of H+ RT at elevated temperatures perhaps by altering the structure of the template-primer to a form less able to protect RT. Template-primer structure strongly influences RT binding affinity (44). These half-lives were determined in buffer containing MgCl2 (Materials and Methods), so that the RNase H activity associated with H+ RT was active during the incubation and could have altered the structure of the template-primer.


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  		Table 1. Half-lives of the DNA polymerase activity of RTs at 50°C
 
Surprisingly, in contrast to the temperature optima in Figure 1, the intrinsic thermal stability of each avian RT at 50°C in the absence of template-primer was slightly less than that of its murine counterpart (Table 1). The half-lives of H– AMV and H– M-MLV RT at 37°C in the absence of template-primer were of much longer duration than at 50°C, but again the avian RT was less stable than the murine enzyme. Thus, factor(s) other than intrinsic thermal stability must be responsible for the ability of avian RTs to function at higher temperatures. There was a substantially greater increase in half-life of each avian RT over that of its murine counterpart observed in the presence of template-primer (Table 1). This differential increase is consistent with the avian RTs spending much more time bound to template-primer, perhaps by virtue of higher binding affinity.

Affinities of RTs for nucleic acids
A number of laboratories have assigned equilibrium binding constants (KD) to HIV RT for various nucleic acids (45–50). In no case was a comparison made of H+ and H– HIV RT. H+ and H– forms of M-MLV RT were shown to have similar binding affinities for DNA/DNA template-primer (44). These KD values were generally in the 2–10 nM range. We used a nitrocellulose filter-binding assay (42,43; Materials and Methods) to determine the equilibrium binding constants of H+ and H– RTs for various nucleic acids at room temperature (Table 2). For single-stranded (dA)246, (dA)246 annealed to (dT)20 at a 1:2 M ratio, and CAT cRNA, H+ and H– AMV RT had very similar dissociation constants that were in the range reported previously for HIV RT. A comparison of binding to RNA/DNA was not done because of the potential influence of the RNase H activity of H+ RT on RNA/DNA structure. Based upon the comparisons that could be made, we conclude that alteration of the AMV RT RNase H active site by eliminating RNase H activity did not change RTs ability to bind nucleic acids. Therefore, differences in binding affinity for template-primer do not explain the higher thermal stability of H– over H+ AMV RT. There were dramatic differences observed, however, in the binding affinities of a given avian RT for DNA, DNA/DNA, RNA and RNA/DNA. Binding to single-stranded cRNA was 4-fold tighter than to single-stranded (dA)246. Introduction of a complementary DNA primer, (dT)20, increased the binding affinity of AMV RT for both DNA and RNA, but particularly for the 3'-poly(A) tail of CAT cRNA (9-fold increase). The order of binding affinity of AMV RT for nucleic acids was RNA/DNA >> RNA {cong} DNA/DNA > DNA. Polynucleotide length influenced binding. AMV RT binding to (dA)20, (rA)20, (dA)20•(dT)12–18, or (rA)20•(dT)12–18 was so weak that binding constants could not be measured accurately (KD >250 nM).


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  		Table 2. Nucleic acid dissociation constants of avian and murine RTs
 
M-MLV RTs bind with much less affinity to cRNA and cRNA•(dT)20 than avian RTs: the KDs were 24–32- and 50-fold higher, respectively (Table 2). This result supports the idea that an avian RT possesses greater apparent thermal stability in the presence of template-primer than its murine RT counterpart (Table 1) because of the ability to bind much tighter to template-primer.

Model template-primer degradation studies with HIV and M-MLV RT indicate that RT prefers to bind at recessed ends of complementary oligonucleotides bound to longer oligonucleotides (51–53). When DNA with a recessed 3' end is bound to RNA, as is the case with CAT cRNA•(dT)20, RT binds with its polymerase active site positioned at the recessed DNA 3' end in preparation for primer extension. When RT is exposed to RNA with recessed ends bound to DNA, RT binds with its polymerase active site positioned at the recessed RNA 5' end, in preparation for engaging its RNase H active site in RNA cleavage (51–53). As discussed below, establishing differences in RT binding affinities for these two types of sites is important in explaining the difference in thermal stability of H– and H+ RT. To test if there is a difference in affinities, binding studies were carried out with CAT cRNA annealed to a complementary DNA 24mer and with CAT cDNA to which a RNA 24mer complement of the DNA 24mer was annealed (Materials and Methods). Both H– AMV and H– M-MLV RT bound with 4.5-fold greater affinity to the RNA/DNA hybrid with a recessed RNA 24mer than to the hybrid with a recessed DNA 24mer (Table 2). In agreement with the binding affinities established with CAT cRNA•(dT)20, binding to these hybrids by H– AMV RT was >10-fold tighter than binding by H– M-MLV RT.

Processivities of RTs
We have already shown that H+ and H– forms of AMV RT and H+ and H– forms of M-MLV RT have similar binding affinities for nucleic acids. We also found that avian RTs bind tighter than murine RTs to nucleic acids. These affinities were determined in the absence of deoxynucleotide substrates. We wished to confirm that the equilibrium binding constants reflect the behavior of the enzymes during cDNA synthesis. Thus, the processivity of H+ and H– avian RT would be expected to be the same, and the processivities of avian RTs might be expected to be much greater than those of the murine enzymes. The processivity of a DNA polymerase can be defined as the number of nucleotides incorporated during each enzyme to template-primer binding event before the enzyme dissociates from the template-primer. The processivities of HIV, M-MLV RT and native AMV RT on cRNA annealed to a DNA primer have been reported to be relatively low, in the range of 50–100 nt (28,45,54). We compared the processivities of native and recombinant H+ and H– ASLV RTs during cDNA synthesis from an RSV cRNA template ~1400 nt long under reaction conditions established to be optimal for copying mRNA. The processivity of H+ M-MLV RT was assessed for comparison. Figure 2 (upper panel) shows that for all ASLV RTs, including H+ and H– RT, the processivity was the same and was substantially greater than 100. A heparin trap was used to restrict cDNA synthesis to a single enzyme to template-primer binding event (see Fig. 2, upper panel, lane F for control where RT was exposed to trap and template-primer simultaneously). In the presence of the trap added after RT was permitted to bind to template-primer, avian RTs synthesized cDNA product 90–900 nt long in 30 s and 90 nt to full length (~1400 nt) in 2 min at 45°C (Fig. 2, upper panel, A–E). Approximately one-half of the cDNA product made by each RT was >400 nt long. At 45°C both H+ and H– avian RT catalyze cDNA synthesis with an average processivity >400 nt and a chain growth rate of 20–30 nt/s. Processivity measured with a longer cRNA template showed the avian RTs do not synthesize cDNA products much beyond 1400 nt long during a single cycle of synthesis (data not shown). Nucleotide concentration influenced processivity. These experiments were carried out in the presence of 1 mM dNTPs. At 0.5 mM dNTPs, there was no change in the processivity (data not shown). At 0.2, 0.1 and 0.02 mM dNTPs, however, the maximum length of cDNA product synthesized by cloned AMV RT from the RSV cRNA 1400-nt template in the presence of trap dropped from ~1400 to ~1000, ~500 and ~200 nt, respectively (data not shown). If multiple initiation events were permitted to occur in the absence of trap (Fig. 2, lower panel), most cDNA products were full-length in 2 min. Exceptions were defined cDNA products ~350 and ~900 nt long that were observed in the presence of trap and persisted in the absence of trap. Apparently the avian RT terminates processive synthesis at a number of sites, but is also capable of continued synthesis uninterrupted to the end of the template. At a few of these pause sites, such as at template positions corresponding to products ~350 and ~900 nt long, the enzyme has greater difficulty reinitiating DNA synthesis. H+ M-MLV RT synthesized little discernible cDNA product longer than primer in the presence of trap (Fig. 2, upper panel, G). In the absence of trap, H+ M-MLV RT synthesized mostly full-length product (Fig. 2, lower panel, G), indicating that the reaction conditions used supported efficient cDNA synthesis. Analysis by denaturing polyacrylamide gel electrophoresis of the length of cDNA product synthesized by H+ or H– M-MLV RT shows their processivities are 20–40 nt (data not shown). We conclude that differences in template-primer binding affinity and processivity do not explain the higher apparent thermal stability of H– over H+ RT, but do play a critical role in making avian RT more thermal stable than murine RT in the presence of template-primer.



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  		Figure 2. Processivity measurements of ASLV RTs and H+ M-MLV RT. DNA was synthesized in the presence (upper panel) and absence (lower panel) of a heparin trap from RSV cRNA annealed to a 5' 32P-labeled DNA 20mer (Materials and Methods). Reaction mixtures were incubated for 0.5 and 2 min and contained cloned H+ AMV RT (A), native AMV RT (B), H– AMV RT (C), cloned H+ RSV RT (D), H– RSV RT (E) or H+ M-MLV RT (G). (F, upper panel) The total inhibition of cDNA synthesis by cloned H+ AMV RT initiated in the presence of both template-primer and heparin, demonstrating the effectiveness of the heparin trap. 32P-labeled markers were 1 kb DNA ladder (lane M1) and 100 bp DNA ladder (lane M2). The arrow indicates full-length product.

 
Changes in template-primer structure produced by RT RNase H
To explain the higher apparent thermal stability of H– over H+ RT, we are left with the possibility that RT RNase H alters template-primer structure in a fashion that reduces RT binding affinity and thus protection from heat inactivation. The results in Table 3 show that exposure of CAT cRNA•(dT)12–18 to the RNase H activity of H+ AMV RT drastically diminishes the ability of this template-primer to protect H– AMV RT from thermal inactivation. The half-life of H– AMV RT at 50°C in the presence of CAT cRNA•(dT)12–18 that received no pre-treatment with RT was 150 min. Pre-exposure to H– AMV RT had little effect on the ability of CAT cRNA•(dT)12–18 to protect H– RT, reducing the half-life only slightly to 135 min. In contrast, pre-treatment of template-primer with H+ AMV RT reduced the half-life of H– RT 9-fold to 19 min.


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  		Table 3. Half-life of the DNA polymerase activity of H– AMV RT at 50°C in the presence of pre-treated template-primer
 
By labeling a CAT cRNA template at its 3' end, we were able to assess the impact the RNase H activity of AMV RT had on the template-primer structure that apparently is protecting the RT. The CAT cRNA used in these studies contained a 3'-poly(A) tail of ~50 nt (Materials and Methods). If the poly(A) tail is removed by RT RNase H one would expect to see a distribution of labeled cleavage products 50 nt or less in length. A high incubation temperature (55°C) was used to assess whether p(dT)12–18•cRNA is indeed a substrate for RT RNase H even at temperatures near the upper limit of RT use. Polyacrylamide gel electrophoresis was used to fractionate any breakdown products generated from 3' 32P-labeled CAT cRNA annealed to p(dT)12–18 after a brief exposure to AMV RT (Fig. 3). As might be expected, incubation with H– AMV RT did not discernibly change the structure of the cRNA (Fig. 3, compare lanes H and I with lanes B and C). In contrast, incubation with H+ AMV RT for even a brief period (15 s) resulted in rapid cleavage of the cRNA poly(A) tail with formation of oligomers 5–50 nt long (Fig. 3, lane D). The product size distribution was the same at 2 min (Fig. 3, lane E). At 15 s and 2 min, only 19 and 8%, respectively, of the radioactivity remained in undigested template. Incubation for longer periods with a greater amount of H+ RT reduced the length distribution to a limit digest of 2–30 nt (Fig. 3, lane G). Similar results were obtained with 8 pmol of H+ M-MLV RT at 37°C (data not shown). So the action of RT RNase H on cRNA•(dT)12–18 at the beginning of a cDNA synthesis reaction results in deadenylation of the cRNA with the formation of single-stranded cRNA and (rA)n•(dT)12–18 hybrids with n varying from 5 to 50 nt. Making the assumption that (rA)20•p(dT)12–18 mimics the structure of many of the cleavage products removed from the 3' end of CAT cRNA•p(dT)12–18 by RT RNase H, the results in Table 1 (last column on the right) indicate that (rA)n•p(dT)12–18 protects RT poorly from heat inactivation. Also, single-stranded RNA protects RT poorly from thermal inactivation based upon the results in Table 3. Consistent with reduced thermal protection of RT by (rA)n•(dT)12–18 or single-stranded RNA, RT binds with much lower affinity to these nucleic acids than to cRNA•DNA (Table 2 and text). We conclude that in the presence of RNase H the structure of the template-primer is changed quite rapidly at the beginning of a reaction to a deadenylated form that does not protect RT well from thermal inactivation.



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  		Figure 3. Deadenylation of CAT cRNA•p(dT)12–18 by AMV RT. CAT cRNA 32P-labeled at the 3' end and annealed to p(dT)12–18 was incubated with H+ and H– AMV RT for various times at 37 or 55°C and then fractionated on a denaturing 20% polyacrylamide gel (Materials and Methods). Template-primer that was not incubated is shown in lane A. Samples were incubated at 4°C for 2 min without RT (lane B); 55°C for 2 min without RT (lane C); 55°C for 15 s (lane D) and 2 min (lane E) with 0.42 pmol H+ AMV RT; 37°C for 5 min without RT (lane F); 37°C for 5 min with 4.2 pmol H+ AMV RT (lane G); 55°C for 15 s (lane H) and 2 min (lane I) with 0.42 pmol H– AMV RT; and 37°C for 5 min with 1 U E.coli RNase H (lane J). 32P-labeled markers were 10 bp ladder, (dT)4 and (dT)6.

 
The results in Figure 3 were obtained in the absence of dNTPs, when RNase H activity is independent of polymerase activity. In the presence of dNTPs, the results were similar. Oligomers 5–50 nt long were again produced and very little intact RNA persisted after 15 s. In addition, some larger breakdown products 70–200 nt long were observed at 15 s that were reduced to smaller species (5–50 nt) in 2 min (data not shown).

Practical implications
A suitable method for judging the useful upper temperature limit of the DNA polymerase activity of RT is an assessment of the effect of increasing reaction temperature on the amounts of full-length cDNA products synthesized by RT from an equimolar mixture of cRNAs of various lengths. The labeled, full-length cDNA products can be separated and quantified on an alkaline agarose gel. As the reaction temperature is increased, full-length cDNA products disappear starting with those derived from longer cRNAs until a temperature is reached where no discernible full-length product of any length is synthesized. The results of such a gel analysis carried out with H– and H+ M-MLV RT at 42–52°C are shown in Figure 4. H– M-MLV RT continues to synthesize cDNAs from cRNAs 1.4–9.5 kb in length up to 50°C, while H+ M-MLV RT does so only up to 45°C. The results of this analysis and a similar analysis of full-length cDNA products synthesized by AMV RTs at temperatures ranging from 45 to 58°C are summarized in Table 4. At temperatures up to 50°C, H+ AMV RT and H– AMV RT synthesized full-length cDNAs from cRNAs 1.4–9.5 kb in length. At temperatures >50°C, only H– AMV RT continued to make full-length products of all lengths.



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  		Figure 4. The effect of temperature on full-length cDNA synthesis from equimolar amounts of cRNAs. cDNAs synthesized by H– and H+ M-MLV RT (400 nM) at 42°C (lane A), 45°C (lane B), 48°C (lane C), 50°C (lane D) and 52°C (lane E) from equimolar amounts (12 nM each) of cRNAs of 1.4 (0.1 µg), 2.4 (0.17 µg), 4.4 (0.31 µg), 7.5 (0.54 µg) and 9.5 kb (0.67 µg) were analyzed by alkaline agarose gel electrophoresis (Materials and Methods). Incubation time was 30 min. 32P-labeled DNA (1 kb ladder) was run as a marker (lane M).

 

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  		Table 4. Functional cDNA synthetic activity of RTs at elevated temperatures
 
The amounts of enzymes used to synthesize cDNA in Figure 4 and Table 4 were established empirically to be those required to synthesize the maximum amount of long cDNA (>5 kb) at temperatures (37°C) and incubation times (60 min) normally used to prepare cDNA (data not shown). At 37°C, both avian and murine RTs are stable for extended periods (Table 1) so that enzyme stability should not be a factor in determining the amount of enzyme required. In spite of this, AMV RT was required only in near stoichiometric amounts (80 nM RT with 60 nM total template), while M-MLV RTs were required in much higher amounts (400 nM). Table 5 shows that when either H– or H+ M-MLV RT is used at 37°C during a 60-min incubation in an amount equivalent to a 5.2-kb cRNA template (50 nM), very little full-length cDNA is synthesized. Changing to a long incubation time partially alleviates this diminishment in full-length cDNA synthesis, as demonstrated by the increase in full-length products synthesized by 50 nM H– M-MLV RT during a 240-min incubation (Table 5). So an RT possessing relatively low processivity and nucleic acid binding affinity must be used in excess amounts or for long incubation periods to synthesized full-length cDNA product from long mRNA. As the incubation temperature of a reaction is increased >37°C, the requirement for excess amounts of M-MLV RT becomes even greater.


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  		Table 5. cDNA synthesis by RTs at different enzyme concentrations at 37°C
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Stabilization of enzymes to thermal inactivation by binding to substrate is a well-documented phenomenon (55) that is thought to take place because of substrate-induced conformational changes in a monomeric enzyme and/or enhanced subunit interaction in a multimeric enzyme (56). Mutations that enhance substrate binding therefore often increase thermal stability. The mutations introduced into AMV RT (D450A) and M-MLV RT (D524G, E562Q and D583N; unpublished data) that eliminated RNase H catalytic activity probably did so by eliminating binding of the divalent metal ion(s) required for activity (57–59). However, we have demonstrated that these mutations do not affect RT template-primer binding affinity or processivity. The apparent thermal stabilization of RT introduced by altering the RNase H active site is a new and different phenomenon caused by virtue of maintaining RTs substrate in a particular structural state.

The thermal protection data in Table 1 demonstrates poly(A)-tailed mRNA mixed with an excess of p(dT)12–18 protects H– AMV RT effectively in the absence of dNTPs at 50°C. The enzyme apparently has a preference for binding tightly to oligo(dT) annealed to the poly(A) tail of mRNA (Table 2). AMV H– RT efficiently initiates cDNA synthesis from mRNA•p(dT)10 or mRNA•p(dT)20 at temperatures in the 50–55°C range, above the predicted Tms of the poly(rA)/oligo(dT) hybrids (G.Gerard, unpublished data). This suggests that bound AMV RT helps stabilize the duplex structure of oligo(dT)•poly(A)mRNA. In a typical cDNA synthesis reaction containing 15 U AMV RT (1.6 pmol), 1 µg of mRNA (1.6 pmol of RNA ends if the average mRNA is 2 kb), and an excess of p(dT)12–18 [10–20 pmol of p(dT)12–18 primer ends bound per mRNA ~(A)200 tail], there is an excess of a single type of RT tight binding site available. This site is composed of DNA with a recessed 3' end bound to a longer RNA. At this type of binding site the RT polymerase domain is positioned at the DNA recessed 3'-terminus in preparation for initiation of cDNA synthesis (51–53). In the absence of RNase H activity, this type of binding site remains available throughout the course of the reaction to protect RT from thermal inactivation. The mRNA poly(A) tail remains intact and is available for unused p(dT)12–18 primers to remain bound, providing DNA recessed 3' end binding sites. The 3'-OH end of the growing cDNA chain also persists as a recessed 3' end binding site. This is not the case in the presence of RT RNase H activity. During initiation of cDNA synthesis from mRNA there is opportunity for RT RNase H to remove the mRNA poly(A) tail in a polymerase-dependent and -independent manner (28,44,60–63). Initiation of cDNA synthesis and RT RNase H-catalyzed cleavage of the mRNA poly(A) tail appear to be almost instantaneous with initiation of cDNA synthesis apparently occurring first (Fig. 3; G.Gerard, unpublished data). The poly(A) tail of mRNA•oligo(dT) is quickly converted by RT RNase H into oligo(rA) fragments (Fig. 3), many of which are too short to remain bound to oligo(dT). Oligo(dT), oligo(rA) and oligo(dT)•oligo(rA) hybrids are bound with much lower affinity by RT and protect the enzyme poorly from heat inactivation during the course of the reaction (Table 1). In addition the RNase H activity of H+ RT catalyzes polymerase-dependent cleavage of RNA as cDNA synthesis proceeds, generating RNA oligonucleotides 6–20 nt long (28,44,60–63). The RNA oligonucleotides that remain bound to cDNA product quickly become the predominant potential RT binding sites in the reaction mixture. The binding affinity studies in Table 2 show that this type of binding site is actually preferred over the type of site present at the 3' end of the growing cDNA chain. RT that disengages from cDNA synthesis because of pausing might be expected to bind preferentially at these RNA recessed ends. This would have several detrimental effects. First, enzyme engaged in productive cDNA synthesis would be diminished. Secondly, at these sites RT binds in such a way that the polymerase domain is positioned at the 5' end of the RNA hybridized to the longer cDNA (51–53), preparing the enzyme for polymerase-independent cleavage of the RNA. Such cleavage would result in the generation of small RNA oligonucleotides that do not remain bound to DNA and in the loss of the hybrid structure bound by RT, diminishing the ability of the template-primer to protect RT from thermal inactivation.

The situation with M-MLV RTs is somewhat different. The enhancement in template-primer thermal protection obtained by eliminating the RNase H activity of M-MLV RT was not nearly as great as obtained with AMV RT (Table 1). Optimal reaction temperatures were lower for the murine RTs (Fig. 1 and Table 4), and the enhancement in the upper temperature limit of effective use was not as great for murine RTs (Table 4). H– M-MLV RT could be used at 50°C, as compared with 45°C for H+ M-MLV, while H– AMV RT could be used at 58°C, as compared with 50°C for H+ AMV RT. These differences were not due to intrinsic thermal stability as the murine RTs actually had slightly greater half-lives than the avian enzymes at 50°C in the absence of nucleic acids (Table 1). The dominant factor in causing these differences is the weaker interaction between murine RT and template-primer. Avian RTs bind with much higher affinity and their polymerases have greater processivity than murine RTs. The KD of AMV H– RT is 50-fold lower than that of M-MLV H– RT to cRNA•(dT)20 (Table 2), and processivities of the avian enzymes are at least 10-fold greater than murine RTs at high dNTP concentration. Therefore, during cDNA synthesis murine RT spends much more time than avian RT in an unbound state not protected by template-primer from thermal inactivation. Also consistent with the data in Table 5, a less processive RT that spends more time cycling between a bound catalytically active state and a non-productive unbound state would be expected to be more dependent on the presence of excess enzyme to synthesize full-length copies of longer mRNAs.

Heteropolymeric RNA-directed DNA polymerase processivity values reported for HIV, M-MLV and AMV RT are relatively low (average of 50–100 nt) (28,45,54). We found the processivity of the polymerase activity of all ASLV RTs to be much higher, averaging >400 nt with an upper limit of ~1400 nt (Fig. 2). This discrepancy can be explained at least in part by the fact that prior processivity measurements were made at dNTP concentrations of between 25 and 150 µM. Nucleotide concentrations in this range match or exceed by several fold KmdNTP values reported for avian and murine RTs (46,64,65). The processivity assays described here were performed at 0.5–1 mM dNTPs, a concentration range reported to be more optimal for synthesis of cDNA from long mRNA (40,66,67). Decreasing the dNTP concentration reduced dramatically the processivity of ASLV {alpha}ß RT polymerase from an upper limit of ~1400 nt at 1 mM dNTPs to ~200 nt at 20 µM dNTPs. In contrast, dNTP concentration appears not to influence the processivity of murine RTs to the same extent. At 25 µM dNTPs, the value reported for M-MLV RT polymerase was 69 nt (28), and we found at 500 µM dNTPs the processivity was ~30 nt for H– and H+ M-MLV RT. The DNA polymerase of avian {alpha}ß RT is therefore much more processive than murine RT polymerase at high dNTP concentrations. While at dNTP concentrations approaching the KmdNTP, avian RT processivity begins to approach that of the murine enzyme. The ß subunit of ASLV {alpha}ß RT possesses an additional nucleic acid binding domain, integrase, not present in HIV or M-MLV RT (19,68). The presence of this domain increases the affinity of ASLV {alpha}ß RT for nucleic acids and increases the processivity of its polymerase relative to the {alpha} form (23,64) that is missing the domain. We speculate that high dNTP concentrations increase the binding affinity of avian {alpha}ß RT for template-primer and thus increase processivity. Perhaps dNTPs at high concentrations are bound by the ASLV RT integrase domain resulting in an increase in binding affinity of the enzyme for template-primer. High concentrations (2.5 mM) of nucleoside triphosphate have been reported to stimulate the catalytic activity of AMV RT integrase (69).

Maintenance of polymerase activity at elevated temperatures has practical implications for the use of RT in copying mRNA. The secondary structure of mRNA is reduced as temperatures are increased, reducing the chances that RT will terminate cDNA synthesis at secondary structural features in the RNA (29,63). The ability to perform cDNA synthesis at elevated temperatures also improves the specificity with which the reaction is primed (70).

We have shown in this study that tight binding by RT to DNA recessed 3' ends in template-primer protects RT from thermal inactivation. One approach demonstrated here to achieving higher incubation temperatures with RT is to maintain this type of binding site in the template-primer during the course of the reaction. An alternative approach to increasing retroviral RT thermal stability is to make amino acid changes in RT that increase binding to template-primer and/or increase the enzyme’s intrinsic thermal stability.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Prasad,V.R. (1993) Genetic analysis of retroviral reverse transcriptase structure and function. In Skalka,A.M. and Goff,S.P. (eds), Reverse Transcriptases. CSHL Press, Plainview, NY, pp. 135–162.

  2. Moelling,K. (1974) Characterization of reverse transcriptase and RNase H from Friend-murine leukemia virus. Virology, 62, 46–59.[ISI][Medline]

  3. Gerard,G.F. and Grandgenett,D.P. (1975) Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. J. Virol., 15, 785–797.[Abstract/Free Full Text]

  4. Kotewicz,M.L., D’Alessio,J.M., Driftmier,K.M., Blodgett,K.P. and Gerard,G.F. (1985) Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene, 35, 249–258.[ISI][Medline]

  5. Roth,M.J., Tanese,N. and Goff,S.P. (1985) Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem., 260, 9326–9335.[Abstract/Free Full Text]

  6. Kotewicz,M.L., Sampson,C.M., D’Alessio,J.M. and Gerard,G.F. (1988) Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H. Nucleic Acids Res., 16, 265–277.[Abstract/Free Full Text]

  7. Gerard,G.F. and D’Alessio,J.M. (1993) Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA. In Burrell,M.M. (ed.), Methods in Molecular Biology, Vol. 16: Enzymes of Molecular Biology. Humana Press, Totowa, NJ, pp. 73–93.

  8. Gerard,G.F., Fox,D.K., Nathan,M. and D’Alessio,J.M. (1997) Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA. Mol. Biotechnol., 8, 61–77.[ISI][Medline]

  9. DiMarzo Veronese,F., Copeland,T.D., Devico,A.L., Rahman,R., Oroszlan,S., Gallo,R.C. and Sarngadharan,M.G. (1986) Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science, 231, 1289–1291.[Abstract/Free Full Text]

  10. Hansen,J., Schulze,T. and Moelling,K. (1987) RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. J. Biol. Chem., 262, 12393–12396.[Abstract/Free Full Text]

  11. Hansen,J., Schulze,T., Mellert,W. and Moelling,K. (1988) Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J., 7, 239–243.[ISI][Medline]

  12. LeGrice,S.F.J., Naas,T., Wohlgensinger,B. and Schatz,O. (1991) Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J., 10, 3905–3911.[ISI][Medline]

  13. Hostomsky,Z., Hostomska,Z., Fu,T. and Taylor,J. (1992) Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis. J. Virol., 66, 3179–3182.[Abstract/Free Full Text]

  14. LeGrice,S.F.J. (1993) Human immunodeficiency virus reverse transcriptase. In Skalka,A.M. and Goff,S.P. (eds), Reverse Transcriptases. CSHL Press, Plainview, NY, pp. 163–191.

  15. Bebenek,K. and Kunkel,T.A. (1993) The fidelity of retroviral reverse transcriptases. In Skalka,A.M. and Goff,S.P. (eds), Reverse Transcriptases. CSHL Press, Plainview, NY, pp. 85–102.

  16. Grandgenett,D.P., Gerard,G.F. and Green,M. (1973) A single subunit from avian myeloblastosis virus with both RNA-directed DNA polymerase and ribonuclease H activity. Proc. Natl Acad. Sci. USA, 70, 230–234.[Abstract/Free Full Text]

  17. Rho,H.M., Grandgenett,D.P. and Green,M. (1975) Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase. J. Biol. Chem., 250, 5278–5280.[Abstract/Free Full Text]

  18. Schiff,R.D. and Grandgenett,D.P. (1978) Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase. J. Virol., 28, 279–291.[Abstract/Free Full Text]

  19. Golomb,M. and Grandgenett,D.P. (1979) Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. J. Biol. Chem., 254, 1606–1613.[Abstract/Free Full Text]

  20. Werner,S. and Wohrl,B.M. (2000) Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase {alpha}ß: localization of the polymerase and RNase H active sites in the {alpha} subunit. J. Virol., 74, 3245–3252.[Abstract/Free Full Text]

  21. Werner,S. and Wohrl,B.M. (2000) Homodimeric reverse transcriptase from Rous sarcoma virus mutated within the polymerase and RNase H active site of one subunit are active. Eur. J. Biochem., 267, 4740–4744.[ISI][Medline]

  22. Kimmel,A.R. and Berger,S.L. (1987) Preparation of cDNA and the generation of cDNA libraries: overview. Methods Enzymol., 152, 307–316.[ISI][Medline]

  23. Werner,S. and Wohrl,B.M. (1999) Soluble Rous sarcoma virus reverse transcriptases alpha, alphabeta and beta purified from insect cells are processive DNA polymerases that lack an RNase H 3' to 5' directed processing activity. J. Biol. Chem., 274, 26329–26336.[Abstract/Free Full Text]

  24. Soltis,D.A. and Skalka,A.M. (1988) The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities. Proc. Natl Acad. Sci. USA, 85, 3372–3376.[Abstract/Free Full Text]

  25. Berger,S.L., Wallace,D.M., Puskas,R.S. and Eschenfeldt,W.H. (1983) Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid. Biochemistry, 22, 2365–2372.[Medline]

  26. DeStefano,J.J., Wu,W., Seehra,J., McCoy,J., Laston,D., Alborne,E., Fay,P.J. and Bambara,R.A. (1994) Characterization of an RNase H deficient mutant of human immunodeficiency viurs-1 reverse transcriptase having an aspartate to asparagine change at position 498. Biochim. Biophys. Acta, 1219, 380–388.[Medline]

  27. Harrison,G.P., Mayo,M.S., Hunter,E. and Lever,A.M.L. (1998) Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5' and 3' of the catalytic site. Nucleic Acids Res., 26, 3433–3442.[Abstract/Free Full Text]

  28. DeStefano,J.J. Buiser,R.G., Mallaber,L.M., Myers,T.W., Bambara,R.A. and Fay,P.J. (1991) Polymerization and RNase H activities of reverse transcriptases from avian myeloblastosis, human immunodeficiency and Moloney murine leukemia viruses are functionally uncoupled. J. Biol. Chem., 266, 7423–7431.[Abstract/Free Full Text]

  29. Wu,W., Henderson,L.E., Copeland,T.D., Gorelick,R.J., Bosche,W.J., Rein,A. and Levin,J.G. (1996) Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse trancriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. J. Virol., 70, 7132–7142.[Abstract/Free Full Text]

  30. Myers,T.W. and Gelfand,D.H. (1991) Reverse transcription and DNA amplification by Thermus thermophilus DNA polymerase. Biochemistry, 30, 7661–7666.[Medline]

  31. Tosh,C., Hemadri,D., Sanyal,A., Pattnaik,B. and Venkataramanan,R. (1997) One-tube and one-buffer system of RT–PCR amplification of 1D gene of foot-and-mouth disease virus field isolates. Acta Virol., 41, 153–155.[ISI][Medline]

  32. Freeman,W.M., Vrana,S.L. and Vrana,K.E. (1996) Use of elevated reverse transcriptase reaction temperatures in RT–PCR. Biotechniques, 20, 782–783.[ISI][Medline]

  33. Gerard,G.F., Schmidt,B.J., Kotewicz,M.L. and Campbell,J.H. (1992) cDNA synthesis by Moloney murine leukemia virus RNase H-minus reverse transcriptase possessing full DNA polymerase activity. Focus, 14, 91–93.

  34. Schwartz,D.E., Tizard,R. and Gilbert,W. (1983) Nucleotide sequence of Rous sarcoma virus. Cell, 32, 853–869.[ISI][Medline]

  35. D’Alessio,J.M. and Gerard,G.F. (1988) Second-strand cDNA synthesis with E.coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E.coli DNA ligase. Nucleic Acids Res., 16, 1999–2014.[Abstract/Free Full Text]

  36. Strauss,E.M., Simon,M.C. and Kimball,P.C. (1980) Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification. J. Virol. Methods, 1, 213–221.

  37. Joliot,V., Boroughs,K., Lasserre,F., Crochet,J., Dambrine,G., Smith,R.E. and Perbal,B. (1993) Pathogenic potential of myeloblastosis-associated virus: implications of env proteins for osteopertrosis induction. Virology, 195, 812–819.[ISI][Medline]

  38. Kunkel,T.A., Roberts,J.D. and Zakour,R.A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol., 154, 367–382.[ISI][Medline]

  39. Houts,G.E., Miyagi,M., Ellis,C., Beard,D. and Beard,J.W. (1979) Reverse transcriptase from avian myeloblastosis virus. J. Virol., 29, 517–522.[Abstract/Free Full Text]

  40. Myers,J.C., Spiegelman,S. and Kacian,D.L. (1977) Synthesis of full-length DNA copies of avian myeloblastosis virus RNA in high yields. Proc. Natl Acad. Sci. USA, 74, 2840–2843.[Abstract/Free Full Text]

  41. McDonell,M.W., Simon,M.N. and Studier,F.W. (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol., 110, 119–146.[ISI][Medline]

  42. Bailey,J.M. (1979) Interpretation of nitrocellulose filter assays of protein-nucleic acid binding. Anal. Biochem., 93, 204–206.[ISI][Medline]

  43. Strauss,H.S., Boston,R.S., Record,M.T.,Jr and Burgess,R.R. (1981) Variables affecting the selectivity and efficiency of retention of DNA fragments by E.coli RNA polymerase in the nitrocellulose-filter-binding assay. Gene, 13, 75–87.[ISI][Medline]

  44. Schultz,S.J., Zhang,M., Kelleher,C.D. and Champoux,J.J. (1999) Polypurine tract primer generation and utilization by Moloney murine leukemia virus reverse transcriptase. J. Biol. Chem., 274, 34547–34555.[Abstract/Free Full Text]

  45. Huber,H.E., McCoy,J.M., Seehra,J.S. and Richardson,C.C. (1989) Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis and template switching. J. Biol. Chem., 264, 4669–4678.[Abstract/Free Full Text]

  46. Yu,H. and Goodman,M.F. (1992) Comparison of HIV-1 and avian myeloblastosis reverse transcriptase fidelity on RNA and DNA templates. J. Biol. Chem., 267, 10888–10896.[Abstract/Free Full Text]

  47. Thrall,S.H., Krebs,R., Wohrl,B.M., Cellai,L., Goody,R.S. and Restle,T. (1998) Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode. Biochemistry, 37, 13349–13358.[Medline]

  48. Kati,W.M., Johnson,K.A., Jerva,L.F. and Anderson,K.S. (1992) Mechanism and fidelity of HIV reverse transcriptase. J. Biol. Chem., 267, 25988–25997.[Abstract/Free Full Text]

  49. DeStefano,J.J., Bambara,R.A. and Fay,P.J. (1993) Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures. Biochemistry, 32, 6908–6915.[Medline]

  50. Kim,B., Ayran,J.C., Sagar,S.G., Adman,E.T., Fuller,S.M., Tran,N.H. and Horrigan,J. (1999) New human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) mutants with increased fidelity of DNA synthesis. Accuracy, template binding and processivity. J. Biol. Chem., 274, 27666–27673.[Abstract/Free Full Text]

  51. Schultz,S.J., Zhang,M., Kelleher,C.D. and Champoux,J.J. (2000) Analysis of plus-strand primer selection, removal and reutilization by retroviral reverse transcriptases. J. Biol. Chem., 275, 32299–32309.[Abstract/Free Full Text]

  52. Wisniewski,M., Balakrishnan,M., Palaniappan,C., Fay,P.J. and Bambara,R.A. (2000) The sequential mechanism of HIV reverse transcriptase RNase H. J. Biol. Chem., 275, 37664–37671.[Abstract/Free Full Text]

  53. DeStefano,J.J., Critofaro,J.V., Derebail,S., Bohlayer,W.P. and Fritzgerald-Heath,M.J. (2001) Physical mapping of HIV reverse transcriptase to the 5' end of RNA primers. J. Biol. Chem., 276, 32515–32521.[Abstract/Free Full Text]

  54. Pop,M.P. and Biebricher,C.K. (1996) Kinetic analysis of pausing and fidelity of human immunodeficiency virus type 1 reverse transcriptase. Biochemistry, 35, 5054–5062.[Medline]

  55. Pace,C.N. and McGrath,T. (1980) Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation. J. Biol. Chem., 255, 3862–3865.[Abstract/Free Full Text]

  56. Brewer,J.M. and Wampler,J.E. (2001) A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1. Int. J. Biol. Macromol., 28, 213–218.[ISI][Medline]

  57. Davies,J.F., Hostomska,Z., Hostomsky,Z., Jordan,S.R. and Matthews,D.A. (1991) Crystal structure of ribonuclease H domain of HIV