©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Strand Displacement Synthesis of the Long Terminal Repeats by HIV Reverse Transcriptase (*)

(Received for publication, July 12, 1995; and in revised form, October 25, 1995)

Gloria M. Fuentes (1) Lorna Rodríguez-Rodríguez (2) Chockalingam Palaniappan (2) Philip J. Fay (2) (3) Robert A. Bambara (1) (2) (4)(§)

From the  (1)Departments of Microbiology & Immunology, (2)Biochemistry, (3)Medicine, and the (4)Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.


INTRODUCTION

HIV (^1)is a retrovirus and the causative agent of AIDS. Retroviruses convert their RNA genomes into proviral DNA by the process of reverse transcription (reviewed in Whitcomb and Hughes(1992)). Reverse transcription starts from a cellular tRNA that binds near the 5` end of the virus to the PBS. Synthesis proceeds to the end, forming the minus strong stop DNA. Transfer of this DNA to the 3` end of either co-packaged RNA molecule is necessary to complete the (-)-strand DNA. This transfer reaction is thought to be facilitated by complementary regions at the ends of the virus and by the RNase H activity that degrades the RNA complementary to the newly synthesized DNA. Second strand synthesis is initiated from a purine-rich sequence tract (PPT) located just upstream of the U3 region. This oligoribonucleotide is created by degradation of the viral genome by the RNase H activity of the RT. Synthesis from the PPT, ending in copying part of the tRNA primer of the(-)-strand DNA, forms the plus strong stop DNA. This DNA needs to be transferred to the 3` end of the (-)-strand DNA to resume synthesis. Transfer in this case is thought to be facilitated by the removal of the tRNA primer which allows the complementary sequences spanning the PBS site, at the ends of the (+)- and(-)-strands, to circularize and complete their synthesis (Fig. 1A). After this circularization step in HIV, polymerization displaces about 637 nucleotides (Ratner et al., 1985) to generate the long terminal repeats containing the duplications of the U3, R, and U5 regions. This whole process is carried out by the enzyme reverse transcriptase (RT), which is encoded by the pol gene and is carried inside the virion. The native enzyme in HIV is a heterodimer composed of a 66- and a 51-kDa subunit (diMarzo et al., 1986; Lightfoote et al., 1986).


Figure 1: Strand displacement substrates. A, the postulated substrate for strand displacement synthesis in the reverse transcription model. B shows the substrates used to study strand displacement in this paper. The substrates were prepared as described under Methods. The template and the 5` end-labeled 19-nucleotide primer were the same in both substrate A and B. The regions labeled as PBS, U5, R, and U3 in the template correspond to the sequences found in the HIV genome. PBS, primer binding site for tRNA. U5 and U3, regions unique to the 5` and 3` end of the LTRs, respectively. R, redundancy common to 5` and 3` ends.



Not only is strand displacement necessary for viral replication, it is also likely to play a role in recombination according to the strand displacement-assimilation model (Junghans et al., 1982). This model proposes that recombination can occur during (+)-strand DNA synthesis. The model assumes that during reverse transcription two (-)-strand DNAs are created, and that (+)-strand synthesis can be initiated at multiple points. According to this model, when a downstream DNA is displaced by upcoming synthesis, this fragment can migrate to, or invade, the second copy of(-)-strand DNA and serve as a primer there. If the fragment of DNA being transferred is not 100% homologous to the new template DNA, it may lead to a recombinant virus. Additionally, strand displacement was proposed to be involved in a new model for the second template switch in reverse transcription (Li et al., 1993). In this model, synthesis from the central PPT displaces the plus strong stop DNA. The displaced DNA can base-pair with the 3` end of the(-)-strand DNA; this would eliminate the need for a second jump or transfer event.

Strand displacement by HIV-1 RT has been documented in vitro by Huber et al.(1989) and Hottiger et al.(1994) for segments up to 50 nucleotides in length, but to date there has been no evidence that this reaction can be extensive enough to carry out the lengthy displacement required in vivo. This activity has also been reported for avian myeloblastosis virus and murine leukemia virus RT (Collet et al., 1978; Matson et al., 1980; Whiting and Champoux, 1994). In murine leukemia virus, there is recent evidence that the enzyme can displace 1,334 base pairs of DNA duplex, which is a much greater distance than what is required during reverse transcription in vivo (Whiting and Champoux, 1994). In this paper, we examine the ability of HIV-RT to displace a 634-nucleotide-long segment of DNA containing the natural LTR sequences in vitro.


EXPERIMENTAL PROCEDURES

Materials

HIV-RT with native primary structure was provided by the Genetics Institute (Cambridge, MA). The enzyme had a specific activity of 40,000 units/mg. One unit is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37 °C using poly(rA)-oligo(dT) as template primer. The enzyme was divided into aliquots, stored at -70 °C, and a fresh aliquot was used for each experiment. HIV NC was chemically synthesized by the Louisiana State University Medical Core Center Laboratories. The sequence of the mature NC was that of the first 55 amino acids of the NC precursor protein described by Khan and Giedroc(1992). Biologically expressed NC was obtained from Enzyco (Denver, CO) and was used as a control. The peptide was kept under reducing conditions, and aliquots were stored in 10% 2-mercaptoethanol. The NC protein was also divided into aliquots, stored at -70 °C, and a fresh aliquot was used for each experiment. Purified recombinant human RP-A was obtained from Dr. Marc S. Wold (University of Iowa). This protein was purified as described by Henricksen et al.(1994). T4 polynucleotide kinase, T4 DNA polymerase, and poly(rA)-oligo(dT) were obtained from Boehringer Mannheim. Escherichia coli SSB and Sequenase were obtained from U. S. Biochemical Corp. The dNTPs were from Pharmacia Biotech Inc. [-P]ATP (3000 Ci/mmol) and the Nensorb columns were purchased from DuPont NEN. The DNA oligonucleotides used as primers and the Affinitips streptavidin capture microcolumns were obtained from Genosys, Inc. All other chemicals were from Sigma.

Methods

Creation of the Strand Displacement Substrate Containing the LTR Sequences

The LTR sequences came from the plasmid pUC-BS-WT provided to us by Dr. Malcolm A. Martin (NIAID, National Institutes of Health). The template was created by amplifying the segment of plasmid DNA containing these sequences using polymerase chain reaction. One of the primers used was biotinylated. After polymerase chain reaction amplification, the nonbiotinylated strand could be obtained by passing the double-stranded product through an Affinitip streptavidin capture microcolumn. The downstream segment of DNA was obtained in the same manner using another set of primers. The sequence of the primers (5` to 3`) used to obtain the template DNA were: biotin-ACTTTCGCTTTCAAGTCCC and TGGAAGGGGCTAATTCACTG. The sequence of the primers (5` to 3`) used to obtain the downstream segment of DNA were: biotin-TGGAAGGGCTAATTCACTG and TGCTAGAGATTTTCCACAC. The sequence of the primers (5` to 3`) used to obtain the ``acceptor'' DNA were: biotin-TGCTAGAGATTTTCCACAC and GGGAGCAGTATCTCGAGAC. The sequence (5` to 3`) of the primer used for the strand displacement experiments was: TCGCTTTCAAGTCCCTGTT. The templates were quantitated by absorbance at 260 nm. The DNA primer used in the experiments was labeled at the 5` end with [-P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase and purified through Nensorb columns. The hybrids were prepared by mixing the upstream and downstream primers with the template in 50 mM Tris-HCl, 0.1 mM EDTA, and 80 mM KCl. The primers were present at a 2.5 to 1 molar ratio over the template. This mixture was heated to 95 °C for 10 min and then slowly cooled to room temperature.

HIV-RT Reactions

The substrate was preincubated with the 4 units of enzyme for 5 min in 50 mM Tris-HCl (pH 8.0), 32 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, and the reactions were started with the addition of 6 mM MgCl(2) and 100 µM concentration of each deoxynucleoside triphosphate (dNTP). The reactions were performed in a final volume of 12.5 µl at 37 °C. The incubation time used in each experiment is specified under each figure legend. The reactions were stopped by adding an equal volume of a 2 times loading buffer (90% formamide (v/v), 10 mM EDTA (pH = 8), 0.1% xylene cyanole, 0.1% bromphenol blue). Trapped reactions were performed by adding 2 µg of poly(rA)-oligo(dT) to start the reaction along with the MgCl(2) and the dNTPs.

HIV-RT Reactions Containing Nucleocapsid Protein

The amount of HIV NC necessary to coat the templates used was calculated based on one molecule of NC binding to every 7 nucleotides (You and McHenry, 1993). The substrate was preincubated with the indicated amount of protein for 5 min before the addition of HIV-RT. The reaction conditions were as described above except that 50 µM ZnCl(2) was included in the reactions.

HIV-RT Reactions Containing E. coli SSB or Human RP-A

The amount of protein necessary to coat the templates used was calculated based on one tetrameric molecule of SSB binding to every 65 nucleotides (Kornberg and Baker, 1992) and one molecule of RP-A binding to every 30 nucleotides (Henricksen et al., 1994). The substrate was preincubated with HIV-RT for 5 min, and the indicated amount of protein was added when the reaction was started. Standard HIV-RT reaction conditions were used.

Gel Electrophoresis

Samples were separated using 5% denaturing sequencing gels containing 7 M urea (Sambrook et al., 1989). The gels were dried and analyzed by autoradiography. Quantitations of labeled species were done by a PhosphorImager (Molecular Dynamics) using the ImageQuant program.


RESULTS

The postulated substrate for strand displacement during reverse transcription is shown in Fig. 1A. The substrates used in this study are shown in Fig. 1B. We designed the templates so they would contain the sequences involved during this process in vivo.

Strand Displacement Slows HIV-RT-directed Synthesis

HIV-RT was allowed to carry out DNA synthesis on the substrates used to study strand displacement (Fig. 1B) in a time course reaction (Fig. 2). To show that all molecules of the substrate B contained an annealed downstream segment of DNA, we employed T4 DNA polymerase, which does not catalyze strand displacement synthesis. The downstream DNA stopped T4 DNA polymerase-directed primer elongation beyond 10 nucleotides on essentially all of the substrate B molecules (Fig. 2, lane 2).


Figure 2: Time course of the strand displacement reaction. The first two lanes, at the left, contain products of extension of substrates A and B with T4 DNA polymerase. The T4 DNA polymerase reactions were carried out using the 10 times buffer supplied by the manufacturer. The arrow points to the position at which T4 polymerase stops synthesis when it reaches the downstream primer in substrate B. The rest of the lanes contain the products of standard HIV-RT reactions on substrates A and B. The reactions were stopped at the times indicated in the figure. The product sizes were determined using a labeled 100-bp ladder (not shown).



In the absence of a downstream primer, HIV-RT takes less than 7 min to elongate most annealed primers to the end of the template. After 1 min, the maximum length of extension on substrate A is approximately 200 nucleotides, which gives a rate of synthesis of 3.3 nucleotides per s. Virtually the same distribution of products was obtained after 1 min in two additional independent experiments (not shown). On substrate B, the first elongated primers reach the end after 15 min. Even at the 60-min time point, there is little synthesis to the end of the template. At the 7-min time point, the maximum length of extension is approximately 120 nucleotides, which gives an approximate strand displacement rate of 0.28 nucleotides per s. At 15 min, the maximum length of extension is approximately 250 nucleotides. This gives a consistent displacement rate of 0.27 nucleotide per s. Two other independent experiments produced essentially the same distribution of products (not shown). This indicates that strand displacement slows RT-directed synthesis by approximately 12-fold.

We studied the effect of ionic strength on strand displacement synthesis by titrating the amount of KCl in the reactions from 32 mM to 80 mM (not shown). Synthesis on substrate A was not affected significantly by the amount of KCl in the reactions over the range examined, but synthesis on substrate B was more efficient at low ionic strength. With substrate B at KCl concentrations above 50 mM, there were virtually no extension products longer than 100 nucleotides after incubation with HIV-RT for 30 min. This inhibition of strand displacement at high ionic strength is expected since high salt stabilizes duplex DNA.

Strand Displacement Synthesis Promotes Dissociation of HIV-RT

Using the PhosphorImager, we noticed that with substrate B the length of the extension products is decreased, but the number of primers being extended remained approximately the same as with substrate A. This suggests that at least one reason why strand displacement synthesis is a slow process is because it promotes the premature dissociation of the RT from the template. The time necessary for frequent rebinding would then contribute to the slowdown in the reaction. To determine whether this is the case, we measured the amount of synthesis that occurs during a single enzyme binding event by using an enzyme trap (Fig. 3). The trap, poly(rA)-oligo(dT), was added in great excess over the template so that it would sequester RT molecules that dissociate from the experimental primer-template, preventing their return for a second round of synthesis. When the trap was added to the reaction before the enzyme, no products were formed, demonstrating that the trap effectively sequestered the RT (Fig. 3, lanes 2 and 5). Experiments were performed by binding the enzyme to the primer-template and then initiating the reaction by simultaneous addition of dNTPs and trap. In the presence of trap, approximately 2% of the enzymes on substrate A synthesized the 644 nucleotides needed to reach the end of the template, without dissociating. Approximately 60% of the enzymes dissociated before synthesizing 100 nucleotides. Under the same conditions, all the enzymes on substrate B dissociated before adding 100 nucleotides. These results show a decrease in processivity, the number of nucleotides added per binding of RT to the polynucleotide substrate, when the enzyme performs strand displacement synthesis.


Figure 3: Strand displacement synthesis in the presence of trap. Trapped HIV-RT reactions were performed for 30 min as described under Methods. The trap used was poly(rA)-oligo(dT). Lanes 1 and 4 are nontrapped reactions, lanes 2 and 5 are trap control reactions, and lanes 3 and 6 are trapped reactions. The product sizes were determined using a labeled 100-bp ladder (not shown).



Even when the strand displacement reactions were carried out for over 3 h in the absence of trap, we did not observe a high proportion of the upstream primer reaching the end of the template (not shown). We thought that extension may not have reached the end of the template because most the enzymes inactivated before synthesis could be completed. To test our hypothesis, we added fresh enzyme to the strand displacement reaction every 30 min and showed that with sufficient time, and a constant replenishment of RT, complete extension of initiated primers can be accomplished (not shown).

Effect of Single-stranded Binding Proteins in Strand Displacement Synthesis

The effect of HIV NC, E. coli SSB, and human RP-A in the strand displacement reaction was tested. Single-stranded binding proteins exist in all cell types and are often used to stimulate DNA replication (Kornberg and Baker, 1992).

NC is a viral protein that normally coats part of the viral genome in vivo. NC promotes strand exchange from double-stranded DNA to single-stranded DNA favoring the most stable complex (Tsuchihashi et al., 1994). NC was titrated into the strand displacement reactions at concentrations ranging from 1.2 µM to 75 nM. These concentrations of NC are enough to cover 200% to 12.5% of the DNA in the reactions. At high concentrations, NC was inhibitory for synthesis, and, at low concentrations, no stimulation of strand displacement was observed (not shown). The effect of NC was tested by adding NC before and after preincubation of the substrate with RT. To investigate whether the effect of NC was due to the melting of the primer away from the template, we fractionated an aliquot of the completed reactions on a native polyacrylamide gel. We found approximately the same proportion of unannealed primers in the presence and absence of NC (not shown).

We then tested whether a protein known to stimulate DNA synthesis by melting template secondary structure could enhance the reaction. E. coli SSB has this capacity, presumably because of its ability to bind and stabilize single-stranded DNA (Kornberg and Baker, 1992). Decreasing concentrations of SSB were added to the strand displacement reactions after preincubation of the substrate with HIV-RT (Fig. 4A). E. coli SSB enhanced the extent of strand displacement synthesis, especially at concentrations of 150 nM and lower (lanes 3-5). In order to quantitate this enhancement, we measured the percent of the extended products longer than 400 nucleotides. There was an approximately 3-fold increase in these products in the presence of E. coli SSB. Furthermore, the percent of extended primers in the presence and absence of E. coli SSB remained constant. This indicates that E. coli SSB affected the reaction by increasing the length of the extension products rather than increasing the amount of extended products. This effect of E. coli SSB was unchanged when the protein was preincubated with the substrate before the addition of the RT. When E. coli SSB was present at 600 and 300 nM (lanes 1 and 2), the enhancement of strand displacement synthesis was diminished. This could be because E. coli SSB aggregates at high concentrations (Kornberg and Baker, 1992), and its ability to enhance strand displacement synthesis could be impaired.


Figure 4: Strand displacement reactions in the presence of single-stranded binding proteins. Standard HIV-RT reactions in the presence of E. coli SSB or RP-A were carried out for 30 min as described under Methods. Only substrate B was used in this experiment. A, the concentrations of SSB in lanes 1-5 were 600, 300, 150, 75, and 37.5 nM, respectively. Assuming each tetramer of SSB covers 65 nucleotides (Kornberg and Baker, 1992), these concentrations of SSB are enough to coat 1000% to 62% of the DNA in the reaction. Lane 6 contained no SSB. B, the concentrations of RP-A used in lanes 1-4 were 35, 70, 140, and 280 nM. If each molecule of RP-A covers 30 nucleotides (Henricksen et al., 1994), these concentrations should coat 12.5, 25, 50, and 100% of the DNA in the reactions. Lane 5 contained no RP-A. The product sizes were determined using a labeled 100-bp ladder (not shown).



Finally, the effect of human RP-A on the strand displacement reactions was tested (Fig. 4B). RP-A is the human analog of E. coli SSB. It binds single-stranded DNA, and it can stimulate the activity of polymerases and helicases (Henricksen et al., 1994). Like E. coli SSB, RP-A enhanced the percent of extended primers that were longer than 400 nucleotides. Using the PhosphorImager, we measured a 4-6-fold increase in the percent of these products in the presence of 35-140 nM RP-A (lanes 1-3). When RP-A was present at 280 nM, virtually no synthesis occurred (lane 4). At 140 nM RP-A, the percent of extended primers was decreased, and, at 70 and 35 nM, the percentage of extended primers in the presence and absence of RP-A remained constant. Therefore, RP-A increases the formation of long products, even when it is present at levels high enough to decrease the total amount of synthesis. The inhibition of polymerases by RP-A has been observed previously (Tsurimoto and Stillman, 1991), possibly because high concentration RP-A blocks the binding of the polymerase to the primer terminus.

The Presence of an Acceptor Template Does Not Stimulate Strand Displacement Synthesis

It is possible that during reverse transcription, two(-)-strand DNAs are synthesized from the two RNAs found in the virion. In vivo, the presence of the second (-)-strand DNA in the reaction may enhance strand displacement synthesis by binding or stabilizing the displaced segment of DNA. In order to test this hypothesis, we designed an 834-nucleotide-long DNA which was complementary to the downstream DNA primer. This DNA was called the acceptor DNA. It was made longer than the template DNA so that the two strands could be distinguished by electrophoretic mobility. The strand displacement reaction was performed in the presence of increasing concentrations of the acceptor DNA (Fig. 5). The presence of acceptor DNA did not stimulate strand displacement synthesis.


Figure 5: Strand displacement synthesis in the presence of an acceptor DNA. Standard HIV-RT reactions were performed for 60 min as described under Methods. The acceptor was added along with the MgCl(2) and the dNTPs to start the reactions. The first lane contains elongation of a labeled primer on the acceptor template. The bands at position T correspond to extension products that transferred to the acceptor template and completed synthesis there. The bands at position 663 correspond to the full-length extension products on the original template DNA. The product sizes were determined using a labeled 100-bp ladder (not shown).



Instead, the primer DNA transferred to the acceptor DNA whether there was a downstream segment of DNA annealed to the template or not. The primer did not have homology to the acceptor DNA until it had been elongated by at least 10 nucleotides. Transfer in the absence of a downstream segment of DNA, substrate A, was first observed when the acceptor DNA was present at a 10-fold excess over the template DNA. Transfer in the presence of a downstream segment of DNA, substrate B, was first observed when the acceptor DNA was present at a 5-fold excess over the template DNA. Furthermore, with substrate B, virtually no full-length products were seen. This transfer phenomenon was not specific for HIV-RT, since Sequenase was also found to perform this reaction (not shown).


DISCUSSION

During HIV replication, a DNA segment approximately 637 nucleotides in length needs to be displaced in order to generate the long terminal repeats. Strand displacement by HIV-RT in vitro has been documented previously for substrates 50 or less nucleotides in length (Huber et al., 1989; Hottiger et al., 1994). Preliminary results from our laboratory (not shown) using similar substrates showed a small but noticeable slowing of primer elongation in the presence of a downstream primer. This suggested that displacement of the natural LTR region, as is necessary in vivo, might be a daunting problem for the RT. When we measured strand displacement synthesis on an LTR substrate in vitro, we found that, indeed, synthesis was slow and inefficient. In fact, the primer elongation rate was reduced by approximately 12-fold from synthesis over an LTR sequence template lacking the long downstream primer. Whiting and Champoux(1994) reported that murine leukemia virus RT was able to displace 1334 base pairs of DNA with approximately a 10-fold reduction in the rate of synthesis. These results show a similarity in strand displacement rates between these two RTs despite the differences in the substrate used.

After infection by HIV-1 in cell culture, it takes 4-8 h before complete polymerization is observed (Kim et al., 1989). Synthesis of two strands of 9.5 kilobases each must occur. This requires a minimum synthetic rate of 0.65-1.31 nucleotides per s. In this study, we observed a polymerization rate of 3.3 nucleotides per s in the absence of a downstream primer. This is comparable to the polymerization rate observed by Klarmann et al.(1993) and Yu and Goodman(1992) of 1-3 nucleotides per s over heteropolymeric templates. In our system, the polymerization rate in the presence of a downstream primer dropped to 0.28 nucleotide per s.

It is still not known whether the (+)-strand is initiated at one or two sites, or if strand displacement is performed by one RT extending the (-)-DNA or by two RTs extending the(-)- and (+)-strands simultaneously (Fig. 1A). If two RTs extend the(-)- and (+)-strand simultaneously, it would take approximately the same time for one enzyme to synthesize through the whole LTR region (637 nucleotides/0.28 nucleotide/s = 38 min) as for the other enzyme to synthesize the rest of the (+)-strand (9000 nucleotides/3.3 nucleotides/s = 45 min). However, if the (+)-strand is initiated at two or more sites, strand displacement may be a rate-limiting step in the reaction. In either case, the rate of strand displacement that we have observed does not conflict with known information on the rate of viral growth in vivo. Nevertheless, it is remarkably slow compared to the synthesis rates of most DNA polymerases, which are hundreds to thousands of nucleotides per s (Kornberg and Baker, 1992).

We observed that the HIV-RT is less processive for synthesis during strand displacement. The need to frequently rebind the primer-template during synthesis contributes to the length of time needed to complete primer elongation. The RT may also have a particularly difficult time rebinding at sequences that promote pausing and are likely positions for dissociation. Klarmann et al.(1993) observed that HIV-RT preferentially initiates synthesis at unextended primers, even when pause products comprise a significant proportion of potentially available primers. This is consistent with our observation that the entire population of primers that can be elongated grow slowly throughout the reaction.

We attempted to enhance strand displacement by including single-stranded binding proteins in the reaction. Since viral NC protein normally coats part of the viral genome in vivo, we tested whether its presence would enhance the rate of synthesis. We did not see an enhancement of strand displacement synthesis over a wide range of tested NC concentrations. Current results with the LTR template showed an inhibition of DNA synthesis at high NC concentrations. We do not know why this inhibition is observed, but it does not seem to result from melting of the primer from the template by NC. On the other hand, E. coli SSB and human RP-A enhanced the extent of strand displacement in vitro. These proteins presumably act by stabilizing the displaced fragment of DNA. This makes displacement more energetically favorable. Stabilization of the single-stranded region may also discourage the partially displaced strand from promoting dissociation of either the RT or the 3` end region of the upstream primer. Hottiger et al.(1994) did not report a stimulation of the HIV-RT strand displacement reaction catalyzed by E. coli SSB. However, the effects of SSB may not have been great for the 50-nucleotide segment displaced in their system. Even though RP-A enhances strand displacement in vitro, it is unknown whether it would do so in vivo. Immunocytochemistry studies have shown that RP-A is found almost exclusively in the nucleus (Kenny et al., 1990). It is widely accepted that reverse transcription occurs in the cytoplasm (Whitcomb and Hughes, 1992). Furthermore, it has been shown that full-length(-)-strand DNA can be found in the cytoplasm (Miller et al., 1995). These findings suggest that RP-A does not influence strand displacement synthesis in vivo.

In another attempt to enhance the strand displacement reaction, we added an additional segment of single-stranded DNA to the reaction that was complementary to the downstream segment of DNA. The strand displacement/assimilation model suggests that the displaced DNA can actively migrate to a complementary piece of DNA. We expected that this process would stimulate the displacement reaction by stabilizing the displaced strand in a double-stranded form. However, the presence of this DNA strand did not stimulate the strand displacement reaction. Instead, the extending upstream primer readily transferred to the new strand sometime after it had been extended by at least 10 nucleotides. This is an interesting observation since it suggests that recombination is possible during (+)-strand synthesis. The process may be due to complex secondary structures that are formed in the LTR template. Strand transfer in vitro is promoted at positions where the RT pauses during synthesis (Wu et al., 1995), and pausing correlates with secondary structure in the template (DeStefano et al., 1992). The transfer was observed in the presence or absence of a downstream DNA, but it appeared to be more efficient in the presence of a downstream DNA primer. During displacement, when the enzyme pauses or dissociates, the already displaced segment of DNA may reanneal to the template, forcing dissociation of the 3` end region of the upstream primer. This process, called branch migration, is likely to occur between episodes of dissociation and reassociation of the RT that occur frequently during strand displacement synthesis. Once the 3` terminus is dislodged, it would be expected to associate readily with the new complementary DNA. This process is so efficient in vitro, that when the complementary DNA is present in excess, virtually all extending primers undergo transfer. We propose that this reaction is a likely means of recombination in vivo.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM 49573, Minority Predoctoral Fellowship 1F 31 GM 17200-01, Cancer Center Core Grant CA11198, and the James P. Wilmot Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-275-3269; Fax: 716-271-2683.

(^1)
The abbreviations used are: HIV, human immunodeficiency virus; PBS, primer binding site; RT, reverse transcriptase; LTRs, long terminal repeats; PBS, primer binding site; U5 and U3, regions unique to the 5` and 3` end of the LTRs, respectively; R, redundancy common to 5` and 3` ends of the LTRs; NC, nucleocapsid protein; SSB, single-stranded binding protein; RP-A, replication protein A; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. Jasbir Seehra, from the Genetics Institute, for the generous gift of HIV-RT used in these studies. We thank Dr. Malcolm A. Martin for providing us with the plasmid pUC-BS-WT used to generate the templates, and Dr. Marc S. Wold for providing us with purified human RP-A. We also thank Pauline Leakey for technical assistance.


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