3086–3092 Nucleic Acids Research, 1996, Vol. 24, No. 15
 1996 Oxford University Press
Strand transfer is enhanced by mismatched
nucleotides at the 3′ primer terminus: a possible link
between HIV reverse transcriptase fidelity and
recombination
Leyla Diaz and Jeffrey J. DeStefano*
Department of Microbiology, University of Maryland, Building 231, College Park, MD 20742, USA
Received February 15, 1996; Revised and Accepted June 18, 1996
ABSTRACT
Strand transfer catalyzed by HIV reverse transcriptase
(RT) was examined. The system consisted of a 142 nt
RNA (donor) to which a 50 nt DNA primer was
hybridized. The primer bound such that its 3′ terminal
nucleotide hybridized to the 12th nt from the 5′ end of
the donor. The 3′ terminal nucleotide on the primer was
either a G, A or T residue. Since the corresponding
nucleotide of the donor was a C, the G formed a
matched terminus and the A or T a mismatched
terminus. The efficiency with which DNA bound to the
donor transferred to a second RNA, termed acceptor,
was monitored. The acceptor was homologous to the
donor for all but the last 9 nt at the 5′ end of the donor.
Therefore, homologous strand transfer could occur at
any point prior to the DNA being extended into the
nonhomologous region on the donor. Strand transfer
occurred approximately twice as efficiently with the
mismatched versus matched substrates. The
mismatched nucleotide was fixed into transfer
products indicating that excision of the mismatch was
not required for RT extension or transfer. Results
suggest that base misincorporations by RT may
promote recombination by enhancing strand transfer.
INTRODUCTION
The human immunodeficiency virus (HIV) has been shown to
contain a high degree of genetic heterogeneity (1). The fidelity of
HIV-reverse transcriptase (RT) is a major contributing factor in the
generation of diversity (2–8). This multifunctional enzyme
converts the single-stranded RNA viral genome to double-stranded
DNA through a series of steps (for a review see 9). The
RNA-dependent DNA polymerase activity synthesizes the negative strand DNA while the RNase H activity hydrolizes the RNA
template. The DNA-dependent DNA polymerase activity then
synthesizes the positive strand DNA to complete synthesis of the
proviral DNA. Much of the genetic variability is thought to arise
from nucleotide misinsertions. HIV-RT has been found to incorporate more mistakes during replication than reverse transcriptases
* To
whom correspondence should be addressed
from avian myeloblastosis virus (AMV) and Moloney murine
leukemia virus (MuLV) (2). One study performed in vitro
estimated the frequency of base misincorporations for the polymerase at 1 in 6900 on an RNA template and 1 in 5900 on a DNA
template (8). It should be noted that, although all studies suggest
that HIV-RT has relatively low fidelity, error frequency estimates
have varied widely. One study suggests that fidelity is several-fold
higher when RNA versus DNA is used as template (6). Another
study implies that RT fidelity in vivo is considerably greater than
the in vitro estimates (10). As is the case with other retroviruses,
HIV-RT contains no 3′–5′ exonuclease, therefore, errors made by
the enzyme are not proofread (3).
Strand transfer occurs when DNA synthesized on one template
is translocated to another region on the same or a different
template. Two such events, occurring at the terminal regions of
the retroviral genome, are an integral part of retroviral replication.
These are the transfer of the minus and plus strand strong-stop
DNAs (11,12). These DNA are initially synthesized at the 5′ end
of their respective viral templates, then transfer to homologous
regions at the 3′ end of the template. Since the virus contains two
copies of the genomic RNA, the transfers could occur either intraor inter-molecularly. It has also been shown that transfer of the
growing DNA strand can occur at internal regions of the viral
RNA (13–16). Research suggests that pausing of the polymerase
at a specific site may promote transfer of the DNA strand to a
homologous region of a different RNA strand (17–19). A base
misincorporation could provide the conditions necessary for
pausing of the polymerase and could set the stage for a strand
transfer event. Results have shown that even though RT can
extend a mispaired 3′ terminus more efficiently than RTs from
other viruses, the frequency is much lower when compared with
the extension of the correct nucleotide (20–22). Pausing of the
polymerase may allow the RNase H activity of RT to cleave the
RNA template, making the interaction between the primer and the
RNA less stable. This could lead to a subsequent dissociation of
the DNA from the original template and binding to a homologous
region on a different RNA strand. Alternatively, pausing may
allow more time for strand invasion to occur (18). In this scenario
the primer is displaced from the template RNA upon binding to
a second homologous RNA template which ‘invades’ the duplex.
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In this report we demonstrate that strand transfer to a
homologous acceptor template occurs more efficiently in the
presence of a mismatched 3’ termini between the growing DNA
strand and template RNA. The mismatched primers were
extended less efficiently than the primer containing the correct
nucleotide. At the same time, strand transfer from the mismatched
primers was enhanced. The mismatch was retained in the vast
majority of transfer products, thus there was no excision of the
non-complimentary base. These results were found to be
independent of enzyme and acceptor template concentration.
and 80 mM KCl. The mixture was heated 65C for 10 min and
then slowly cooled to room temperature. After hybridization 6×
native gel loading buffer [40% (w/v) sucrose, 0.25% (w/v) xylene
cyanol and bromophenol blue] was added and the mixture was
electrophoresed on a non-denaturing 8% polyacrylamide gel as
described below. The hybrid complex was located by
autoradiography, excised and eluted in a buffer containing 50 mM
Tris–HCl (pH 8.0), 80 mM KCl, 6 mM MgCl2 and 1 mM
dithiothreitol.
MATERIALS AND METHODS
Gel electrophoresis. Denaturing 8% polyacrylamide sequencing
gels (19:1, acrylamide–bisacrylamide) containing 7 M urea or
non-denaturing native gels (29:1, acrylamide–bisacrylamide)
were prepared and subjected to electrophoresis as described (23).
Materials
Recombinant HIV-RT with native primary structure was
graciously provided by Genetics Institute (Cambridge, MA). The
enzyme had a specific activity of ∼40 U/µg. One unit of RT is
defined as the amount required to incorporate 1 nmol dTTP into
nucleic acid product in 10 min at 37C using poly(rA)–oligo(dT)
as a template–primer. As we have previously reported, the
enzyme preparations contained very low levels of single strand
nuclease activity. We found that this activity could be inhibited by
including 5 mM AMP in the assays (18). The AMP, at this
concentration, did not affect the polymerase or RNase H activity
of the RT (data not shown). Aliquots of HIV-RT were stored
frozen at –70C and a fresh aliquot was used for each experiment.
T4 Ligase, T4 polynucleotide kinase and Sequenase version 2.0
were obtained from United States Biochemical Corp. Klenow
fragment, restriction enzymes, T7 RNA polymerase rNTPs and
dNTPs were obtained from Boehringer Mannheim Biochemicals.
Superscript was obtained from Gibco BRL. The oligonucleotide
DNA primers were synthesized by Genosys Inc. All other
chemicals were from Sigma. Radiolabeled compounds were from
New England Nuclear.
Methods
Standard strand transfer and primer extension reactions. In the
standard reaction primer–donor template (2 nM) and acceptor
template (20 nM, unless otherwise indicated) were preincubated
for 3 min in a volume of 10.5 µl at 37C. Acceptor was omitted
in primer extension assays designed to measure donor-directed
extension only. Reactions were initiated by the addition of 2 U
(∼35 nM final concentration) of HIV-RT in 2 µl of 50 mM
Tris–HCl (pH 8.0), 1 mM dithiothreitol and 80 mM KCl. The
final concentrations of reactions components were 50 mM
Tris–HCl (pH 8.0), 5 mM AMP, 6 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM EDTA (pH 8.0), 100 µM dNTPs and 80
mM KCl. Samples were incubated for 32 min at 37C unless
otherwise indicated, and reactions were terminated by addition of
12.5 µl of gel loading buffer (90% formamide, 10 mM EDTA pH
8.0, 0.1% xylene cyanol, 0.1% bromophenol blue).
Hybridizations. The 50 nt deoxyoligonucleotides for the matched
and mismatched substrates (see Fig. 1) were labeled with 32P at
the 5′ end using T4 polynucleotide kinase. The labeled primer
was hybridized such that the 3′ end terminal nucleotide was
positioned opposite the 12th nt from the 5′ end of the RNA
transcript. The hybrids were prepared by mixing primer and
transcript at a 4:1 ratio in 50 mM Tris–HCl, 1 mM dithiothreitol
Transcription reactions. Run-off transcription was performed as
described (24). For the donor template pBSM13∆, prepared as
described previously (17), was cleaved with MvaI and T7 RNA
polymerase was used to prepare RNA transcripts 142 nt in length.
For the acceptor template, pBSM13+ was cleaved with MvaI and
T7 RNA polymerase was used to prepare a transcript 189 nt in
length. RNA for both the donor and acceptor templates was gel
purified on denaturing polyacrylamide gels. The electrophoresed
RNA was located on the gel by ultraviolet shadowing, excized,
and eluted in a buffer containing 150 mM NaCl, 50 mM Tris (pH
8.0), 1 mM EDTA and 0.1% SDS. The RNA was recovered from
the eluate as previously described (25).
Isolation of transfer products. The strand transfer reactions were
subjected to electrophoresis on an 8% denaturing polyacrylamide
gel. The strand transfer products were located by autoradiography, excised from the gel, and recovered as described above.
The recovered DNA was amplified by PCR for 30 cycles as
described (26). The reactions were carried out in buffer containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2 and 50
mM dNTPs. One primer, (5′-GGGCGAATTCGAGCTCGGTACCCGGGGATC-3′) was complimentary to nt 78–108 on the
transfer products while the other (5′-TACGCCAAGCTCGGAATTAA-3′) was identical to nt 8–28 on the 5′ end of the
products. The PCR reactions were then subjected to
electrophoresis on a non-denaturing polyacrylamide gel.
Products were located using ultra violet light after staining the
gels with ethidium bromide (23). The products were excised and
eluted in 150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 0.1% SDS
and recovered by precipitation in ethanol. The PCR products were
then treated with the Klenow fragment in the presence of 100 µM
dNTPs. These blunt-ended products were ligated into pBCSK
previously cleaved with EcoRV. The ligation mixture was
transformed into Escherichia coli XL-1 Blue competent cells.
Clones containing the insert were located by blue–white color
selection and sequenced using Sequenase version 2.0 according
to the manufacturer’s instructions. The primer used for sequencing (5′-TAATACGACTCACTATAGGG-3′) was complimentary
to the T7 promoter on the plasmid.
Quantification of nucleic acids. Donor and acceptor templates
and DNA primers were quantitated spectrophotometrically by
measuring absorbance. Quantification of transfer and primer
extension products was accomplished by scanning the dried
polyacrylamide gel with a phosphoimager (BioRad GS525). The
3088 Nucleic Acids Research, 1996, Vol. 24, No. 15
Figure 1. Configuration of substrates. (A) The general configuration of the substrate used to analyze strand transfer is shown. The system consisted of a heteroduplex
in which a 142 nt RNA (donor) was hybridized to a 50 nt DNA primer such that the 3′ terminal nucleotide of the DNA bound to the 12th nucleotide from the 5′ end
of the RNA. A second RNA (acceptor), which was 189 nt in length (acceptor), was identical to the donor RNA for the first 133 nt from the 3′ end of the RNAs. Primers
initially bound to the donor could undergo homologous strand transfer to the acceptor within the region termed ‘Transfer Zone’. Numbers refer to the lengths of primer
extension products elongated to the indicated positions on the RNAs. (B) The nucleotide sequence of the donor template RNA and primer DNAs downstream of and
including the primer binding region is shown. Note that three primers were used, one with a matched (G residue) and the others mismatched (A or T residue) 3′ terminus.
amount of labeled heteroduplex substrate recovered from native
gels was determined by specific activity.
RESULTS
Construction of substrates for testing HIV-RT-catalyzed
strand transfer
The strand transfer system used in these experiments is shown in
Figure 1A. The system consisted of a 142 nt RNA strand to which
a 50 nt 5′-32P-labeled DNA primer was hybridized. This RNA is
referred to as the ‘donor’ template, which is the template on
which DNA synthesis initiates. In order to observe strand transfer,
a second RNA template termed ‘acceptor’ was employed. The
acceptor was the template to which DNAs initially hybridized to
the donor would transfer. The acceptor was 189 nt in length and
was homologous to the donor for all but the last 9 nt at the 5′ end
of the donor. Full-length donor-directed DNA extension products
were 61 nt in length while DNAs which transferred to, and were
subsequently fully extended on the acceptor were 108 nt long.
This difference in length allowed us to easily distinguish transfer
events from donor-directed extension using denaturing
polyacrylamide gels (see Methods). Primer DNAs extended to
the end of the donor could not undergo homologous strand
transfer and subsequent extension on the acceptor since the last
9 nt of these DNAs would be mismatched on the acceptor
template (see above). Although nonhomologous recombination
could potentially occur, this type of recombination is rare
occurring at 1/100–1/1000 the frequency of homologous recombination (27). Consequently, we would expect that most of the
observed transfer events resulted from transfer of DNAs which
transferred before being extended into the region of the donor that
was not homologous to the acceptor.
Three different 50 nt DNA primers were used in our
experiments (see Fig. 1B). The DNAs were identical in sequence
for the first 49 nt from the 5′ end. The 3′ terminal nucleotide (50th
from the 5′ end) was a G in the case of the ‘matched’ substrate,
and an A or T in the ‘mismatch’. The matched substrate was
completely complementary to the RNA while in the mismatches,
Figure 2. Analysis of isolated primer DNA–donor RNA hybrid. Shown is an
autoradiogram of an experiment run on a native polyacrylamide gel. Samples
contained primer DNA and/or previously isolated (see Methods) heteroduplex
hybrid as indicated. Nucleic acids were labeled at the 5′ end of the DNA with
32P. In some cases samples were incubated at 37C for 30 min or 100C for 5
min prior to electrophoresis. Final reagent concentrations in the incubations
were the same as those in strand transfer assays.
the 3′ terminal A or T residue of the DNA was opposite a C on
the RNA strand. Note that the last five residues at the 3′ end of the
DNA in the A mismatch substrate were As. Frameshift mutations
(±1) within homopolymeric runs of As and Us (Ts on DNA
template) are among the most common error observed during
HIV-RT-directed DNA synthesis on RNA (6) or DNA (28)
templates. Such mutations are likely generated by slippage of the
primer–template within the region of the run (29).
In these experiments, our objective was to quantitate RT-catalyzed strand transfer events. That is, the release of the DNA
primer from the donor RNA and its subsequent association with
the acceptor. The DNA oligonucleotides used to prime the donor
RNA were complementary to both the donor and acceptor
templates. Therefore, any DNAs that were not associated with the
donor (free single-stranded DNA) at the start of the reactions could
hybridize with the acceptor without being transferred. Thus, for
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accurate determinations, it was essential that nearly all of the DNAs
were initially bound to the donor RNA. We isolated hybrid
DNA–donor RNA substrates on native polyacrylamide gels and
processed them such that the DNA remained associated with the
donor RNA under all experimental conditions (shown in Fig. 2). The
far left lane in Figure 2 shows a sample with free unhybridized DNA
primer from the matched substrate. This DNA migrated to the same
position on the native gel after being incubated at 37C for 30 min
under reaction conditions (2nd lane from left). The isolated hybrid
substrate (4th from left) migrated as three distinct bands with the vast
majority of the material running just above the primer DNA. The
hybrid was dissociated after 5 min at 100C generating free primer
(far right lane). The mismatched substrates and primers gave similar
results (data not shown).
The presence of a 3′ terminal mismatch increases the
efficiency of strand transfer
In these experiments strand transfer was expressed as a ‘percent
efficiency’. The transfer efficiency was defined as the amount of
transfer products (T) divided by the amount of full-length
donor-directed (F) plus transfer products times 100 [(T/F + T) ×
100]. The number reflects the proportion of DNA primers
extended to the end of the acceptor versus those extended to the
end of the donor. This representation of the data, as opposed to
simply determining the gross level of transfer products, expresses
transfer relative to total DNA extension. Therefore, differences in
the total amount of primers extended with the match versus
mismatch substrate are compensated for.
Figure 3A is an autoradiogram of a typical strand transfer
experiment using the matched or A mismatched substrate. In the
standard assay 2 nM substrate and 20 nM acceptor template were
used. Under these conditions, transfer products were detected
∼4–8 min into the reactions and increased thereafter. At the final
data point (32 min) the gross level of transfer products with the
matched substrate was about half that with the mismatch. Figure
3B shows a composite graph for six independent experiments of
the efficiency of transfer versus time using the A mismatch or
matched substrate. Between 8 and 32 min the transfer efficiency
was two to three times as great with the mismatched substrate,
reaching ∼17% as opposed to 7% with the match. Between 32 and
64 min only a slight increase in transfer efficiency was observed
(data not shown).
We also studied strand transfer with a substrate that had a 3′
terminal T:C mismatch. Results with this substrate (T mismatch) and
the matched substrate are shown in Figure 3C. Once again the
transfer efficiency was significantly greater with the mismatched
substrate. Transfer was somewhat more efficient with the A versus
T mismatch, however, the same general trend was observed.
Figure 3. (A) Strand transfer with the matched and A mismatched hybrid
substrates. Shown is an autoradiogram of a standard strand transfer assay
performed as described under Methods. Reactions were with the matched or A
mismatched substrate for the indicated time in the presence or absence of
acceptor template (as indicated). The positions of the 50 nt DNA primer,
full-length donor template-directed extension products (F) and transfer
products (T) are indicated. (B and C) Strand transfer with the matched and A
(B) or T (C) mismatched hybrid substrates. Shown are plots of the efficiency
of strand transfer (see Results) versus time for the matched or mismatched
substrates. Six separate experiments of the type shown in (A) were used to
construct the plot for (B) while two experiments were used to construct (C).
Error bars span the standard deviations at the particular time points.
3090 Nucleic Acids Research, 1996, Vol. 24, No. 15
varied little (summarized in Table 1). The efficiency of transfer
decreased when the ratio of acceptor to substrate was decreased
below10:1 (Fig. 4). This is consistent with previous results
addressing the efficiency of transfer versus acceptor concentration (17,30). However, at any given acceptor concentration, the
ratio of the efficiency with the mismatch versus the match was
constant at ∼3:1. The data suggest that the observed differences
in transfer efficiency between the matched and mismatched
substrates resulted from the structure of the substrates and not a
more complex interaction involving the substrate and enzyme
and/or acceptor.
Analysis of the nucleotide sequence of transfer products
Figure 4. Efficiency of strand transfer at various acceptor concentrations using
the matched and A mismatched substrates. Shown is a plot of the efficiency of
strand transfer versus the ratio of acceptor template to hybrid substrate. Strand
transfer reactions (see Methods) were with the matched or A:C mismatched
substrates for 16 min in the presence of various amounts of acceptor template
(x-axis). The concentration of hybrid substrate was held constant at 2 nM.
Efficiencies were calculated from the levels of transfer and full-length
donor-directed products as described under Results. The levels of these
products were determined from phosphoimager analysis of gels of the type
shown in Figure 3A.
Table 1. Strand transfer with different amounts of enzyme
Enzyme units
Transfer product (fmol)
Transfer efficiency (%)
match
mismatch
match
mismatch
0.5
0.15
0.39
1.0
6.8
1
0.15
0.52
1.1
6.6
2
0.19
0.91
1.2
8.9
4
0.25
0.74
1.5
6.5
Reactions were performed with the matched or A mismatched hybrid substrate
as described under Methods. Incubations with RT were for 16 min. Samples
were electrophoresed on a denaturing polyacrylamide gel and the levels of
transfer and full length donor-directed products (see Fig. 3) were determined
using a phosphoimager. These values were used to calculate the transfer efficiency as described under Results.
The increased transfer efficiency of the mismatch was
independent of enzyme and acceptor concentrations
The above results suggest that a mismatched primer–template
enhances strand transfer. If this enhancement is solely a function
of the primer–template structure, then it should be qualitatively
independent of the concentration of enzyme and acceptor. To test
this we performed strand transfer assays with the A mismatch and
matched substrates for 16 min using various amounts of enzyme
and acceptor. Varying the amount of enzyme between 0.5 and 4 U
affected the level of transfer products with products increasing as
the level of enzyme increased. However, the efficiency of transfer
Since the conclusions drawn from these experiments are based on
the presence of a mismatched terminus in one of the templates, it
was important to show that the mismatch was incorporated into
transfer products. Although HIV-RT can extend mismatched
primer templates (20,22,31), it was possible that extension on the
donor or acceptor templates occurred only after the mismatched
nucleotide was excised. Excision could occur due to a low level
intrinsic exonuclease activity possessed by HIV-RT, or contamination of the RT enzyme preparation with nucleases. The
former is unlikely since the level of 3′–5′ exonuclease activity in
HIV-RT is probably very low (3). Under the conditions employed
in these experiments the preparation of HIV-RT used showed very
low nuclease activity (see Materials). Therefore, we would expect
that mismatches would be fixed into extension products. To test
this, we used PCR to amplify transfer products produced using the
A mismatched substrate (see Methods). Products from PCR were
inserted into a vector and used to transform E.coli. The DNA from
20 separate clones was sequenced and the results are summarized
in Table 2. Three separate classes of transfer products were found.
Eighteen of the 20 had retained the A:C mismatch at the
mismatched substrate primer terminus (fixed and +1 frameshifts)
and of these, three had an additional insertion of a G following the
run of As (+1 frameshift). Such an insertion may occur by a
primer–slippage mechanism as described by others (29). Two of
the 20 sequenced cloned had lost the terminal mismatched A
residue (excised), presumably due to exonuclease activity.
Whether the exonuclease activity was a contaminate or intrinsic
RT activity is not known. However, these results indicate that the
vast majority of transfer products produced with the mismatched
substrate were extended without excision of the 3′ terminal
mismatch.
The kinetics of nucleotide incorporation are reduced
with the mismatched substrate
It has been shown that HIV-RT can extend mismatched primer–
templates (20,22,31). The efficiency of extension relative to a
correctly matched primer–template varied greatly depending on
the nature of the mismatch (i.e. G:U, C:U, G:T, A:C etc.). Some
mismatches were extended only ∼40-fold less efficiently (based
on the ratio of Vmax/Km for matched versus mismatched
substrates), while in some cases extension of the mismatch was
several thousand times less efficient. In general, differences in
efficiency resulted from large variations in the Km value (the
denominator in the above equation) for addition of the next
correctly base-paired nucleotide. These values were often 2–3
orders of magnitude greater for the mismatched versus matched
primer–templates. Thus, the rate of mismatch extension often
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Table 2. Results from DNA sequencing of transfer products generated
from the A mismatched substrate
Strand transfer products were isolated from denaturing polyacrylamide
gels, amplified by PCR, and cloned and sequenced as described under
Methods. Refer to the Results section for an explanation of the ‘Mismatch fate’. In the heteroduplex structure the primer DNA is shown
above the acceptor template RNA. For the ‘fixed’ and ‘+1 frameshifts’
the first 5 nt shown on the DNA strand correspond to the 5 nt at the 3′
end of the primer. In the ‘excised’ structure the 3′ terminal A residue was
removed from the DNA primer before extension.
varies over a wider range of dNTP concentration than extension
of a correctly paired substrate.
We measured extension of the primer DNA on the matched or
mismatched substrates used in our experiments. Assays were
performed in the absence of acceptor template employing the
conditions used in the strand transfer reaction (100 µM dNTPs).
A graph of a representative experiment is shown in Figure 5.
Results are expressed as % primers extended versus time. The
matched substrate was rapidly extended with over 25% of the
total primers elongated within the first 30 s of the reaction. Only
∼8% of the mismatched substrates were extended over the same
period. Extension with the matched and mismatched substrates
had essentially reached a maximum level by 8 min. In the
experiment shown, ∼60 and 40% of the total primers were
extended with the matched and mismatched substrates respectively. Although the proportion of extended primers varied
somewhat from experiment to experiment, the extension kinetics
for the mismatches were generally similar and always significantly slower than the matched substrate The results indicate that,
under the conditions used in these experiments, primer extension
was substantially delayed on the mismatched substrates.
Superscript, a commercially available form of MuLV-RT
lacking RNase H activity, extended ∼90% of the primers (data not
shown). The fact that only a portion of the total primers were
extended even on the matched substrate may suggest that HIV-RT
has difficulty initiating DNA synthesis on this particular substrate. Therefore, RT RNase H activity may cause a significant
portion of the primers to dissociate from the template prior to
extension (18). This would explain why the MuLV enzyme
extended a higher proportion of primers.
We also performed strand transfer experiments on the A
mismatched substrate at various nucleotide concentrations (data
not shown). We found that the efficiency of tranfer with both the
matched and mismatched substrates increased as the concentration of nucleotides decreased from 200 to 10 µM dNTPs.
However, the increase was modest, with transfer efficiency ∼1/3
greater at 10 versus 200 µM. We were unable to make
Figure 5. Primer extension on the matched and mismatched substrates. Shown
is a plot of the % of the total DNA primers in each reaction that were extended
on the matched and A or T mismatched hybrid substrates versus time. Primer
extension assays were performed in the absence of acceptor template as
described under Methods. The graph is from a single experiment. Similar
results were obtained in other experiments.
comparisons <10 µM since the extension of the mismatch was
very low below this point.
DISCUSSION
We have shown that the presence of a mismatched nucleotide at
a 3′ primer terminus enhances strand transfer from the mismatched substrate to a homologous acceptor template. Given the
high misincorporation rate of HIV-RT, and the lack of significant
proofreading activity it is likely that mismatched termini occur
with relative frequency during RNA-directed DNA synthesis (see
Introduction). This report suggests that such events may generate
genetic diversity not only through the possible fixation of
nonparental nucleotides, but also by stimulating strand transfer
events.
In these experiments we examined the effect on strand transfer
of two types of mismatch. One of these mismatches (A:C) would
result from a primer–slippage-type mechanism occurring within
runs of the same nucleotide (29). The insertion within the primer
strand of an additional nucleotide complementary to the nucleotides within the run on the template strand is among the most
common errors occurring during HIV-RT-directed DNA synthesis (6,29). A mismatched C:A primer–template was extended
∼1300 times less efficiently than a matched substrate by HIV-RT
(20). Although in those experiments, unlike ours, the C was on the
primer strand and A on the template strand. AMV-RT extended
an A:C primer–template mismatch ∼300 times less efficiently
(based on Vmax/Km) than the matched G:C pair (21). Since the
kinetics of extension may vary depending on the surrounding
nucleotide sequence, the particular RT used, and the orientation
of the mismatch, it is not possible to draw a direct correlation
between the AMV-RT work or previous HIV-RT work and our
substrates. It was clear from our experiments (Fig. 4) that
extension of the mismatched template was less efficient than the
3092 Nucleic Acids Research, 1996, Vol. 24, No. 15
match, but a quantitative evaluation of efficiency was not done.
We also evaluated the effect of a T:C mismatch on strand transfer.
Since the efficiency of extension under the conditions employed
in our experiments was approximately the same for the A:C and
T:C mismatches, it was not possible to determine if increased
transfer efficiency correlates with a decrease in extension
efficiency. The similarity between the extension efficiencies of
the two mismatches may result from the concentration of dNTPs
used in our experiments which are well above RTs Km for
nucleotides. Since the efficiency of mismatch extension as
assessed by Vmax/Km indicates that the lower efficiency of
mismatch extension is due mostly to elevated Km values (see
Results), differences in extension kinetics would be less pronounced at high dNTP concentrations. We were unable to
evaluate transfer with mismatched substrates at very low
nucleotide concentrations due to the low level of extended primer
under these conditions.
We found that the A:C mismatch was fixed into transfer
products by two different mechanisms. Of the 18 sequenced
transfer products in which the mismatch was fixed, 15 resulted
from extension directed from the next nucleotide (G) downstream
of the mismatched C on the template strand. This mechanism
results in ‘in frame’ DNA products with a G to A substitution on
the primer strand. The other three fixed transfer products were
DNAs in which extension was directed from the mismatched C
residue on the template. This mechanism produces insertion
‘mutants’ with +1 frameshifts. Frameshift mutants in open
reading frames are more likely to produce nonfunctional proteins
than are nucleotide substitutions. It is noteworthy that substituted
DNA products were produced with much greater frequency (15
as compared with 3) than frameshifts. It would also be interesting
to know whether the type and proportion of mutations produced
during strand transfer are different from those generated during
extension of the donor.
The increase in transfer efficiency with the mismatch versus
matched templates was modest (2–3-fold), but clearly significant
(see Fig. 2). With the two mismatched templates >10% of the
extended primers had transferred to the acceptor template by the
32 min time point. This proportion seems unrealistically high
since estimates for retroviral strand transfer in vivo are on the
order of 1 event per 25 000 nt copied (16). Some possible reasons
for the high in vitro rate include the small sizes of the acceptor and
donor templates and the high ratio of acceptor to donor in our
reactions. The former explanation would lead to more efficient
transfer since it would be easier for regions of homology to align
appropriately on a very small template. The considerably greater
length of the normal retroviral genome probably makes alignment
more difficult. The latter explanation is clearly illustrated in
Figure 4. The efficiency of transfer decreased as the ratio of
acceptor to donor decreased. Although our in vitro system does
not represent in vivo recombination in a quantitative sense, it is
likely that there are mechanistic similarities.
We did not attempt to elucidate the mechanism by which the
mismatched terminus enhanced strand transfer. However, one
possibility, consistent with the reduced extension of the mismatched versus matched substrates (Fig. 5), is that the mismatch
promotes pausing of the polymerase at the primer terminus. It has
been shown that pausing during RNA-directed DNA synthesis
can promote strand transfer (17,18). Pausing allows greater time
for the RT RNase H activity to cleave the RNA beneath the
terminal region of the DNA primer. Extensive cleavage may
weaken the interaction between the DNA and RNA allowing for
invasion of the heteroduplex by a homologous acceptor RNA, or
dissociation and subsequent binding of the DNA strand to a
complementary acceptor (18). Further experiments will be
necessary to elucidate the mechanism by which mismatches can
enhance strand transfer and to confirm this phenomena in vivo.
ACKNOWLEDGEMENTS
We wish to thank Drs Jasbir Seehra and John McCoy, representing Genetics Institute, for the generous gift of HIV-RT. This work
was supported by NIH grant GM-51140-01.
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