From the Department of Microbiology, University of Maryland, College Park, Maryland 20742
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ABSTRACT |
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A system to study the fidelity of internal strand
transfer events was constructed. A donor RNA, on which reverse
transcriptase (RT)-directed DNA synthesis was initiated, shared
homology with an acceptor RNA, to which DNAs initiated on the donor
could transfer. The homology occurred over a 119-base internal region
of the donor which coded for the N-terminal portion of the
-lac gene. Polymerase chain reaction (PCR) was used to
amplify DNA synthesis products. The PCR products were then digested
with PvuII and EcoRI and ligated into a vector
which had this same region excised. Transformed Escherichia
coli were screened for the ability to produce a functional
-galactosidase protein by blue-white phenotype analysis with white
colonies scored as those with errors in
-lac. Products synthesized on the donor were used to assess the error rate of human
immunodeficiency virus-RT while products transferring to and
subsequently extended on the acceptor (transfer products) were used to
monitor transfer fidelity. Human immunodeficiency virus-RT made
approximately 1 error per 7500 bases copied in the assay. Nucleocapsid
protein (NCp), although stimulating strand transfer 3-fold, had no
effect on RT fidelity. Transfer products in the absence of NCp had
essentially the same amount of errors as donor-directed products while
those produced with NCp showed a slight increase in error frequency.
Overall, strand transfer events on this template were highly accurate.
Since experiments with other templates have suggested that transfer is
error prone, the fidelity of strand transfer may be highly sequence
dependent.
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INTRODUCTION |
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The human immunodeficiency virus (HIV)1 has been shown to contain a high degree of genetic heterogeneity (1). The high error rate of HIV reverse transcriptase (RT) (2-8), and recombination between viral genomes (for reviews, see Refs. 9 and 10) are major contributing factors in the generation of diversity. Retroviruses are single-stranded plus sense RNA viruses which go through a double-stranded DNA intermediate during their life cycle (11). The virion contains two copies of genomic RNA which are not necessarily 100% homologous. Recombination, occurring by a process referred to as strand transfer (also strand switching, template switching, or template jumping), can result in the integration of genetic information from both genomic copies. The resulting provirus codes for new genomic RNA that is a chimera of the parental genomes and, therefore, not identical to either.
Strand transfer occurs when DNA synthesized on one template is
translocated to another region on the same or a different template. Two
such events, the transfer of minus and plus strand strong-stop DNAs,
are an integral part of retroviral replication (for a review, see Ref.
12). These DNAs are initially synthesized at the 5 ends of the viral
RNA (
strong stop) or minus strand DNA (+ strong stop). Subsequently,
each is transferred to a homologous region located at the 3
end of the
respective templates. As a consequence of the diploid nature of
retroviruses, strand transfer from internal regions of the RNA genome
can also occur during the synthesis of minus strand DNA. Such
recombinational events are proposed to occur by a "forced copy
choice" mechanism (13, 14). The forced copy choice model postulates
that some viral genomic RNAs are truncated and, therefore, unable to
produce a completed copy of minus strand DNA. When DNA synthesis
reaches the end of the truncated RNA, the nascent DNA is "forced"
to switch to the homologous region of the second copy of genomic RNA
within the diploid virion to complete minus strand synthesis. Others
have shown that recombination of this type may not require broken RNAs
(15). In such a situation, the template switch may not always be
forced. A slightly modified version of this model termed "copy
choice" could describe all recombinations occurring during minus
strand DNA synthesis. Note also that recombination may occur during the
synthesis of plus strand DNA as proposed in the "strand
displacement-assimilation model" (16, 17). Although reports have
suggested that plus strand recombination may occur (18), other results
have indicated that recombination during the synthesis of minus strand
DNA is the predominant pathway (9, 19).
Internal strand transfer has been shown to occur both in
vivo (20-22) and in vitro (23, 24). Models for strand
transfer have been proposed although specific details of the mechanism remain unclear (10, 24, 25). It has been suggested that strand transfer
may be error prone (26, 27). This is based on the finding that HIV-RT
tends to incorporate additional non-template-directed bases at the 3
end of a growing DNA strand after reaching the 5
terminus of the
template (26-28). Such events could be particularly relevant to strong
stop transfers or forced copy choice type transfers, all of which can
happen at template termini. It has also been shown, using an in
vitro system designed to test the fidelity of internal transfer
within a hypervariable region of the nef gene, that strand
transfer was more error prone than RNA-directed DNA synthesis (29). In
contrast, results using an in vivo system based on Moloney
murine leukemia virus suggested that strand transfer was highly
accurate (30). We investigated the fidelity of strand transfer using a
system in which transfer occurred between RNAs containing homologous
sequences from the
region of
-galactosidase. Reverse
transcriptase-derived DNA products were PCR-amplified and tested in an
-complementation assay. Errors occurring during RNA-directed DNA
synthesis or strand transfer were scored based on the inability of
mutated products to complement
-galactosidase activity, resulting in
white rather than blue colonies in the assay. Results showed that
strand transfer and RNA-directed DNA synthesis were of approximately
equal fidelity. The presence of viral nucleocapsid protein (NCp)
enhanced strand transfer but had little effect on fidelity. This
suggests that transfer on this substrate is a highly accurate process.
Taken together with results from others, our work implies that the
fidelity of strand transfer may be highly sequence dependent.
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EXPERIMENTAL PROCEDURES |
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Materials
Recombinant HIV-RT, having properties described (31), was
graciously provided to us by Genetics Institute (Cambridge, MA). This
enzyme had a specific activity of approximately 40,000 units/mg (1 unit
of RT is defined as the amount required to incorporate 1 nmol of dTTP
into nucleic acid product in 10 min at 37 °C using oligo(dT)-poly(rA) as primer-template). As we have previously reported,
the enzyme preparations contained very low levels of single strand
nuclease activity (24). We found that this activity could be inhibited
by including 5 mM AMP in the assays. 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
70 °C and a fresh aliquot was used for each experiment. HIV-1 NCp
was from Enzyco (Denver, Colorado). This 55-amino acid protein
corresponded to the p7 portion of HIV-1MN (32). RNase
(DNase-free), T3 RNA polymerase, calf intestinal phosphatase, rNTPs,
and dNTPs were obtained from Boehringer Mannheim Biochemicals. T4
polynucleotide kinase, Sequenase, Klenow polymerase, and T4 DNA ligase
were from U. S. Biochemical Corp. Taq polymerase and
placental RNase inhibitor were from Promega. Restriction enzymes were
from Boehringer Mannheim, New England Biolabs, or Life Technologies. Oligonucleotides were synthesized by Genosys Inc. All other chemicals were from Fisher Scientific or Sigma. Radiolabeled compounds were from
NEN Life Science Products.
Methods
Strand Transfer and DNA Synthesis Reactions with
HIV-RT--
Donor template RNA (2 nM, see Fig.
2B), was hybridized to 5 32P-labeled primer DNA
as described below. This template-primer was preincubated for 5 min at
37 °C in the presence or absence (as indicated) of 10 nM
RNA acceptor template and presence or absence of NCp (2 µM final concentration), in 21 µl of buffer (see
below). Four µl of HIV-RT (10 units (approximately 85 nM final concentration)) in 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 80 mM KCl (buffer A) was
added to initiate the reactions. Reactions included the following
reagents at the indicated final concentrations: 50 mM
Tris-HCl (pH 8), 80 mM KCl, 6 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
EDTA (pH 8), 5 mM AMP, 100 µM
ZnCl2, 100 µM dNTPs, and 0.4 units/µl RNase
inhibitor. Reactions were incubated for 40 min at 37 °C and stopped
by addition of 25 µl of 2 × formamide dye (90% formamide, 10 mM EDTA (pH 8.0), 0.1% xylene cyanol, 0.1% bromphenol
blue) containing 0.5 µg of RNase (DNase free). Samples were heated to
65 °C for 10 min to digest the RNA and then for 2 min at 90 °C.
Samples were electrophoresed on 8% denaturing polyacrylamide gels as
described below. Wet gels were exposed to film and transfer or
full-length donor-directed product were excised and eluted (24). Some
gels were dried and used for product quantitation by phosphoimagery
using a Bio-Rad GS-525 PhosphorImager.
RNA-directed DNA Synthesis with the Klenow Fragment of DNA Polymerase I-- These assays were performed essentially as described above with the following changes: 1) 5 units of Klenow were used in place of RT; 2) the final [KCl] was 10 mM; 3) AMP was excluded from the reactions.
RNA-DNA Hybridization--
For strand transfer reactions the DNA
primer was hybridized to donor RNA by mixing primer:RNA transcript at
approximately a 5:1 ratio of 3 termini in buffer A. The mixture was
heated to 65 °C for 5 min then slow cooled to room temperature.
Gel Electrophoresis-- Denaturing 8% polyacrylamide sequencing gels (19:1 acrylamide:bis-acrylamide), containing 7 M urea, native 8% polyacrylamide gels (29:1 acrylamide:bis-acrylamide), and 1% agarose gels were prepared and subjected to electrophoresis as described (33).
Preparation of RNAs--
Run-off transcription was done as
described in the Promega Protocols and Applications Guide (1989). For
the donor template, plasmid pBSMCS, prepared as described below, was
cleaved with BglI and T3 RNA polymerase was used to prepare
run-off RNA transcripts approximately 189 nucleotides in length. For
the acceptor template, plasmid pBS
PvuII1146
was cleaved with PvuII and T3 RNA polymerase was used to
prepare run-off transcripts approximately 179 nucleotides in length.
The transcription reactions were extracted with
phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with
ethanol. The RNA was gel-purified on denaturing polyacrylamide gels,
located by ultraviolet shadowing, and recovered as described previously
(24). The amount of recovered RNA was determined spectrophotometrically
from optical density.
Polymerase Chain Reaction (PCR) and DNA Sequencing--
Strand
transfer products from reactions performed with acceptor template, or
full-length donor-directed products from reactions performed without
acceptor, were excised and eluted from denaturing gels as described
above. The eluted DNA was amplified by PCR using the following
primers: 5-CCTCTTCGCTATTACGCCAG-3
and
5
-GCTCGAATTCGCCCTATAGTGAGTC-3
. The first is identical to the
primer used to prime the donor RNA while the second overlaps the
EcoRI site on the donor and acceptor (see Fig.
2B). Reactions were performed in 100 µl of 10 mM Tris-HCl (pH 9 at 25 °C), 50 mM KCl, 2 mM MgCl2, 0.1% Triton X-100, 200 µM dNTP, 100 pM of each primer, and 5 units
of Taq polymerase. Thirty-five cycles of 94 °C (1 min),
50 °C (1 min), then 72 °C (1 min) were performed. Products were
extracted, precipitated, and digested as described in Fig.
2B. Fifty units of EcoRI per PCR reaction was
used and after 1 h, 2.5 units of calf intestinal phosphatase was
added and incubation was continued for an additional hour. The calf
intestinal phosphatase was inactivated according to the manufacturer's
instructions and the DNA was recovered by precipitation. Thirty units
of PvuII for 1 h was used to digest each reaction. The
samples were then electrophoresed on an 8% native polyacrylamide gel
as described above. Products were located by UV shadowing and recovered
as described above, and quantitated by spectrophotometry. These
products were ligated into vector pBS
PvuII1146 which had been previously
cleaved with PvuII and dephosphorylated with calf intestinal
phosphatase, then cleaved with EcoRI. The linear vector was
recovered from a 1% agarose gel with a Qiagen gel extraction column.
The vector (50 ng) and insert (0.05 pm) were ligated for 16 h at
16 °C in a volume of 10 µl. Two µl of the ligation was used to
transform Escherichia coli XL-1 Blue competent cells
(Stratagene) according to the manufacturer's protocol. Cells were
plated in the presence of
5-bromo-4-chloro-3-indolyl-
-D-galactoside and
isopropyl-
-D-thiogalactopyranoside. Colonies were
analyzed and scored as blue or faint blue or white. For DNA sequencing, mini-preps were prepared from white or faint blue colonies and the DNA
was sequenced using Sequenase (U. S. Biochemical Corp.) according
to the manufacturer's protocol. The sequencing primer was
5
-GGAAACAGCTATGACCATGA-3
.
Construction of Plasmids pBSMCS and
pBS
PvuII1146--
Both plasmids were derived from
pBSM13+ (Stratagene Inc.). Plasmid pBS
MCS (see Fig. 2A)
was made by cleaving pBSM13+ with EcoRI (position 881) and
HindIII (position 932) and filling in both ends with Klenow.
After ligation with T4 DNA ligase a transformant containing
recircularized plasmid which had lost the portion of the multiple
cloning site (MCS) between the EcoRI and HindIII (confirmed by sequencing) was expanded and the plasmid was isolated using a Qiagen Mega-Prep kit. Plasmid
pBS
PvuII1146 was produced through multiple
steps as described in Fig. 1. This
plasmid has a 7-base pair (1149-1155 of pBSM13+) deletion that
disrupts the PvuII site at position 1146, leaving a single
site for this enzyme at 764.
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RESULTS |
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System Used to Study Strand Transfer--
The templates used to
study strand transfer and the plasmids used to make those templates are
shown in Fig. 2. The donor RNA (produced from pBSMCS (Fig. 2A), on which RT initiates
DNA synthesis, was primed with a specific 20-nucleotide DNA
oligonucleotide labeled at the 5
end with 32P. Extension
to the end of the donor would produce a 152-nucleotide product while
homologous transfer of the growing DNA to the acceptor (produced from
pBS
PvuII1146) and subsequent extension yields a 199-base product (Fig. 2B). The region of homology
(transfer zone) between the donor and acceptor encompassed 119 bases
corresponding to a region of
-lac near the N terminus of
this protein. Transfer or full-length donor-directed products were
isolated from denaturing polyacrylamide gels (see Fig.
3) and used for PCR as described under
"Methods." The products were processed as shown in Fig. 2B (see "Methods" for details). The final 119-base pair
product, which was dephosphorylated at the EcoRI cleaved
end, was ligated into plasmid pBS
PvuII1146
(Fig. 2A) which had this same fragment excised and was
dephosphorylated at the PvuII-cleaved end. The plasmid and
insert were dephosphorylated to decrease the likelihood of ligation
products consisting of multiple inserts and/or plasmids. Such products
would generally produce white colonies due to the orientation of the
ligated products and not the sequence of the insert. After
transformation, colony color was analyzed with white or faint blue
colonies scored as those carrying plasmids with mutations in the
-lac region. We note that not all mutations will be
manifested as white or faint blue colonies. Frameshifts, which disrupt
the reading frame of
-lac, are generally detected while
base changes that result in amino acid changes may be detected depending on their effect on
-galactosidase activity. Base changes in the wobble base position that do not change amino acids will not be
detected.
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Nucleocapsid Protein Stimulates Strand Transfer-- An autoradiogram from a typical strand transfer experiment is shown in Fig. 3. Full-length donor-directed (F) or transfer products (T) were excised from gels and processed as described under "Methods." Donor-directed products were taken from assays without acceptor template. The presence of NCp (2 µM) in the reactions stimulated strand transfer about 3-fold. The efficiency of transfer, defined as the amount of transfer product divided by the sum of transfer plus full-length products × 100 ((T/(T + F)) × 100) was about 12% (average of three experiments) without and 36% (average of two experiments) with NCp. Note also that when NCp was used, several of the pause sites (sites on the template where premature termination occurs) evident in the absence of acceptor faded when acceptor was added. This suggests that the paused products are "chased" into transfer products by binding to the acceptor and subsequently being extended (24). The "chasing" was not observed without NCp although transfer clearly occurred. However, in the presence of higher amounts of acceptor template (40 nM), several of the pause sites decrease in intensity and the level of transfer products increases even in the absence of NCp (data not shown). Others have also shown that the level of strand transfer is proportional to the amount of acceptor (36). It is likely that chasing was not detected in Fig. 3 due to the low level of transfer products without NCp and not because it only occurs in the presence of NCp. The fact that several different paused products appear to transfer suggests that strand transfer occurs from several locations on the template.
Transfer and Donor-directed Products Showed Approximately the Same
Level of Errors in the Presence or Absence of NCp--
Transfer and
donor-directed products were processed as described in Fig.
2B. Analyses from three independent experiments are shown in
Table I. In each experiment, 1500 to several thousand colonies derived
from a given DNA source were scored. Error frequencies for transfer and
donor-directed products produced using standard conditions did not vary
significantly. As was previously noted, products produced with Klenow
had significantly less errors. We also performed assays using
suboptimal concentrations of dATP during HIV-RT synthesis. The error
frequency increased about 10-fold when 1 µM dATP was used
as opposed to 100 µM in the standard assay (data not
shown). These results indicate that a decrease in fidelity can be
detected by the assay. The only notable deviation for HIV-derived products was a small increase in the error frequency for transfer products in the presence of NCp. The third column shows the results after subtraction of background (see definition in table legend) or
subtraction of the error frequency obtained using Klenow (in parentheses). These values reflect the highest and lowest error frequencies, respectively, for HIV-RT in the assay. The first value
assumes an error frequency of zero for RNA polymerase and Taq while the second assumes a zero value for Klenow. Based
on these values the detectable error frequency of RT in this assay was
between 1.3 × 104 and 2.4 × 10
4
or 1 error per approximately 4200-7700 bases. This value is close to
that obtained for RNA-directed DNA synthesis by Ji and Loeb (8), but is
significantly higher than values from others (6, 29). Also shown in
Table I are calculations for the transfer frequency. These numbers
express the frequency of transfer per base over the 119-base region of
homology (transfer zone) between the donor and acceptor. The inverse of
this value reflects the average number of bases within the transfer
zone synthesized per strand transfer event. Without NCp there was about
one transfer event per 990 bases and one per 330 bases with NCp.
Sequence Analysis of Mutants--
Several of the mutants from
donor-directed products produced in the absence of NCp or Klenow
products were sequenced (Fig. 4). It is
difficult to assign specific errors to RT since the background in the
assay was relatively high (see above). Errors may have resulted from
RNA polymerase, Taq, or improper insertion. The latter group
consisted mostly of plasmids with 2 inserts in which the
PvuII cut blunt end of one insert had ligated to a modified EcoRI end of a second insert. The 4 base 5 overhang
generated by EcoRI was apparently cleaved off by a
contaminating nuclease generating a blunt end. This accounted for about
one-fourth of the mutated plasmids observed with Klenow (data not
shown). The other mutant plasmids from Klenow consisted of single base
substitutions and deletions, presumably resulting from errors made by
RNA polymerase, or Taq, or Klenow. Some of the observed
errors were unique to assays with HIV-RT. Among them were frameshifts
within a run of A's (bases 65-68) and multiple substitution mutants
at positions 61 and 86.
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DISCUSSION |
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We have shown that internal strand transfer occurring over a
defined region of RNA is highly accurate. This conclusion was based on
the error frequency of HIV-RT DNA synthesis products produced by primer
extension on a single template, or those which transferred to and were
extended on a second acceptor template. Each type of product showed
similar levels of errors (Table I). If strand transfer were inaccurate,
then transfer products should have more errors than those that had not
undergone transfer. Due to the assay background and variability, very
small increases in error frequency could not be reliably detected.
However, if 2% or more of transfer events were inaccurate this could
have been easily detected. A 2% per event error rate for transfer
would have increased the error frequency by about 2 × 104 per base. This calculation is based on 2 additional
mutant colonies per 100 total colonies or 2% additional mutants. Since
each insert corresponds to 99 synthesized nucleotides (see
"Results") the error frequency would be 2/(100 × 99) or about
2 × 10
4. This level of error would increase the
2.8 × 10
4 value for full-length products without
NCp from Table I (column 2) to 4.8 × 10
4 for the
transfer products. Thus, if errors occurred in only 1 in 50 transfer
events they would have been easily detected. There was some increase in
error frequency for transfer products versus donor-directed
products produced in the presence of NCp. An increase from 2.7 × 10
4 to 3.4 × 10
4 represents one
additional error per about every 14,000 bases copied or a 0.7% per
event error rate for transfer. Since this increase is relatively small
it may have resulted from normal experimental variation. Several
additional experiments would have to be performed to substantiate this
difference. What was clear from the results is that from a per base
perspective, strand transfer on this particular template was
considerably more accurate than RT-directed DNA synthesis. The latter
had a maximum error frequency after background subtraction of 2.4 × 10
4 in the assay (see "Results"). This corresponds
to one error per about 4200 nucleotides. Clearly, errors resulting from
strand transfer were well below this value. That does not mean that
strand transfer is 100% accurate, but that base misincorporation
errors resulting from HIV-RT's infidelity occur at a significantly
higher frequency than those resulting from erroneous strand transfer. This may be especially true in vivo where the strand
transfer frequency using an spleen necrosis virus based system was
estimated to be about 4% per kilobase during a single round of
replication (9). If the frequency of an inaccurate strand transfer
event was 1%, using this value of 4% to estimate the frequency of
recombination per kilobase leads to an error frequency per base of
4 × 10
7 (0.01 × 0.04/1000) or 1 error per
2.5 × 106 bases on average. Our experiments suggest
an error frequency per strand transfer event of less than 1%. If this
scenario is reasonable, the contribution of strand transfer to base
misincorporations would be essentially negligible.
We should note that the frequency of strand transfer events in our
in vitro system is 1-2 orders of magnitude greater than the
4% per kilobase value sited above. We observed frequencies corresponding to average rates of about 1 transfer event per 1000 or
330 nucleotides for reactions in the absence or presence of NCp,
respectively (Table I). The high rate observed in our experiments may
result in part, from the small sizes of the donor and acceptor templates and the high ratio of acceptor to donor (5:1) (36). The
relatively large size of the retroviral genome would likely make
alignment of homologous regions more difficult than with the small
templates used here. In addition, one might expect that the large
number of bases of the genome may increase the likelihood of erroneous
transfer events, essentially by increasing the potential targets for
transferring DNAs. For example, a nascent DNA could potentially
transfer to a region on the acceptor genome hundreds or even thousands
of bases away from the region homologous to that used for synthesis of
the DNA. Such events are referred to as "nonhomologous" strand
transfer since the DNA transfers to a region of a second template
different from the region on which it was synthesized. In this type of
transfer there is often, but not always, a small region of
complementarity between the acceptor RNA and 3 terminus of the
transferring DNA (37, 38). Experiments performed in vivo
using a system based on spleen necrosis virus and Moloney murine
leukemia virus indicate that nonhomologous recombination occurs at only
1/100th to 1/1000th the frequency of homologous recombination (37).
Nonhomologous transfer events may be important for viral transduction
(38, 39); however, homologous transfer events, being more frequent and
less likely to produce defective provirus, presumably contribute more
to the genetic diversity of the viral population.
The size of the templates used in our experiments limits the potential
for evaluating nonhomologous transfer. In fact, any transfers resulting
in the deletion or insertion of about 10 or more bases would have been
missed by our assays as a result of the way the insert was isolated (by
gel purification in which a region of about ±10 bases was excised).
Therefore, the assay we used assesses mutations occurring during
homologous strand transfer. Our conclusions suggest that such transfers
are highly accurate. Others, however, have found strand transfer with
HIV-RT to be somewhat inaccurate (26, 29). Using an in vitro
system designed to test the fidelity of internal transfer within a
hypervariable region of the nef gene, Wu et al.
(29) reported that strand transfer was more error-prone than
RNA-directed DNA synthesis. The overall error frequencies per base for
DNA synthesis and strand transfer were 2.8 × 105
and 6.2 × 10
5, respectively. The value for
RNA-directed DNA synthesis is about 10-fold lower than the value
calculated from our experiments but is comparable to the values
suggested by Boyer et al. (6). Although the error frequency
in strand transfer more than doubled in the above experiments, the
overall gross increase was 6.2 × 10
5-2.8 × 10
5 or 3.4 × 10
5. We note that such
an increase would not be reliably detectable in our assays and would
represent a tenuous increase over the error frequency which we
determined. There were important differences between our experiments
and those of Wu et al. (29). Although both used color
changes due to loss of
-complementation to detect errors, in the
experiments of Wu et al. (29) synthesis and transfer occurred over a hypervariable region of the nef gene which
was inserted within the multiple cloning site of plasmid pBSM13+, between the
-lac promoter and the majority of the coding
region for
-lac. The insert was in-frame with the
downstream
-lac gene. Mutations occurring during DNA
synthesis or strand transfer within the insert region would disrupt
-lac only if the mutations resulted in a termination
codon or frameshift. Therefore, the assay was designed to detect
mutations resulting in frameshifts only. Since our assay detected both
frameshifts and some point mutations (see "Results"), we would
likely detect a higher proportion of the total errors. However, since a
high proportion of the errors made by HIV-RT on RNA are frameshift
errors (6), this still cannot explain the 10-fold difference between
the experiments. One very important difference is the sequence of the
RNA used in the experiments. We performed synthesis and transfer over
the N-terminal region of the
-lac gene while Wu et
al. (29) used a portion of the HIV genome. It is quite possible
that there are significant sequence-dependent differences
in error frequencies. This may also explain the increase in errors in
strand transfer products. Perhaps there are specific sequences which
tend to transfer inaccurately. In support of this Wu et al.
(29, 40) noted that although transfer occurred from several regions of
the template, a particular run of 4 C's had a high error frequency
only after strand transfer. Perhaps our template did not contain a
hypermutable transfer sequence. Finally, it is possible that strand
transfer on our template occurred essentially from a single location
that just happened to promote high fidelity transfer. However, as was
noted under "Results," variations in pause site intensities in the
absence or presence of acceptor suggested that transfer occurred from
several locations on the template, although the relative contribution
of each site to transfer was not assessed.
Results from others have suggested that strand transfer of minus strand
strong stop DNA may be inaccurate due to non-template-directed base
insertions at the 3 terminus of the transferring DNA (26). Mutations
of this type would have resulted in frameshifts in our assay system and
were not detected. Perhaps the nature of strand transfer events
occurring from template termini (i.e. strong stop transfers)
differs from those occurring from within internal regions of a
template.
It was interesting that the error frequency calculated in our
experiments was very close to the 1 error per 6900 base value determined by Ji and Loeb (8). When the error rate for Klenow was used
as background we calculated a minimum error frequency of 1 per 7700 bases (see "Results"). Although the approach used by Ji and Loeb
(8) differed from ours, the sequence and length of RNA used in the two
studies were comparable. Hence it is not surprising that similar values
were obtained. Many of the mutations observed by Ji and Loeb (8) were
also observed in our experiments, specifically frameshifts within a
particular run of adenosines (bases 65-68 in Fig. 4). There were also
differences, particularly in the observation of guanosine to thymidine
transversions of which we detected only one (base 64). In contrast, a
significantly lower error frequency for RNA-directed DNA synthesis,
especially with respect to base substitutions, was observed using a
larger region of -lac that contained the sequences used
in our experiments (6). We, like Ji and Loeb (8), observed a
substantially higher proportion and number of base substitutions in
comparison to frameshifts while fewer substitutions were observed by
Boyer et al. (6). We note that the methodologies used in the
three studies were considerably different. Our strategy required
several enzymatic steps and a ligation to score both transfer and
donor-directed products, while Boyer et al. (6) employed the
least manipulation in their approach. Due to the many steps, the
background in our experiments was relatively high, perhaps accounting
for up to one-half of the total errors (see "Results"). This made
it difficult to be sure that a particular error was due to RT and not
one of the other enzymes used in the process. However, in comparing
Klenow and RT products, some errors were observed multiple times and only with RT, implying that they resulted from this enzyme (Fig. 4).
All things considered, the lower background of the assays used by Boyer
et al. (6) and Ji and Loeb (8) make these approaches more
informative with respect to analyzing specific errors made by
HIV-RT.
In conclusion, our work indicated that strand transfer on the template used in these experiments was highly accurate, while experiments using a different template (29) showed that some transfer events are error prone. Taken together the results imply that the fidelity of strand transfer may be highly sequence dependent.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jasbir Seehra and John McCoy, Genetics Institute, for the kind gift of HIV reverse transcriptase, and Leyla Diaz and Jason Cristofaro for their help in preparing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51140-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
University of Maryland, Bldg. 231, College Park, MD 20742. Tel.:
301-405-5449; Fax: 301-314-9489; E-mail: jd146{at}umail.umd.edu.
1 The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; RNase H, ribonuclease H; NCp, nucleocapsid protein; Taq, Thermus aquaticus; PCR, polymerase chain reaction; NCp, nucleocapsid protein; MCS, multiple cloning site.
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