From the Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland 20742
Received for publication, October 4, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Interactions between human immunodeficiency virus
(HIV) reverse transcriptase (RT) and structures mimicking intermediates proposed to occur during recombination (strand transfer) were investigated. One mechanism proposed for strand transfer is strand exchange in which a homologous RNA (acceptor) "invades" a donor RNA·DNA duplex (replication intermediate) on which DNA
synthesis is occurring. The acceptor displaces the donor of the duplex
and binds to the DNA. During exchange a transient trimeric structure forms. A model structure was designed with a replication intermediate to which an acceptor RNA was bound. The acceptor was bound to the
5'-end of the DNA over a 54-base region, whereas the donor associated
with the DNA 3'-end over a 28-base region. The dimeric constituents of
the trimer (acceptor RNA·DNA and donor RNA·DNA) were also
constructed. The acceptor RNA·DNA formed a branched structure in this
case. Results showed that RT could cleave the RNA portion of all the
structures examined. Association with junction substrates was less
stable as determined by off-rates. On the trimer, RT cleaved both RNAs
but showed a clear preference for cleaving the donor RNA region. This
preference was accentuated by HIV nucleocapsid protein (NC). Results
suggest that during recombination RT generally associates with the
donor-RNA portion of the trimer and the acceptor RNA is protected but
not immune from cleavage. The partial protection likely allows the
acceptor RNA to more easily complete strand exchange and shield this
RNA to provide a means to salvage replication if the DNA were to
dissociate from the cleaved donor RNA.
Strand transfer is an essential step in retroviral replication and
has been shown to occur from terminal regions and internal regions of
RNA templates. Minus strand DNA strand transfer is proposed to
potentially occur by two different mechanisms (1). In one, DNA being
synthesized on an RNA template (referred to as the donor in this case)
dissociates from the RNA and binds to a homologous region on a second
RNA (referred to as the acceptor) where synthesis continues. A second
mechanism proposes that the acceptor RNA actively participates in the
displacement of the DNA strand by associating with the DNA and
dislodging the donor. Because retroviruses are diploid, the donor and
acceptor RNAs would represent the two genome copies in the capsid.
Results from previous experiments suggest that viral nucleocapsid
protein (NC)1 stimulates the
binding of a complementary RNA acceptor template to the DNA displacing
the donor RNA in the process (2). Consistent with the second mechanism
above, this implies that strand transfer could proceed through a
trimeric complex consisting of the nascent DNA bound to both the RNA
template it was being made on (donor) and a second homologous RNA
(acceptor). Such a structure has been shown to occur during DNA
transfer in vitro (2, 3). Models developed in
vivo (4) and in vitro (5, 6) also indicate the likely
existence of a trimeric intermediate as one of the mechanisms used for
strand transfer during recombination. Little is known about what
activities RT manifests on such a structure. The binding and
orientation of RT to such a structure would become important, because
both the RNA templates are accessible to the enzyme. The fate of the
acceptor template is important, because degradation of the acceptor
prior to transfer would eliminate a potential pathway to continue DNA
synthesis if the nascent DNA were to dissociate from the donor template.
In this report, experiments were designed to characterize the activity
of RT on strand transfer intermediates with the goal of determining if
RT has a preference for associating with the donor or acceptor RNA on a
trimeric strand transfer intermediate and if the RNase H activity of RT
cleaves these RNAs. Three separate templates were designed to mimic the
entire strand transfer intermediate (trimeric structure) or various
parts of this structure. One template consisted of the donor RNA bound
to the DNA (donor RNA·DNA), which mimics the "replication
intermediate." The second template consisted of the acceptor RNA
bound to the DNA such that the bases at the 3'-end of the DNA are not
hybridized to the acceptor (acceptor RNA·DNA). This mimics the
binding of the acceptor RNA to the DNA of the replication intermediate.
The third structure, the strand transfer intermediate, contains both
the donor and acceptor RNAs bound to DNA. Previous results have
indicated that RT is anchored to nucleic acid substrates by a 3'
recessed DNA terminus or a 5' recessed RNA terminus (7-13). With this
in mind, RT would be expected to bind preferentially to the donor RNA
of the trimer, because the 3'-end of the DNA strand is recessed on the
donor RNA. In contrast, there is no 3' recessed DNA end on the acceptor RNA and the 5'-end of the acceptor is not associated with the DNA
suggesting that RT would not efficiently cleave the acceptor RNA.
Results showed that RT cleaved both RNAs of the trimer but with a clear
preference for cleaving the donor RNA region. In the presence of HIV
nucleocapsid protein (NC), this preference was even stronger. The
findings imply that during recombination RT generally associates with
the donor-RNA portion of the trimeric recombination intermediate and
the acceptor RNA is protected but not immune from cleavage. The partial
protection likely allows the acceptor RNA to more easily complete
strand exchange and shield this RNA to provide a means to salvage
replication if the DNA were to dissociate from the cleaved donor RNA.
Materials--
Wild-type recombinant HIV-RT was graciously
provided to us by Genetics Institute (Cambridge, MA). Aliquots of the
enzyme were stored at Construction of Substrates--
Run-off transcription was
performed according to the instructions of the enzyme manufacturer. The
donor RNA
(5'-GGGAACAAAAGCUAAUUCGCCCUAUAGUGAGUCGUAUUACAAUCACUGG) was
made from HaeIII cleavage of pBSM13(
The donor and acceptor RNAs were then dephosphorylated using
calf-intestinal alkaline phosphatase. The alkaline phosphatase was
heat-inactivated at 65 °C for 10 min. Reactions were extracted with
phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and precipitated with
ethanol. The RNA precipitates were labeled at the 5'-end with
32P using T4 polynucleotide kinase according to the
manufacturer's instructions. A DNA oligonucleotide 93 bases in length
(5'-GAGGGATCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTGGCCAGTGAATTGTAATACGACTCACTATA) was also radiolabeled at the 5'-end. Two other DNAs were also used in
the construction of dimeric substrates, a second 93-base DNA
(5'-GAGGGATCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTGATCCGGGCCCTGTAATACGACTCACTATA) was used to construct the acceptor RNA·DNA (+10 hybrid) substrate (see "Results"), and a 64-nucleotide DNA
(5'-GAGGGATCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTG) was used to construct the acceptor RNA·DNA (recessed 3'-DNA) substrate.
The RNA·DNA hybrid structures were made by mixing together the
radiolabeled RNAs and the radiolabeled 93-mer DNA at a ratio of 1:2.
The mixture was heated at 70 °C for 5 min and slowly cooled down to
room temperature. After hybridization, 6× native gel loading buffer
(40% (w/v) sucrose, 0.25% (w/v) xylene cyanol and bromphenol blue)
was added, and the mixture was electrophoresed on a non-denaturing 6%
polyacrylamide gel (29:1 acrylamide:bisacrylamide) as described previously (15). The hybrid complex was located by autoradiography, excised, and eluted in a buffer containing 50 mM Tris-HCl
(pH 8) and 1 mM DTT. The eluate was centrifuged and
filtered as described above and used directly for experiments.
The trimeric complex consisted of the donor and acceptor RNA bound to
the 93-nucleotide DNA. The three radiolabeled components in the trimer
had equal specific activities. The trimer was constructed by first
making a hybrid of the donor RNA and the DNA at a ratio of 1:0.5. The
hybrid was made as described above. To this hybrid, acceptor RNA was
added in a solution containing 50 mM Tris-HCl (pH 8.0), 20 mM KCl, 1 mM DTT, 100 µM
ZnCl2, and 6 mM MgCl2. The reaction
was incubated at 37 °C for 15 min in the presence of 5 µM NC. Proteinase K was then added to the mixture and
placed at room temperature for an additional 20 min. The reaction was stopped by the addition of 6× native dye, and the sample was then run
on a 6% native non-denaturing polyacrylamide gel. Other methods, including mixing all three components together at once or making the
acceptor RNA·DNA first, were also tried in both the presence and
absence of NC, but the above method gave the highest yields and most
reproducible results. The trimeric complex was located by
autoradiography, excised, and eluted at 4 °C in a buffer containing 50 mM Tris-HCl (pH 8) and 1 mM DTT. The eluate
was centrifuged and filtered as described above and used directly for experiments.
Quantification of Nucleic Acids--
Donor and acceptor RNA
templates were quantified spectrophotometrically by measuring the
absorbance. The molecular weight of the RNA was used to determine the
molar concentrations of the RNA templates. The amount of radiolabeled
heteroduplex and trimeric substrates recovered from native gels was
determined by specific activity. Quantification of RNase H cleavage
products was accomplished by scanning the dried polyacrylamide gels
with a phosphorimaging device (Bio-Rad, GS 525).
RNase H Cleavage Assays--
In the standard RNase H cleavage
assay, 5 nM of either the heteroduplex or the trimeric
substrate was incubated in a volume of 10.5 µl containing the
following components at their final concentrations: 50 mM
Tris-HCl (pH 8), 80 mM KCl, 7.1 mM
MgCl2, 0.2 unit/µl RNasin, 1 mM DTT, 0.1 mM EDTA (pH 8), and 6 mM AMP at 37 °C.
Reactions were initiated with the addition of 2 units (0.4 pmol) or in
some 0.2 unit (0.04 pmol) of HIV-RT in a 2-µl solution containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 80 mM KCl. In RNase H assays performed in the presence of NC,
the NC was allowed to preincubate with the substrate for 2 min before the initiation of the reaction. In these reactions, the amount of NC
used is indicated in the figures. All reactions were terminated at the
indicated times with 12.5 µl of 2× gel loading buffer (90% formamide, 10 mM EDTA (pH 8), 0.1% xylene cyanol, and
0.1% bromphenol blue). Samples were electrophoresed on 8%
polyacrylamide (19:1 (w/w) acrylamide:bisacrylamide)-7 M
urea gels as described below. The gels were dried and used for cleavage
product quantification by phosphorimaging using a Bio-Rad GS-525
phosphorimaging device.
Cleavage "Trap" Assays--
In assays designed to allow only
a single binding event between the enzyme and template ("trap
assay"), 2 units (or as indicated) of enzyme was allowed to
preincubate with 5 nM template for 3 min in 10.5 µl of
buffer containing 50 mM Tris-HCl (pH 8), 80 mM
KCl, 0.2 unit/µl RNasin, 1 mM DTT, 0.1 mM
EDTA (pH 8), and 5 mM AMP at 37 °C. The reactions were
initiated with 2 µl of a supplement in the above buffer
containing 37.5 mM MgCl2, and 2.5 µg of
poly(rA)-oligo(dT20) primer-template was used as the trap. The reactions were incubated at 37 °C for the indicated times and
were terminated by the addition of 12.5 µl of 2× gel loading buffer
and electrophoresed as described above.
Off-rate (koff)
Determination--
Experiments designed to determine the dissociation
rate constant were performed by pre-binding the enzyme (2 units) with
the substrate (5 nM) in a volume of 8.5 µl of buffer
containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT,
10 mM KCl, 0.2 unit/µl RNasin, 0.1 mM EDTA
(pH 8), and 5 mM AMP at 37 °C. Two microliters of a
solution containing 2.5 µg of poly(rA)-oligo(dT20) trap
in the above buffer was added to the reaction and allowed to incubate further. At various times after trap addition, 2 µl of a solution containing 37.5 mM MgCl2 in the above buffer
was added to initiate the cleavage reaction, and samples were incubated
for 5 min. The reactions were terminated by the addition of 12.5 µl
of 2× gel loading buffer, and electrophoresis was performed as
described above. For the time zero point, trap and
MgCl2 were added at the same time. Determinations of
koff values were performed by fitting the data
for the relative amount of cleaved RNA versus time to an
equation for exponential decay (f(x) = ae Gel Electrophoresis--
Denaturing 8% polyacrylamide gels
(19:1 acrylamide:bisacrylamide), containing 7 M urea, and
native 6% polyacrylamide gels (29:1 acrylamide:bisacrylamide) were
prepared and subjected to electrophoresis as described (15).
Construction of Substrates--
The substrates used for RNase H
cleavage assays were described under "Experimental Procedures" and
are depicted schematically in each autoradiogram. Five different
structures were used. The main structure from which the others were
derived is referred to as the "trimer" (Fig.
1). It consisted of three nucleic acid strands, the donor and acceptor RNAs, and a DNA strand. The donor RNA
and acceptor RNA were transcribed in vitro and radiolabeled at the 5'-end with 32P. The DNA oligonucleotide was also
5'-end-labeled. The donor RNA was 50 nucleotides in length, acceptor
RNA approximately (see "Experimental Procedures") 99 nucleotides in
length, and the DNA 93 nucleotides in length. The acceptor RNA·DNA
structure was designed such that a hybrid region of 54 bases formed
between the 3'-end of the RNA and an internal portion of the DNA that
started 30 bases from the 3'-end of the DNA. The donor RNA was designed
such that a hybrid region of 28 bases formed between the 3'-ends of the
DNA and donor. This design essentially left one unhybridized base (29th
base from the 3'-DNA-end) between the donor and acceptor RNAs. There
were also 10 bases at the 5'-end of the DNA that were not hybridized to
RNA. Note that the 28-base hybrid formed between the donor RNA and the
DNA is longer than the presumed 18 bases between the polymerase and
RNase H active sites of HIV-RT. However, it is still within the realm
of what is likely to exist in vivo. Because RNase H activity
is generally only about one-tenth the polymerase activity (20), hybrid
regions between the nascent DNA and genomic RNA are likely to range
from as long as about 30 bases down to sizes too small to remain stably
associated (21, 22). In addition to the timer, dimeric constituents
consisting of the acceptor (acceptor RNA·DNA) or donor (donor
RNA·DNA) bound to the DNA were also examined. Finally, two additional
structures based on the acceptor RNA·DNA were also used. In one the
hybrid region between the acceptor and DNA was increased to 64 bases by
moving the junction 10 bases toward the 3'-end of the DNA (termed acceptor RNA·DNA (+10 hybrid)). In the second the DNA was shortened by 29 bases at the 3'-end to produce a 64-nucleotide DNA. The resulting
structure (termed acceptor RNA·DNA (recessed 3'-DNA)) eliminated the
branch point of the original acceptor RNA·DNA structure and produced
a recessed 3'-DNA terminus.
To ensure that the appropriate structures were used, it was important
to determine that the DNA remained associated with the RNA for both the
duplexes and the trimeric structures. For all preparations of
substrates obtained, this was verified by examination of the recovered
material by native gel electrophoresis. As can be seen from Fig.
2, the various complexes and
single-stranded nucleic acids migrated to unique positions on the
native gel. This allowed recovery of pure material and confirmation of
the integrity of the isolated complexes. It should be noted that the acceptor RNA·DNA and trimer structures represent "model
substrates" of intermediates occurring during strand transfer. An
authentic trimeric transfer intermediate would contain an acceptor RNA
that was complementary to the 3'-end of the DNA. Various conditions were used in an attempt to isolate "authentic" structures by first forming donor RNA·DNA, then adding acceptor RNA that was
complementary to the complete DNA of the donor RNA·DNA. In all cases
an acceptor RNA·DNA structure devoid of donor RNA was resolved on the
native gels. Regardless, the model substrates that were produced should yield valuable information concerning the recognition of strand transfer intermediates by RT and the mechanism of RNase H cleavage on
the substrates.
Cleavage of RNA in Heteroduplex Structures--
RNase H cleavage
assays done on heteroduplex structures provide information about the
binding site of RT. When the donor RNA was bound to the DNA in the
heteroduplex (donor RNA·DNA), results were as expected given the
distance between the polymerase and RNase H active sites on RT and the
3'-5' directional cleavage activity of RT (data not shown) (7, 8, 21,
22). The enzyme initially cleaved the RNA ~18 bases behind the 3'-end
of the DNA, and the larger initial cleavage products were subsequently chased into smaller products over time by the 3'-5' nuclease activity of the RT. This results in a shortening of the hybrid region between the DNA and RNA and eventually can cause dissociation of the nucleic acids.
In the heteroduplex structure mimicking the binding of the acceptor RNA
to the DNA, cleavage of the acceptor RNA was observed (Fig.
3, acceptor RNA·DNA). This was in
contrast to what had been reported previously. Work by others using RNA
bound to single-stranded circular DNA suggested that RT could cleave
structures with the 5'-end of the RNA not bound to the DNA. However,
this was providing that the unbound 5'-RNA end was less than 12 nucleotides in length (11). Based on these results alone it would be
predicted that the acceptor RNA·DNA duplex would be resistant to
cleavage, because the acceptor RNA has an unbound 5'-end that is 45 nucleotides away from the hybrid region (see "Discussion" also).
Despite this results indicated that the RNA was cleaved. The product
sizes at the early time points were diverse indicating that RT was
initially bound to the structure such that the RNase H active site
contacted the RNA at several positions. The shortest (and most
prominent) RNA observed corresponded to cleavage 15 bases behind the
junction of the RNA·DNA hybrid (60-nucleotide product). Products
longer than 60 nucleotides were processed over time to produce more of the 60-base product, whereas small amounts of 56-nucleotide and shorter
products were observed later in the reaction. Assays in which E. coli RNase H was added after RT digestion indicated that the
60-nucleotide RNA was still bound to the DNA (data not shown). Overall
results suggested that, after somewhat random (although with a
preference for sites near junction) initial cleavages, subsequent
cleavages proceed toward the RNA·DNA junction. Processing at the
junction to produce a small unstable hybrid region appeared to be
slow.
To compare the cleavage of the acceptor RNA·DNA substrate to cleavage
of a more standard substrate with a 3' recessed DNA termini, a
64-nucleotide DNA that bound the RNA and terminated at the end of the
hybrid region was constructed (acceptor RNA·DNA (recessed 3'-DNA)).
The DNA was identical to the first 64 bases at the 5'-end of the 93 base DNA used in the acceptor RNA·DNA substrate but was missing the
29 bases at the 3'-end. This configuration should direct RT cleavage
~18 bases behind the DNA 3' terminus (
To determine how the position of the junction affected cleavage a
substrate was constructed in which the position of the junction was
advanced by 10 nucleotides toward the 3'-end of the DNA. This produced
an acceptor RNA·DNA substrate with a 64-base as opposed to a 54-base
hybrid region. Results with this substrate are shown in Fig. 3
(acceptor RNA·DNA (+10 hybrid)). At early time points, as with the
original acceptor RNA·DNA, an array of products were seen with the
distribution shifted toward products produced by cleavages nearer the
junction. Prominent products 56 (
In the above experiments cleavages were carried out under conditions
that allowed multiple binding and rebinding events between RT and the
substrates. This makes it difficult to determine if the cleavages
occurring even at early time points were initial or secondary
cleavages. To more clearly determine the initial cleavage positions
trap assays were performed with the substrates (Fig.
4). In these assays RT is preincubated
with the substrate then reactions are initiated by addition of
MgCl2 and poly(rA)-oligo(dT) trap. The trap sequesters
enzymes that dissociate from the substrate thus limiting cleavage to a
single binding event (21). Because RT can catalyze both primary
(initial) and secondary cuts in one binding event, the technique does
not show only initial cleavage positions, however, secondary cuts occur
slowly, so at early time points primary cleavages represent the
majority of the observed products (7, 10, 12, 13). A trap control assay
in which RT was preincubated with trap was performed for each substrate (lanes E). A low level of cleavage was observed in these
lanes in comparison to the starting material (lane F, shown
for acceptor RNA·DNA only) indicating that the trap was not 100%
effective. However, the level of trap read-through was very low and did
not interfere with interpretations of the results. For the substrate with the recessed 3' termini (acceptor RNA·DNA (recessed 3'-DNA)) the
The stability with which RT bound to the junction versus
substrate with recessed 3' terminus was also investigated. To eliminate possible sequence-specific effects, the acceptor RNA·DNA and the acceptor RNA·DNA (recessed 3'-DNA) rather than the donor RNA·DNA were used. A modified version of the trap assay was used to measure the
rate of dissociation (koff) of RT from the
substrate. For these experiments the KCl concentration was reduced to
10 mM to decrease the rate of dissociation. Using 80 mM KCl RT dissociates rapidly in the absence of
MgCl2 (16). Divalent cation must be omitted in the
preincubation phase of the assay, because it would stimulate cleavage
of the substrate. For this assay, RT was preincubated with substrate
then a supplement containing poly(rA)-oligo(dT) trap was added.
Incubation was continued for a fixed time then a supplement containing
MgCl2 was added to initiate cleavage and incubations were
continued for 2 min. During the incubation with trap RT molecules that
dissociate from the substrate are sequestered. The greater the time
between trap and divalent cation addition the more dissociation occurs.
This results in a progressive decrease in cleavage that can be used to
determine the half-life of the complex (see "Experimental
Procedures"). Fig. 5 (A and
B) shows the results of a typical
koff experiment. Clearly dissociation is
considerably slower for acceptor RNA·DNA (recessed 3'-DNA). This
indicates that the presence of a recessed 3'-DNA terminus increases the
stability of RT binding relative to the junction substrate. Values for
koff were 0.015 ± 0.003 and 0.008 ± 0.001 (average of three experiments ± S.D.) for acceptor
RNA·DNA and acceptor RNA·DNA (recessed 3'-DNA), respectively,
indicating that RT dissociated about twice as quickly from the
substrate with no 3' recessed terminus.
Cleavage of RNA in the Trimer Structure--
RNase H cleavage
assays performed on the trimer structure indicated that both the donor
RNA and the acceptor RNA were cleaved (Fig.
6). The design of the trimeric structure
allowed for the identification of cleavage product derived from both
donor and acceptor RNAs in the same reactions. Cleavage products from
the acceptor were longer than the donor RNA allowing resolution in individual reactions. This allowed all three nucleic acids to be
radiolabeled in the trimer. In addition, the specific activity of each
labeled component was approximately the same allowing a direct
comparison of the amount of each RNA that was cleaved. Trimer
preparations typically showed a low level of RNA breakdown products
that migrated below the donor RNA in the substrate (time point 0 in
Fig. 6). These products did not interfere with interpretation of the
results. The observed cleavage products in Fig. 6 indicated that RT
bound each RNA in the same manner as it bound the RNAs in the duplex
structures (see above). Note that the amount of enzyme used in the
assay shown in Fig. 6 was low in comparison to cleavages with the
substrates above (0.1 unit per reaction as opposed to 2 units in the
assays for the other substrates). This accounts for the slower rate of
cleavage observed and allows a more careful determination of the rate
of cleavage of the individual RNAs. The results showed that the 50-base
donor RNA was cleaved faster than the 99-base acceptor (see Fig. 10
below). This suggests that RT associates more frequently with the
region of the trimer that has a recessed 3' terminus. This is despite
the fact that this region contains only about one-half as much
RNA·DNA hybrid in comparison to the region bound to the acceptor.
A second experiment was performed to measure the off-rate of RT from
the two RNAs in the trimer. The experiment was performed as described
above with 10 mM KCl in the reactions. Consistent with the
more rapid dissociation of RT from the branched versus 3'
recessed substrates above, RT dissociated more rapidly from the
acceptor region of the trimer (Fig. 7,
A and B, koff equals 0.005 and 0.014 for donor and acceptor regions, respectively). Taken
together, results with the trimer indicate that RT associates with both
regions of the trimer but binding is more stable to the donor
region.
The observed cleavages of both RNAs in the trimer could occur in many
ways. A single enzyme molecule could cleave both RNAs in succession
without dissociating from the trimer. There could be a preference as to
which RNA is bound first, or it may be random. Alternatively, RT may
bind without preference to either RNA on the trimer and then dissociate
after cleaving that RNA. If both RNAs were bound with nearly equal
preference, then equal levels of donor and acceptor RNA cleavage would
be observed throughout the reaction. Finally, two enzyme molecules
could bind each trimer molecule in a cooperative fashion, one on the
donor and a second on the acceptor. The latter mechanism is unlikely,
because results have shown that RT does not bind cooperatively to
substrates.2 However, at high
enzyme concentrations it is possible that more than one RT molecule
could be bound to a single trimer simply due to a substantially greater
number of enzyme versus substrate molecules. This could lead
to cleavage of both RNAs even if RT preferred one RNA to the other. To
eliminate this possibility reactions were performed with a limiting
amount of enzyme under conditions allowing only a single binding event
(trap reaction) between the enzyme and the substrate as described
above. In trap assays it was observed that both the donor and acceptor
RNAs of the trimer were cleaved even at low enzyme concentrations when only a fraction of the total substrate was cleaved (Fig.
8). The fact that only a small fraction
of the substrate was cleaved at low enzyme amounts implies that the
reactions were not overloaded with enzyme, such that more than one
molecule was bound to the substrate. This conclusion assumes that RT is
not binding cooperatively. Therefore, these results suggest that either
one enzyme molecule cleaves both RNAs in succession or RT binds the
RNAs randomly and dissociates after cleavage. In other words, at any
given time during the reaction, RT may be bound to the donor RNA of one
trimer molecule and acceptor RNA on another. After cleaving the RNA
that is bound, RT dissociates without cleaving the second RNA.
In an attempt to differentiate between these possibilities, a time
course trap reaction was performed (Fig.
9, A and B). In this experiment reactions were terminated at time points ranging from 2 to 160 s. Both RNAs were cleaved even at the earliest time point
with more cleavage of donor versus acceptor. Both RNAs
showed an increase in cleavage between 2 and 10 s and no further
significant increase (Fig. 9B). This suggests that cleavage
is due to individual enzyme molecules bound to either RNA and not to a
single molecule cleaving the RNAs in succession (see "Discussion").
If this mechanism is correct, given that the level of donor cleavage
was greater than for acceptor, then the implication is that RT
associates better with the donor portion of the trimer as was noted
above.
Effect of NC on Cleavage of RNA in the Trimer Complex--
It has
been well documented that NC affects the stability and formation of
hybrid structures and can stimulate strand transfer by promoting the
formation of a trimeric intermediate (see Introduction). Therefore, it
is possible that NC would affect the binding and orientation of RT to
the trimer complex. To determine if NC influenced RT binding to the
trimer, RNase H assays were performed in the presence of NC. Two
different amounts of NC were used corresponding to one molecule
of NC for every seven nucleotides of nucleic acid (a final
concentration of about 0.17 µM) and about one NC per two
base (about 0.6 µM). A ratio of 1:2 NC:nucleotide was
previously determined to be optimal for strand transfer reactions
in vitro, although the actual in vivo ratio is
~1 NC per 7 nucleotides (23). In the presence of NC the site of
cleavage did not change, but there was a decrease in the amount of
cleavage for the acceptor RNA (Fig. 10,
A and B). Cleavage inhibition increased as the
concentration of NC increased but was evident at both concentrations
tested. The level of inhibition was small for cleavage of the donor RNA and was only observed at the higher NC concentration (Fig.
10B). The results are consistent with NC decreasing the
binding of RT by competing with the enzyme for binding sites on the
substrate (2). Notable is the large difference between inhibition on the donor and acceptor. Even without NC the donor was cleaved more
rapidly than the acceptor (see Fig. 6 also), and this difference was
greatly accentuated in the presence of NC.
In these experiments the binding of RT to a trimeric substrate and
its constituent dimer components was investigated. The trimer was
designed to mimic a strand transfer intermediate in which the acceptor
RNA is "invading" the donor RNA·DNA complex (see Introduction).
Overall the experiments showed that the two RNAs involved in a
recombination event that proceeds through the formation of a trimer are
recognized differently by RT. RT binds the acceptor RNA, especially in
the presence of NC, relatively poorly. Binding and cleavage of the
donor RNA is clearly favored. The partial protection of the acceptor as
well as cleavage of the donor may serve to promote more effective
strand transfer as well as provide a salvage pathway for replication
should the DNA and donor RNA dissociate due to RT RNase H cleavage.
HIV-RT bound both the donor and acceptor RNAs of the trimer while
showing a preference for associating with the donor region (Figs.
6-10). Cleavage of the acceptor was not anticipated because other
experiments indicated that the 5'-end of an RNA molecule must be bound
or in close proximity to the DNA to observe RT-directed RNA cleavage
(11). Results showed that cleavage can be independent of the presence
of a DNA 3'-end and is directed by the 5'-RNA end providing the unbound
5'-RNA end was short (less than 12 bases). In our experiments cleavage
was clearly not directed by the 5'-RNA terminus, because it was too far
away (45 bases from the RNA·DNA hybrid). In fact initial cleavages
were only "loosely" directed by the position of the junction
suggesting that RT binds the junction substrate with some tendency to
bind nearer the junction (Figs. 3 and 4). The apparent contradiction
between these and the previous results may be due to the difference in
the substrates used. Palaniappan et al. (11) used a
substrate in which RNA primers were annealed to large circular DNA
templates. The presence of DNA termini on the substrates used in our
experiments may have promoted the association of RT with the acceptor.
A second possibility is that the difference in the size of the DNA
templates used in the experiment affected binding. The circular DNAs
employed by Palaniappan et al. were several thousand
nucleotides long. This could have resulted in binding competition for
RT, because the RNA·DNA hybrid regions were relatively small. It has
been shown that RT has some affinity for single-stranded nucleic acid
(24, 25). In the absence of a bound 5'-RNA end the single-stranded
region of the substrate could have out-competed the hybrid region for
RT binding. This argument is strengthened by the fact the binding to
the junctioned acceptor RNA·DNA was less stable than binding to a
comparable substrate with a recessed 3'-end (Fig. 5). Finally, it is
possible that the particular substrate used in our experiments allowed cleavage due to an undefined sequence or structural property of the
substrate. This seems unlikely because another substrate with a similar
configuration but unrelated sequence was also cleaved by RT (data not shown).
As was noted in the results, cleavage of both the donor and acceptor
RNA in a trimeric complex could occur in several ways. The results
presented here support a mechanism where an RT molecule binds a given
substrate on either the donor or acceptor with a preference for binding
donor. After cleavage of the bound RNA, RT most likely dissociates
without cleaving the second RNA. Several lines of evidence
support this mechanism: 1) Results indicate that RT shows no
cooperativity in binding substrate.2 This indicates that
two RTs would not associate with a single substrate under conditions in
which the concentration of enzyme is limiting. Under such conditions
cleavage of both RNAs was still observed in a trap assay (Fig. 8).
Therefore, cleavage must have resulted from a single RT molecule
cleaving both RNAs in succession or to the mechanism noted above. 2) In
trap assays that limited binding to a single event, cleavage of both
RNAs was observed at early time points, and the extent of cleavage did
not increase after a short time (Fig. 9). This makes it unlikely that
cleavage occurred by one RT molecule cleaving both RNAs in succession. If this were the case an increase in the level of cleavage over time
should have been observed for one of the RNAs. There was an initial
increase between 2 and 10 s in the trap reactions but no further
increase. Others have shown that it takes as long as 10 s for
single RT molecules to catalyze an initial cleavage event on an
RNA·DNA hybrid, although most cleavages occur within a few seconds.
Following initial cleavages RT remains associated with the RNA·DNA
hybrid for up to several minutes catalyzing further cleavages of the
initial product in a 3'-5' direction (21, 22). Given that no
significant increase in total cleavage products was observed beyond
10 s in trap assays, it is unlikely that RT molecules cleaved one
RNA then migrated to and cleaved the second RNA. If this occurred it
would have to have taken place within the first 10 s of the trap
reactions. Because the previous results indicate that RT remains
associated with the RNA potentially for several minutes, cleavage of
two RNAs in succession within 10 s seems highly unlikely.
Because these data support non-successive cleavage of the two RNAs this
implies that RT must have some preference for binding to the donor over
the acceptor RNA on the trimer. A greater proportion of donor
versus acceptor RNA was cleaved in the trap assay indicating that RT associated preferentially with the donor (Fig. 9). In addition,
cleavage in assays with low amounts of enzyme showed the donor RNA of
the trimer being degraded more rapidly than the acceptor (Fig. 10). The
koff values from experiments with the trimer (Fig. 7) as well as dimeric substrates (Fig. 5) support this notion, because RT bound more stably to substrates with 3' recessed termini, consistent with the role of these termini in stabilizing binding (26).
However, the tendency to cleave acceptor or donor in the trimeric
substrate likely depends on the overall affinity
(Kd) of RT for that particular region rather than
the off-rate. Given that the rate of cleavage by RT is several times
greater than the off-rate (20), RT will presumably cleave either RNA
nearly 100% of the time that the enzyme binds to it. Therefore, RT
tends to associate more frequently with donor RNA rather than the
acceptor in the trimer. Whether this is due to binding stability or to the overall configuration of the timer is not clear.
Cleavage in the presence of NC showed the same trend as experiments
without NC. Cleavage of both RNAs occurred with faster cleavage of the
donor RNA. The level of cleavage was slightly reduced on the donor RNA
by NC, whereas cleavage on the acceptor was significantly inhibited
(Fig. 10). This decrease likely resulted from NC competing with RT for
binding to the substrate as has been suggested by others (2, 27). The
greater inhibition of acceptor cleavage may result from RT binding this
region of the substrate more weakly as indicated above. The weaker
binding may make it more difficult to out-compete NC for binding sites.
The nature of the trimeric substrate used in this work did not allow a
direct comparison to what might occur on an authentic strand transfer
intermediate. As was noted under "Results," the acceptor RNA 5'-end
would be complimentary to the 3'-end of the DNA in the authentic
intermediate. The effect of the complementarity would possibly be to
further promote dissociation of the donor RNA from the intermediate.
Other experiments using a replication intermediate similar to the donor
RNA·DNA duplex showed that an acceptor RNA could displace the donor
in the presence of NC (see the introduction and Ref. 2). This occurred
in the absence of RT, indicating that no RNase H activity was required
for the strand exchange. However, at the same time, the current work
clearly shows that the acceptor would not be immune to degradation
during this process. Extensive degradation before completion of strand exchange could result in dissociation of the acceptor from the trimer
complex. But the acceptor could still potentially bind the nascent DNA
strand if the donor dissociated, because the complementary regions near
the 3'-ends of the acceptor RNA and DNA would be preserved, but
shortened. In this regard NC may play two important roles in the
exchange process, promotion of the association of the acceptor and DNA
as well as limiting RNase H cleavage (see Fig. 10), which would favor
exchange over acceptor dissociation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C, and a fresh aliquot was used for each
experiment. T4 polynucleotide kinase was from United States
Biochemical. HIV-1 nucleocapsid protein (NC) was obtained from Enzyco
(Denver, CO). SP6, T3 RNA polymerases, calf intestinal alkaline
phosphatase, and rNTPs were obtained from Roche Molecular Biochemicals.
PvuII was obtained from American Allied Biochemical.
HaeIII, placental RNase inhibitor, and Escherichia
coli RNase H were obtained from Promega. Deoxyoligonucleotides
were synthesized by Genosys Inc. (The Woodlands, TX). Proteinase K was
obtained from Sigma. All other chemicals were from Fisher Scientific or
Sigma. Radiolabeled compounds were from PerkinElmer Life Sciences.
) (14), and T3 RNA polymerase was used to prepare run-off transcripts 50 nucleotides in
length. For the acceptor RNA
(5'-GAAUACUCAAGCUUAUGCAUGCGGCCGCAUCUAGAGGGCCCGGAUCAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAG), the vector plasmid pGEM-11ZF+ (Promega) was cleaved with
BamHI, and plasmid pBSM13+ was cleaved with EcoRI
and HindIII. All termini were filled in with Klenow
polymerase. The EcoRI/HindIII insert from pBSM13+
was isolated and ligated into the plasmid vector pGEM-11ZF+ and
transformed into E. coli XL1 blue cells. Blue white screening was used to isolate white colonies that contained the pGEM-11ZF+ plasmid with the insert. The plasmid with the insert was
isolated and used to obtain the acceptor RNA. The plasmid was cleaved
with PvuII, and SP6 RNA polymerase was used to prepare run-off transcripts 262 nucleotides in length. Reactions for both the
donor and acceptor RNA were extracted with phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and precipitated with ethanol. The acceptor RNA
was further cleaved to obtain an ~99-base pair template as follows:
To the acceptor RNA, a DNA oligonucleotide 20 bases long was hybridized
by mixing the RNA and the specific primer deoxyoligonucleotide (5'-acctcgagggatcagcttgc) at a 1:5 (RNA:DNA) ratio in 50 mM
Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 80 mM KCl. The DNA bound to bases 94-114 from the 5'-end of
the RNA. The mixture was heated to 65 °C for 5 min and then slowly
cooled to room temperature. The RNA was then treated with E. coli RNase H (4 units) at 37 °C for 30 min in 50 mM
Tris-HCl (pH 8.0), 1 mM DTT, 10 mM
MgCl2, and 80 mM KCl. The final length of the
RNA is approximate, because cleavage can theoretically occur anywhere
within the 20-base hybrid region. In practice most RNAs differed by
only a few bases in length. The precipitates from the donor RNA and the
acceptor RNA after its treatment with E. coli RNase H were
resuspended in 1× formamide dye (45% formamide, 5 mM EDTA
(pH 8), 0.05% xylene cyanol, and 0.05% bromphenol blue). The two RNA
were gel-purified on denaturing polyacrylamide gels. The
electrophoresed RNA was located by ultraviolet shadowing, excised, and
eluted overnight by the crush and soak method (15) in a buffer
containing 50 mM Tris-HCl (pH 8), 80 mM KCl,
and 1 mM DTT. The eluate was separated from the
polyacrylamide by centrifugation and subsequent filtration through a
0.45-µm disposable syringe filter. The RNA was recovered by
precipitation in ethanol with 300 mM sodium acetate.
bx, where a = 1, f(x) = y (relative
cleavage), x = time, and b = koff value). In these experiments a slight
modification was made. The value at 10 s was set equal to the
starting relative value of 1 rather than the time 0 value. This allowed
a significantly better fit of the data to the equation. This is
probably due to the addition of MgCl2 along with the trap
for time 0. Divalent cation greatly stabilizes binding of RT to the
substrate (16). Because HIV-RT likely exists in a closed (tight
binding) and open (weak binding) conformation (17-19), those bound in
open conformation may rapidly switch to closed conformation upon
Mg2+ binding while they would dissociate in the presence of
trap and absence of Mg2+. This would result in a
disproportionately high cleavage amount at the time 0 point as was observed.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of substrates. Shown are
schematic diagrams of the trimer and its constituent dimeric parts
(acceptor RNA·DNA and donor RNA·DNA) as well as other substrates
derived from the acceptor RNA·DNA (acceptor RNA· DNA (recessed
3'-DNA) and acceptor RNA·DNA (+10 hybrid)). The positioning and
orientation of HIV-RT on the donor RNA·DNA is also shown.
Numbers indicate the lengths of the various regions in
nucleotides. Asterisks indicate positions of the
32P label.
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Fig. 2.
Autoradiogram of the isolated substrates
separated on a native gel. Radiolabeled nucleic acids were
hybridized and run on a 6% native polyacrylamide gel. The nucleic
acids used for each sample are indicated above the
lanes. On the far right isolated trimer
preincubations at 37 or 95 °C were run. Approximate migration
positions of the acceptor and donor RNA·DNA (4th and
5th lanes from left, respectively) are indicated by
brackets on the left.
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Fig. 3.
Autoradiogram of time-course cleavage of the
acceptor RNA·DNA, acceptor RNA·DNA (recessed 3'-DNA), and acceptor
RNA·DNA (+10 hybrid) complexes. Isolated complexes (as
indicated), 5'-end-labeled on the acceptor RNA, were incubated for the
indicated time with 2 units of HIV-RT as described under "Experiment
Procedures." Digestion times were 0 or 15 s, or 1, 2, 4, 8, or
16 min, as indicated. Size markers on the left and
right are in nucleotides and were based on the position of G
residues located by RNase T1 cleavage (lanes A) of the
substrate RNA. Schematic diagrams of the various substrates are shown
at the top of the figure above the
lanes with that substrate along with approximate positions
of prominent early cleavage products (arrows). The
numbers above the arrows refer to the number of
nucleotides behind either the junction (acceptor RNA·DNA and acceptor
RNA·DNA (+10 hybrid)) or 3' recessed terminus (acceptor RNA·DNA
(recessed 3'-DNA)) that the cleavage occurred. Refer to Fig. 1 for a
more detailed drawing of the substrates. Lanes A, partial
digestion of template RNA with RNase T1; lanes B, partial
base hydrolysis of the template RNA for 15 s; lanes C,
partial base hydrolysis of the template RNA for 1 min.
18 position). Results of a
cleavage assay are shown in Fig. 3 (acceptor RNA·DNA (recessed
3'-DNA)). Early time points revealed initial cleavages 19 (64-nucleotide product) and 16 (61-nucleotide product) bases behind the
3'-end. There were also some nonspecific cleavages that produced longer
products but the
19 and
16 cleavages were clearly the most
prominent. At later points cleavages at
11 (56 bases) were observed
as the levels of the larger products decreased. The results indicate
that cleavage on the standard substrate with a recessed 3' terminus
leads to the expected results with a large proportion of initial
cleavages occurring from RT molecules bound at the 3' terminus.
Subsequent 3'-5' processing results in many of these products being
processed to smaller products that likely destabilize the RNA·DNA
hybrid. In contrast, on the acceptor RNA·DNA the RNA·DNA junction
only loosely directed cleavage and processing was partially inhibited
(see above).
21 relative to the junction), 54 (
19), 50 (
15), 44 (
9), and 43 (
8) nucleotides were observed.
Most products were processed to the 44- and 43-base products at latter
time points. This was in contrast to the original acceptor RNA·DNA
where processing to smaller products was slowed. Results indicate the
junction structure does not directly inhibit processing to shorter products.
19 and
16 (64 and 61 nucleotides, respectively) products identified
above were clearly the dominant cuts at the 2- and 5-s time points, in
agreement with initial cleavage being directed by the position of the
3' recessed end. The preference was not as striking on the acceptor
RNA·DNA substrates, however, cleavage at the
15 (60 nucleotide
product) position relative to the junction was most prominent on the
original acceptor RNA·DNA. On the acceptor RNA·DNA (+10 hybrid),
cleavages at
21 (56 bases) and
15 (50 bases) were most prominent.
This suggests that RT has some preference to bind and cleave near the
junction.
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Fig. 4.
Autoradiogram of time-course cleavage of the
acceptor RNA·DNA, acceptor RNA·DNA (recessed 3'-DNA), and
acceptor RNA·DNA (+10 hybrid) complexes in a trap assay.
Isolated complexes (as indicated), 5'-end-labeled on the acceptor RNA,
were incubated with 2 units of HIV-RT as described under
"Experimental Procedures" for the trap assay. After RT pre-binding,
reactions were initiated with MgCl2 in the presence of the
poly(rA)-oligo(dT) trap. Digestion was continued for the indicated time
in seconds. Lanes D, complete digests of the substrates with
5 units of RT for 20 min in the absence of trap; lanes E,
trap control in which RT was pre-mixed with the trap before addition to
the substrate and incubated for 80 s; lane F, control
incubated in the absence of RT. Other labels were as indicated in Fig.
3.
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Fig. 5.
A and B, off-rate
(koff) determination for acceptor RNA·DNA and
acceptor RNA·DNA (recessed 3'-DNA) complexes. Isolated complexes
(as indicated), 5'-end-labeled on the acceptor RNA, were incubated with
2 units of HIV-RT as described under "Experimental Procedures" for
the "Off-rate (koff) determinations."
Panel A shows an autoradiogram from a typical experiment.
After RT pre-binding, poly(rA)-oligo(dT) trap was added and the
incubations continued for the time indicated. Digestion was then
initiated by the addition of MgCl2, and reactions were
continued for 5 min. Positions of the full-length and cleaved acceptor
RNA are indicated. Lane A, control incubated without enzyme;
lane B, trap control in which RT was pre-mixed with the trap
before addition to the substrate and incubated for 100 s;
lanes C, complete digest of the material with 5 units of RT
for 20 min in the absence of trap. Other markings are as indicated in
Fig. 3. Panel B shows a plot of relative cleavage
versus time derived from the experiment in A.
Values are relative to the 10-s time point in the assay, which was set
equal to 1 (see "Experimental Procedures"). The
koff values from this experiment were 0.018 and
0.009 s 1 for acceptor-DNA and acceptor-DNA (recessed 3'),
respectively. Overall values of 0.015 ± 0.003 and 0.008 ± 0.001 s
1 (average of three experiments ± S.D.) were
determined for acceptor RNA·DNA and acceptor RNA·DNA (recessed
3'- DNA), respectively.
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Fig. 6.
Autoradiogram of time-course cleavage of the
trimer. Isolated trimer, 5'-end-labeled on all three nucleic acid
strands to approximately the same specific activity, was incubated with
0.2 unit of HIV-RT as described under "Experimental Procedures."
Digestion was continued for 0, 15, or 30 s, or 1, 2, 4, 8, 12, or
16 min. Positions of the starting material and cleaved acceptor or
donor RNAs are indicated. A schematic diagram of the trimer substrate
is shown at the top of the figure along with approximate
positions of prominent early cleavage products (arrows).
Arrow designations are as described in Fig. 3.
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Fig. 7.
A and B, off-rate
(koff) determination for the trimer.
Isolated trimer, 5'-end-labeled on all three nucleic acid strands
to approximately the same specific activity, was incubated with 2 units
of HIV-RT as described under "Experimental Procedures" for the
off-rate (koff) determinations. Panel
A shows an autoradiogram from a typical experiment. After RT
pre-binding, poly(rA)-oligo(dT) trap was added, and the incubations
continued for the time indicated. Digestion was then initiated by the
addition of MgCl2, and reactions were continued for 5 min.
Lane A, control incubated without enzyme; lane B,
trap control in which RT was pre-mixed with the trap before addition to
the substrate and incubated for 100 s; lane C, complete
digest of the material with 5 units of RT for 20 min in the absence of
trap. Other markings are as indicated in Fig. 6. Panel B
shows a plot of relative cleavage versus time derived from
the experiment in A. Values are relative to the 10-s time
point in the assay, which was set equal to 1 (see "Experimental
Procedures"). The koff values from this
experiment were 0.014 and 0.005 s 1 for the acceptor and
donor RNAs, respectively. The experiment was repeated with and yielded
similar results.
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Fig. 8.
Autoradiogram of cleavage of the trimer in a
trap assay with varying RT. Isolated trimer, 5'-end-labeled on all
three nucleic acid strands to approximately the same specific activity,
was incubated with the indicated amount of HIV-RT as described under
"Experimental Procedures" for the trap assay. After RT pre-binding,
reactions were initiated with MgCl2 in the presence of the
poly(rA)-oligo(dT) trap. Digestion was continued for 80 s. Lane
A, control incubated in the absence of RT; lane B,
substrate incubated with 0.5 units of E. coli RNase H for 20 min in the absence of trap; lane C, trap control in which RT
was premixed with the trap before addition to the substrate and
incubated for 80 s; lane D, complete digests of the
substrates with 5 units of RT for 20 min in the absence of trap. Other
markings were as indicated in Fig. 7.
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Fig. 9.
Autoradiogram and graph of a time-course
cleavage of the trimer in a trap assay. Panel A, isolated
trimer, 5'-end-labeled on all three nucleic acid strands to
approximately the same specific activity, was incubated with 2 units of
HIV-RT as described under "Experimental Procedures" for the trap
assay. After RT pre-binding, reactions were initiated with
MgCl2 in the presence of the poly(rA)-oligo(dT) trap.
Digestion was continued for the indicated time in seconds, and samples
were electrophoresed on a denaturing polyacrylamide gel to generate the
autoradiogram. Lane A, control incubated in the absence of
RT; lane B, complete digests of the substrates with 5 units
of RT for 20 min in the absence of trap; lane C, trap
control in which RT was pre-mixed with the trap before addition to the
substrate and incubated for 5 min. Other markings are as
indicated in Fig. 6. Panel B, graph of the percentage total
substrate cleaved versus time derived from the experiment
shown in panel A. This experiment was repeated with similar
results.
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Fig. 10.
A and B, autoradiogram and
graph of a time course cleavage experiment with the trimer in the
presence of absence of NC protein. A, isolated trimer,
5'-end-labeled on all three nucleic acid strands to approximately the
same specific activity, was incubated with 0.2 unit of HIV-RT in the
presence or absence of NC protein at 0.17 (1 NC per 7 nucleotides total
substrate) or 0.6 µM (1 NC per 2 nucleotides total
substrate) as described under "Experimental Procedures." Digestion
was continued for the indicated time, and samples were electrophoresed
on a denaturing polyacrylamide gel to generate the autoradiogram. Other
markings are as indicated in Fig. 6. B, graph of relative
substrate remaining versus time derived from the experiment
shown in panel A. This experiment was repeated with similar
results.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank the Genetic Institutes for the kind gift of HIV-RT.
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FOOTNOTES |
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* This work was supported by NIGMS National Institutes of Health Grant GM51140.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.
Present address: Dept. of Cancer Immunology and AIDS, Dana-Farber
Cancer Inst. and the Dept. of Pathology, Division of AIDS, Harvard
Medical School, Boston, MA 02115.
§ To whom correspondence should be addressed: Dept. of Cell Biology and Molecular Genetics, University of Maryland, Bldg. 231, College Park, MD 20742. Tel.: 301-405-5449; Fax: 301-314-9489; E-mail: jd146@umail.umd.edu.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M210201200
2 A. Raja and J. J. DeStefano, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: NC, nucleocapsid protein; RT, reverse transcriptase; HIV, human immunodeficiency virus; RNase H, ribonuclease H, DTT, dithiothreitol.
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