From the McGill University AIDS Centre, Lady Davis
Institute-Jewish General Hospital,
Montréal, Québec H3T 1E2, Canada,
¶ Max-Planck-Institut für Biochemie,
D-82152 Martinsried, Germany, and
Istituto di
Strutturistica Chimica, CNR,
I-00016 Monterotondo Stazione, Rome, Italy
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ABSTRACT |
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In this study, we have analyzed the
interdependence between the polymerase and RNase H active sites of
human immunodeficiency virus-1 reverse transcriptase (RT) using an
in vitro system that closely mimics the initiation of
(+)-strand DNA synthesis. Time course experiments show that RT pauses
after addition of the 12th DNA residue, and at this stage the RNase H
activity starts to cleave the RNA primer from newly synthesized DNA.
Comparison of cleavage profiles obtained with 3'- and 5'-end-labeled
primer strands indicates that RT now translocates in the opposite
direction, i.e. in the 5' direction of the RNA strand. DNA
synthesis resumes again in the 3' direction, after the RNA-DNA junction
was efficiently cleaved. Moreover, we further characterized complexes
generated before, during, and after position +12, by treating these
with Fe2+ to localize the RNase H active site on the DNA
template. Initially, when RT binds the RNA/DNA substrate, oxidative
strand breaks were seen at a distance of 18 base pairs upstream from
the primer terminus, whereas 17 base pairs were observed at later
stages when the enzyme binds more and more DNA/DNA. These data show
that the initiation of (+)-strand synthesis is accompanied by a
conformational change of the polymerase-competent complex.
Retroviral RTs1 are
multifunctional enzymes possessing RNA- and DNA-dependent
polymerase activities and a ribonuclease H (RNase H) activity that
degrades the RNA strand of RNA/DNA hybrids (1, 2). Like other
retroviruses, human immunodeficiency virus type 1 (HIV-1) uses a
cellular tRNA primer to initiate reverse transcription from a
complementary primer-binding site (PBS) near the 5'-end of the viral
RNA (3-6). Despite changes of binding and kinetic properties, observed
concomitant with synthesis of the first DNA strand (7), i.e.
( RT-DNA/DNA complexes, which are generated during (+)-strand synthesis,
have been relatively well characterized (12-15). The crystal structure
of HIV-1 RT complexed to an 18-base primer/19-base template DNA
homoduplex (12) suggests that the first 7 DNA/DNA base pairs near the
polymerase active site adopt an A-type conformation, whereas the region
further upstream is in the preferred B-conformation, both structurally
distinct segments being separated by a kink.
Little information is currently available regarding the interaction
between RT and the RNA/DNA primer/template combination that is
initially bound during (+)-strand synthesis. A short segment near the
3'-end of viral genomic RNA, termed the polypurine tract (PPT), is
resistant to RNase H degradation and, unlike the rest of the genomic
RNA, remains intact during synthesis of the ( Here, we demonstrate that the RNA primer is cleaved precisely after the
12th DNA residue has been incorporated. This is an unexpected result,
since the relative positions of the polymerase and RNase H active sites
would not allow DNA synthesis and RNase H degradation to occur at the
same time on the same strand. In contrast, the spatial relationship
between both active sites facilitates temporally coordinated activities
on opposite strands, in a distance of about 18 base pairs, when RT is
complexed with DNA/RNA primer/templates (9). We now show that RT pauses
after addition of the 12th DNA residue during the initiation of
(+)-strand synthesis. At this point, another RT molecule (not the one
that accomplishes DNA synthesis) binds the substrate in an RNase
H-competent binding mode to cleave the RNA primer. The directionality
of the latter reaction, i.e. in 5' direction with respect to
the RNA strand, distinguishes this binding mode from the
polymerase-competent complex. In the polymerase-competent mode, RT
binds its RNA/DNA substrate in the same orientation as that described
for DNA/DNA, RNA/RNA, and DNA/RNA primer/templates. This has been shown
by treating stalled complexes with Fe2+, which allowed us
to localize the RNase H active site on the DNA template (15). Together,
our data provide a detailed model for the early steps of (+)-strand DNA synthesis.
Nucleic Acids and HIV-1 RT--
Oligonucleotides used in
this study were derived from the polypurine tract located near the
3'-end of the HIV-1 genome (HXB-2 isolate). DNA oligonucleotides as
well as RNA and chimeric DNA-RNA primers were synthesized on an Applied
Biosystems 392/8 synthesizer using the standard phosphoramidite method,
followed by purification on 12% polyacrylamide, 7 M urea
gels containing 50 mM Tris borate, pH 8.0, 1 mM
EDTA. 5'-End labeling of oligonucleotides (DNA, RNA, or chimera) was
conducted with [ Primer/Template Sequences and Generation of Stalled
Complexes--
Primer/template sequences were prehybridized prior to
incubation with RT. A mixture containing the template strand (120 nM) and 32P-labeled primer (100 nM)
in a buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM NaCl was heated to 95 °C for 2 min followed by
incubation at 72 °C for 10 min and cooled for 20 min to room
temperature. Complete hybridization was confirmed on native
polyacrylamide gels. The pre-annealed primer/template substrate (100 nM) was incubated with HIV-1 RT (150 nM) in a
buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM
NaCl. Appropriate dNTP/ddNTP combinations (100 and 200 µM, respectively), which permitted control of the extent
of DNA synthesis, were included in the preincubation mixture to form
ternary RT-primer/template-dNTP complexes. 20-µl reactions were
initiated with MgCl2 at a final concentration of 6 mM and allowed to proceed for 15 min at 37 °C. Nucleic
acids were subsequently precipitated with ethanol, and reaction
products were finally analyzed on 15% polyacrylamide, 7 M
urea gels, followed by exposure overnight.
A list of different primers and the template used to generate registers
3, 4, 6, 11, 12, 13, 14, 15, 16 and 17 are as follows: DNA template
57D,
5'-CGTTGGGAGTGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAGA-3'; the chimeric primer 3D-17R and the homologous DNA primer 20D, 5'-UUAAAAGAAAAGGGGGGACT-3' (RNA residues are written in
italics) and 5'-TTAAAAGAAAAGGGGGGACT-3'. Both primers were employed to generate registers 3, 4, 6, 11, and 12 as shown in Fig. 1. The chimeric
RNA-DNA oligonucleotides 12D-17R,
5'-UUAAAAGAAAAGGGGGGACTGGAAGGGCT-3', 14D-17R,
5'-UUAAAAGAAAAGGGGGGACTGGAAGGGCTAA-3', and 16D-17R,
5'-UUAAAAGAAAAGGGGGGACTGGAAGGGCTAATT-3', were used to
generate registers 12-16. Register 12 was obtained by annealing the
12D-17R primer to the DNA template and incubating the
preformed substrate with RT and ddATP (500 µM) as
described above. In the latter case, Mg2+ was omitted from
the reaction mixture to prevent incorporation of the chain-terminating
stop-nucleotide as well as RNase H degradation of the chimeric primer.
Register 13 was then generated by addition of Mg2+ (6 mM) to allow incorporation of ddATP. The presence of
Mg2+ also resulted in RNase H cleavages at the RNA-DNA
junction. Registers 14 and 15 were generated with primer 14D-17R and
ddTTP, and registers 16 and 17 were generated with primer 16D-17R and
ddCTP using the same procedure.
Time Course Experiments--
Time course experiments were
similarly performed. In these experiments, we lowered the ratio of
RT-primer/template to monitor appearance of all reaction products
during a time course of 60 min. The pre-annealed primer/template
substrate (100 nM) was preincubated for 5 min at 37 °C
with HIV-RT (50 nM) in a buffer containing 50 mM Tris-HCl, pH 7.8, and 50 mM NaCl.
Polymerization and RNase H degradation was then initiated
simultaneously by addition of MgCl2 at a final
concentration of 6 mM. Reactions were performed at 37 °C
and stopped at different time points by adding 1-µl aliquots of the
reaction mixture to 9 µl of 95% formamide containing 40 mM EDTA. Most of the time course experiments were performed
with the DNA template 57D and the chimeric 3D-17R primer. We also used the pure RNA primer 17R, 5'-UUAAAAGAAAAGGGGGG-3', the
chimeric 12D-17R primer and its isolated DNA segment 12D,
5'-ACTGGAAGGGCT-3', for comparative purpose (see Fig. 5). Additionally,
to analyze whether the reaction profile depends on the template
sequence, we employed another DNA template with a randomly chosen
sequence upstream from the PPT binding site:
5'-CAGTGATCTCGAGCTACATGATCGTCACCCCCCTTTTCTTTTAAAAAGTGGCTAAGA-3'.
Fe2+- and ONOOK-dependent
Cleavages--
Stalled RT-nucleic acid complexes that contained
differentially extended primer strands were prepared as described
above. RT was used in a final concentration of 150 nM, and
the ratio of primer/template (radiolabeled at the 3'-end or 5'-end) was 120/100 nM.
Preformed complexes in a volume of 16 µl were incubated with a
mixture of 2 µl of Fe(NH4)2SO4·6H2O (400 µl) and 2 µl of dithiothreitol (50 mM). Reactions were
allowed to proceed for 5 min at 37 °C and were stopped with 40 µl
of a solution containing 0.1 M thiourea, 200 ng of tRNA, 10 mM EDTA, and 0.6 M sodium acetate. Samples were
subsequently precipitated with ethanol and loaded on a 12% polyacrylamide-urea gel. The size of the
Fe2+-dependent cleavage fragments were assigned
by a T-ladder generated after modifying the DNA with
OsO4/bipyridin followed by cleaving the sugar-phosphate
backbone with piperidine. Reactions with ONOOK were performed as
described previously (15). Briefly, RT-primer/template complexes were
prepared in a buffer containing 80 mM sodium cacodylate, pH
7, and 20 mM NaCl. Cleavage reactions were conducted by
adding 1 µl of a stable alkaline ONOOK solution (pH 12, 90 mM) to the sample solution buffered at pH 7. Reaction
products were analyzed as described above.
In order to analyze the interplay between RT polymerase and RNase
H active sites on a PPT-derived RNA/DNA primer/template substrate, we
first generated a series of stalled complexes, termed registers,
through use of various dNTP/ddNTP combinations. Instead of employing a
pure RNA primer that, because of the sequence 5'-CAGT-3' immediately
flanking the primer, leads to termination of DNA synthesis at positions
+1, +2, +3, and +4, we devised a chimeric DNA-RNA primer (Pr + PPT
3D-17R) that yields chain termination at positions +4, +6, +11, and +12
(Fig. 1). This approach allowed us to
study early events of the initiation reaction when RT is complexed with its initially bound RNA/DNA substrate as well as with chimeric replication intermediates. We also devised a DNA primer (Pr + PPT 20D)
to generate a homologous DNA/DNA substrate for comparative purposes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-strand DNA, complexes with the initially bound RNA/RNA duplex and
the newly synthesized DNA/RNA substrates share certain common features.
RNase H cleavages on the RNA strand of DNA/RNA primer/template
combinations occur at a constant distance of 18 bp upstream of the
nascent primer terminus (8, 9). Analogously, RNase H-induced cleavages
within the tRNA/RNA duplex, designated as RNase H* activity (10), were
observed at the same distance from the 3'-end of the primer, although
these cuts are restricted to stalled complexes (11). Together, these
data provide strong evidence that RT binds to both RNA/RNA and DNA/RNA
substrates with the same orientation, and the number of bp between the
two active sites is 18 in each case.
)-strand DNA. The PPT
fragment then serves as a primer for (+)-strand polymerization, whereas
the (
)-strand DNA is used as a template to guide synthesis. Later
after initiation, the RNA primer is removed by RNase H cuts at the
DNA-RNA junction and adjacent positions (16, 17).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and T4 polynucleotide kinase;
and 3'-end labeling of the DNA template was performed with
[
-32P]ddATP using terminal transferase (Boehringer
Mannheim) according to the manufacturer's recommendation. The chimeric
3'-end-labeled primer (3D-17R) was generated by extending
the pure RNA PPT-primer (17R) with HIV-1 RT in the presence of dATP,
dCTP, and [
-32P]dTTP using reaction conditions as
described below. All end-labeled nucleic acids were again
electrophoretically purified to obtain homogeneous products.
Heterodimeric HIV-1 RT (p66/p51) and the RNase H-deficient p66
(E478Q)/p51 mutant enzyme were prepared and purified essentially
as described (37).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RNA/DNA primer/template combination used to
study early events during the initiation of (+)-strand synthesis.
We devised a DNA template (T-PPT 57D) and a chimeric DNA-RNA primer (Pr + PPT 3D-17R) that serve as substrates in this in vitro
study. The chimeric primer contains 17 RNA nt at its 5'-end
(bold) and three DNA residues at its 3'-end that would have
been incorporated during the first three steps of DNA synthesis. Thus,
incorporation of 1, 3, 8, and 9 nt to the chimeric DNA-RNA primer
yields complexes termed registers 4, 6, 11, and 12. The numerical order
of registers reflects the number of DNA residues of the primer strand.
Register 3 represents the RT-RNA/DNA complex in the absence of added
dNTPs. The run-off product, generated in the presence of the four
dNTPs, contains 27 DNA residues.
Characterization of Stalled RT-RNA/DNA and RT-DNA/DNA
Complexes--
The polymerization and RNase H cleavage products of
differentially arrested RT-nucleic acid complexes are shown in Fig.
2. As expected, the 5'-end-labeled DNA
primer was precisely elongated by 1, 3, 8, and 9 nt (left panel,
lanes 4, 6, 11, and 12). The presence of all four dNTPs
yielded a product of 44 nt in length (lane 27). The results
obtained with the homologous RNA/DNA substrate are more complex (Fig.
2, right panel), since the RT polymerase and RNase H
activities both use the same strand as substrate. Consistent with
previous reports (16-18), RNase H cuts are seen at the DNA-RNA
interface and further upstream, adjacent to the junction, at four
consecutive positions, and between positions 7 and
8.
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Precise extension with reasonable yield was observed only in register 4 when a single nt was added to the chimeric primer (Fig. 2, right panel, lane 4). This product was termed 4D-17R, with respect to the number of DNA and RNA residues of the extended primer. The expected reaction product in register 6, i.e. 6D-17R, is visible, but another shorter product, termed 1D-17R (see below), is also seen just above the RNase H cuts. Similar observations were made in registers 11 and 12 (lanes 11 and 12). In either case, the expected reaction products were less pronounced than the shorter ones, i.e. 2D-17R and 3D-17R.
Primary and Secondary Initiation Reactions--
To characterize
further the origin of the shorter reaction products, we next followed
their formation in time course experiments. These were performed with
dNTP/ddNTP combinations to yield registers 6 and 11 (Fig.
3A). The 6D-17R product
appeared within the first few minutes and increased only slightly with
longer reaction times (Fig. 3A, left panel). RNase H cuts at
the junction (17R) and further upstream (16R and 15R) were seen clearly
after 3 min, and the above-mentioned 1D-17R product, which migrated a
little slower than the 17R cleavage band, first appeared between 12 and 20 min and further increased over longer incubation times. This result
shows an order of product formation, i.e. first elongation to yield the 6D-17R product followed by RNase H cleavages and finally
the formation of the 1D-17R product. RT thus initiates a second round
of (+)-strand synthesis using the cleaved RNA fragment as a primer.
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We will now use the term "primary reaction" and "secondary reaction" to distinguish between these two types of initiation events (schematically illustrated in Fig. 3C). During the primary reaction, incorporation of ddATP results in chain termination at position +6, whereas, during the secondary initiation reaction, ddATP is added at position +1 to yield a complex, termed register 1'. Equivalent patterns were seen in registers 11 and 12 (Fig. 3A, right, and B, left); here, the stop-nucleotides (ddCTP and ddTTP) were added at positions +2 and +3 to yield the secondary 2D-17R and 3D-17R products.
The presence of dGTP should, in principal, allow elongation of RNA fragments that are generated by RNase H cleavages at the DNA-RNA junction and adjacent positions. Thus, the shorter secondary products may represent a heterogeneous mixture of chimeric strands, each of which contains a different number of DNA residues at the 3'-end. It can hardly be deduced from the above experiment which of the RNA fragments is preferentially used during the secondary initiation reaction. However, the observation that the 3D-17R product co-migrates exactly with the unextended primer indicates that the 17R cleavage product is most efficiently used (Fig. 3B, left panel). If shorter cleavage products would have been extended, the 3D-17R fragment would have migrated somewhat faster, due to the increased number of DNA residues in the chain-terminated product (see Fig. 2; the pure DNA oligonucleotide migrates faster than the chimeric DNA-RNA strand of the same length).
Product Formation in the Presence of All Four dNTPs-- The above results showed that the initiation of (+)-strand synthesis is a complex process that involves primary and secondary initiation reactions, superimposed on the primer removal reaction. In order to understand better the natural reaction pathways, we next analyzed the order of product formation in the presence of all four dNTPs. The time course (Fig. 3B, right panel) shows that RT pauses at position +12, before DNA synthesis resumes to yield the run-off product (27D-17R). All reaction products, including the cleavage products (17R, 16R, and 15R), are already seen after the 1st min. The primer removal reaction appears to be equally efficient in the presence or absence of chain terminating nucleotides, as the enzyme encounters the template around position +12. For example compare Fig. 3A register 11, Fig. 3B register 12, and run-off synthesis. Thus, these data do not provide any information regarding the temporal relationship between the polymerase and RNase H active sites. The primer may be randomly cleaved at any point after initiation, immediately after synthesis of the run-off product, or alternatively the primer may already be removed once the 12th nt has been added. The above data do not enable us to distinguish among these various scenarios, since cleaved 5'-end-labeled RNA fragments migrate at the same position in each case.
We therefore followed formation and processing of the initially
synthesized product, using a 3'-end-labeled primer (Fig.
4). Putative secondary reactions are not
detectable in this experiment, since the radiolabel is attached to the
third DNA residue. The accumulation of the 12D-17R product, the pausing
site, was again seen at early stages after initiation, i.e.
1 and 3 min. A relatively small fraction of this product is further
extended to yield the unprocessed run-off product, which is later
cleaved, as shown by the time-dependent decrease of this
band. However, it seems that most of the 12D-17R reaction intermediate
is prematurely cleaved to yield the 12D product. The 12D product then
accumulates between 3 and 20 min and is later extended to yield the
processed run-off product. Taken together, these data demonstrate that
the primer is not randomly cleaved at any stage after initiation. The
appearance of the single 12D product shows that the RNA primer is
efficiently and precisely cleaved at the RNA-DNA junction after the
12th DNA residue has been incorporated.
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Whether secondary initiation reactions also occur in the absence of
chain-terminating stop-nucleotides cannot be answered on the basis of
the above time course experiments. Synthesis of a secondary (+)-strand
may be initiated after pausing and the following primer removal. Our
data point to the existence of three different primer 3'-ends at
position +12 that can potentially be recognized by the polymerase
active site, i.e. the DNA 3'-end of the elongated
unprocessed primer (12D-17R), the DNA 3'-end of the elongated processed
primer (12D), and the RNA 3'-end of the processed primer (17R). To
determine which of these three 3'-ends might preferentially be used for
DNA synthesis, we used a stoichiometric mixture of primer/template
combinations with an uncleaved chimeric primer containing 17 RNA and 12 DNA residues and a nicked substrate with a 17-mer RNA primer and a
12-mer DNA primer. The former represents the substrate prior to primer
removal, whereas the latter mimics the substrate after the RNase H cut at the RNA-DNA junction. The efficiency of DNA synthesis from each of
the three available 3'-ends was compared in a competition experiment,
using three separate reaction mixtures with 5'-end-labeled primers and
an RNase H-deficient RT (Fig.
5A). The data show the following order of efficiency: 12D > 12D17R 17R. After a
60-min reaction, 55% of the 12D primer and 45% of the chimeric
12D-17R primer were extended to yield the final run-off product. In
contrast, only 25% of the 17R was found to be extended, and most of
the extended fraction accumulated after incorporation of the first nucleotide. These data show that secondary initiation reactions are
clearly suppressed in the absence of a chain-terminated primary product
and that RT preferentially elongates the 3'-end of the newly
synthesized and processed DNA fragment. The additional pausing site at
position +1 does not necessarily correlate with putative difficulties
of RT to strand-displace the annealed 12D fragment, since accumulation
of this product is also seen in the absence of the 12D strand. Fig.
5B shows that initiation with the pure 17R primer resulted
in dual pausing at position +1 and, in agreement with the data
obtained with the 3D-17R primer, also at position +12. Moreover,
synthesis of full-length (+)-strand DNA is still very efficient in this
circumstance, indicating that the primer removal reaction is not
required for polymerization steps after RT has reached position
+12.
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The Number of Base Pairs Accommodated between the Two Active Sites
of HIV-1 RT with RNA/DNA and DNA/DNA Substrates--
We next analyzed
the possible structural requirements for the specific pausing site at
position +12. Pausing may be caused by the particular sequence or
secondary structures of the single-stranded DNA template that inhibit
the translocation of the enzyme. However, the specific pausing site
after addition of the 12th nt is not observed with an homologous
DNA/DNA substrate (Fig. 6), which indicates that pausing rather depends on the structure of the complexed
chimeric substrate and its interaction with RT. This conclusion is
additionally supported by the observation that different template
sequences, shown under experimental procedures, do not alter the
pausing profile as long as DNA synthesis was initiated with the RNA
primer (data not shown). To further approach this problem, we wished to
define structural characteristics of complexes generated before,
during, and after position +12. Studying RT-DNA/DNA complexes, we have
recently shown that the interaction between the DNA template and the
RT-associated RNase H can be monitored in the presence of
Fe2+ and that Fe2+ binds to one of the
metal-binding sites of the RNase H domain, thereby generating a high
local concentration of hydroxyl radicals that might serve as active
species to cause an oxidative strand break (15).
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Here, we have used this tool to determine the number of base pairs
between both active sites depending on the bound substrate, i.e. RNA/DNA or DNA/DNA. Although the purine-rich sequence
of the dsDNA substrate used in this study differs markedly from the PBS-derived sequence used previously (15), the number of bp between the
RT active sites was identical, i.e. 17 in each case (Fig.
7A, lane 1). In contrast, when
RT was complexed with the homologous RNA/DNA substrate, a specific cut
was seen on the DNA template at a distance of 18 bp upstream of the
primer terminus (lane 2). Lower concentrations of cleaved
products were seen when RT was bound to the RNA/DNA substrate,
suggesting a diminished efficiency of cleavage, but the cleavage
profile was exactly the same. However, the major product is shifted
exactly by a single nucleotide when RNA/DNA is bound to RT. The small
percentage of side products, i.e. less than 10%, can be
explained by the oxidative cleavage mechanism that involves diffusible
hydroxyl radicals as active species (15).
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To provide additional information in regard to differences between
RT-DNA/DNA and RT-RNA/DNA complexes, we also wished to study the
protein-nucleic acid interface using hydroxyl radicals generated via
Fe[EDTA]2 as well as via ONOOK, as described previously
(15, 19). However, a clear footprint could not be obtained, presumably
reflecting the ability of RT to bind these substrates in both a
polymerase-competent mode as well as one that facilitates removal of
the primer (see "Discussion"). Despite the lack of a clear
footprint, a faint band located on the DNA template strand, 7 bp
upstream of the primer 3'-end, was seen when both complexes were
treated with ONOOK (Fig. 7A, lanes 3 and 4). This
cut was also seen as part of the ONOOK-dependent footprint
on the DNA/DNA substrate derived from the PBS sequence (15). This
particular reaction is thus a feature of polymerase-competent complexes
and can be used as a second marker, in addition to the Fe2+
cut, to determine differences in the number of base pairs that fit in
the substrate-binding channel. It is interesting to note that the ONOOK
cut appeared at the same position, regardless whether DNA/DNA or
RNA/DNA was used as substrate. This observation indicates that the 1-bp
variance is not homogeneously distributed over the entire duplex.
Rather, it shows 7 bp between the primer 3'-end and the ONOOK cut,
whereas a 1-bp difference (10 versus 11 bp) was seen
upstream between the ONOOK and the Fe2+ cleavage sites
(Fig. 7B).
Number of Base Pairs between Both Active Sites in Registers
4-17--
The above data indicate that the number of base pairs
located between the active sites of RTs might change when the enzyme has passed the initially bound RNA/DNA substrate and starts to accommodate more and more the newly synthesized DNA/DNA substrate. To
address this issue, we next investigated registers 4, 6, 11, and 12 (Fig. 8, A and B)
using the radiolabel now found at the 5'-end of the DNA template. In
register 4, we observed the same cleavage positions on the DNA
templates as seen in the previous experiment (see Fig. 7), revealing
distances of 17 (Fig. 8A, lane 1) and 18 bp (lane
2), respectively, from the primer terminus. Thus, 3'- or 5'-end
labeling does not influence the positions of the Fe2+ cuts,
indicating an efficient and specific arrest of RT. Consistently, the
DNA template of RT-DNA/DNA complexes in registers 6, 11, and 12 was
always cleaved at a fixed distance of 17 bp from the primer terminus
(lanes 3, 5, and 7), whereas the cleavage
profiles on the corresponding RNA/DNA substrates are more complex. In
register 6 (lane 4), the cut at position 12 was located 18 bp upstream of the 3'-end of the primer generated during the primary
initiation event, whereas the cut at position
17 was located 18 bp
upstream of the 3'-end of the primer generated during the secondary
initiation event (see schematic in Fig. 8B). In
registers 11 and 12, only a single cut was seen, corresponding to a
distance of 18 bp upstream of the primer terminus of the secondary
product (lanes 6 and 8). This is in full
agreement with our analysis of product formation (Fig. 2) and confirms
the existence of a secondary initiation complex.
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To analyze whether the number of base pairs between both active sites
will decrease to 17 nt at later stages after initiation, we next
characterized stalled complexes in registers 12-17 (Fig. 9, A and B,
schematically). These were generated using chimeric primers with 12, 14, and 16 DNA residues, respectively. The preformed complexes,
containing 5'-end-labeled DNA templates, were incubated in the absence
of Mg2+, to ensure that the RNA primer remains intact,
which also excludes any secondary reactions. The next correct ddNTP was
additionally added in the reaction mixture to stabilize the
polymerase-competent complex. Under these reaction conditions,
Fe2+ cuts were not observed in register 12, and only a very
faint band, corresponding to a number of 17 bp, is seen in register 14. However, register 16 shows a clear cut at a distance of 17 nt from the
primer terminus (lane 16), which corroborates the data
obtained with pure DNA/DNA substrates. When these complexes were
preincubated with Mg2+, which initiates the primer removal
step and allows the addition of the single stop-nucleotide, the
specific Fe2+ cuts on the DNA template clearly re-appeared
at distances of 18 and 17 nucleotides from the primer terminus
(lanes 13 and 15).
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Taken together, the presence or absence of a specific Fe2+
cut on the DNA template, as well as differences in its precise
location, indicates structural differences between the various
RT-nucleic acid complexes that are generated during the initiation of
HIV (+)-strand synthesis. These data will be discussed in the context of our time course experiments that show the complex reaction pathway
of initiation, pausing, primer removal, and finally continuation of DNA synthesis.
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DISCUSSION |
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In this study we investigated the interplay between the polymerase
and RNase H active sites of HIV-1 RT during the initiation of
(+)-strand DNA synthesis. Based on time course experiments and
biochemical studies focused on the characterization of the various
RT-nucleic acid complexes involved in this process, i.e. RT
bound to RNA/DNA and DNA/DNA primer/templates as well as complexes containing chimeric replication intermediates, we provide a detailed model for the initiation of retroviral (+)-strand synthesis (Fig. 10).
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Mechanism of the Initiation of (+)-Strand Synthesis and Primer Removal-- In order to determine the orientation of RT on its initially bound RNA/DNA substrate, we treated stalled complexes with Fe2+, which allowed us to localize the RNase H active site on the DNA template (15). Complexes at early stages after initiation, with up to 6 newly synthesized DNA residues, show oxidative Fe2+ cuts on the DNA template at a constant distance of 18 bp upstream from the primer 3'-end. The specific Fe2+ cuts on the DNA template that appear in accord with the extent of DNA synthesis identified the position of the RNase H active site when the polymerase active site lies in the vicinity of the primer 3'-end (Fig. 10, step 1). This result is consistent with biochemical data obtained with DNA/RNA, RNA/RNA, and DNA/DNA primer/template combinations (Refs. 8, 9, 11, 15, and, for recent review, Ref. 20) and demonstrates that RT binds all of its various nucleic acid substrates with the same orientation.
The RNase H domain remains positioned over the template strand, as RT starts to generate more and more DNA/DNA. Particularly, in the complex with 16 newly synthesized DNA residues, the specific Fe2+ cut on the DNA template is seen opposite the DNA-RNA junction of the primer strand. Thus, the RNase H active site as part of a polymerase-competent complex cannot interact simultaneously with the primer strand.
The time course of (+)-strand DNA synthesis shows that RT pauses after the 12th DNA residue has been added (step 2). At this point, the RT-associated RNase H activity starts to cleave the RNA primer from newly synthesized DNA (step 3), which, we propose, is accomplished by another RT molecule that is not involved in DNA synthesis.
This proposal is strongly supported by the RNase H cleavage profiles
obtained with 5'- and 3'-end-labeled primer strands. A single cut at
the DNA-RNA junction is only observed with the 3'-end-labeled primer,
whereas the cleavage profile obtained with the 5'-end-labeled primer
shows a time-dependent increase in shorter products. The
shorter 5'-end-labeled fragments are thus identified as products that
follow the initial cut at the junction. The primer removal reaction is
thus accomplished in the 3' 5' direction, whereas DNA synthesis
proceeds in the opposite 5'
3' direction. These data support the
view that positioning of RT is reversed during the primer removal
reaction (see step 3 and Refs. 22 and 23). In the latter studies, it
has been suggested that RT binds close to the 5'-end of the RNA primer,
such that the RNase H active site is positioned over the RNA-DNA
junction. This is further supported by recent studies, showing that
mutations in the "primer grip" region of HIV-1 RT selectively
diminish this 5'-directed RNase H activity (24-26). Thus, the addition
of 12 residues to the RNA primer may extend the growing strand
sufficiently to facilitate binding of another competing RT molecule to
the 5'-end of the primer that, in turn, initiates removal of the latter.
The observation that specific Fe2+ cuts on the DNA template are not seen in the complex with 12 DNA residues may provide additional support for the existence of an RNase H-competent complex at this stage. However, we cannot exclude the possibility that the lack of Fe2+ cuts may be attributable to altered RT-nucleic acid interactions in the polymerase-competent binding mode.
DNA synthesis finally resumes from the newly synthesized DNA fragment after the DNA-RNA junction, and adjacent positions are efficiently cleaved (steps 4 and 5). The time course experiments indicate that a fraction of the intact primer is also extended and later cleaved after synthesis of the run-off product (step 6). We further show that the enzyme is also capable of elongating the cleaved PPT primer, particularly when chain-terminating stop-nucleotides were incorporated in the primarily synthesized DNA strand (step 7). We note that such secondary initiation reactions may critically limit the efficiency of nucleoside analog RT inhibitors, since reverse transcription could continue as long as cellular dNTPs are incorporated into the second strand.
A similar mechanism, designated "the (+)-strand primer recycling model," has recently been suggested for yeast Ty retrotransposon reverse transcription (27). It has been proposed that synthesis of a secondary (+)-strand might displace the primary (+)-strand strong stop product, thereby facilitating removal of the tRNA primer. Accordingly, the secondary (+)-strand species does not contain any tRNA sequences but is subsequently used to facilitate the second strand transfer. This model is consistent with the observation that Ty elements do not inherit tRNA sequences, although the (+)-strand strong stop DNA that contains the tRNA copy has been identified as the most abundant product in Ty 1 reverse transcription (28, 29). We have now demonstrated secondary initiation reactions for the first time, using a cell-free assay. Our data support the general principal of a primer recycling model which may in fact be important for reverse transcription of retrotransposons. However, numerous studies from different laboratories have shown that retroviruses inherit their tRNA sequences via the second strand transfer (30-32) which indicates that the primary synthesized (+)-strand product is involved in the second strand transfer. Therefore, a primer recycling mechanism may not be of relevance in regard to retroviral replication. This view is consistent with our in vitro data that show that secondary initiation reactions occur efficiently only when DNA synthesis of the primarily synthesized DNA strand is blocked after addition of a stop-nucleotide. In the presence of all four dNTPs, the polymerase active site preferentially extends the 3'-end of the newly synthesized DNA strand rather than the 3'-end of the cleaved RNA primer (Fig. 5).
Implications for a Conformational Change of the Bound Nucleic Acid-- When RT initially binds the RNA/DNA substrate, oxidative Fe2+ cuts were seen at a distance of 18 bp upstream from the primer terminus, whereas 17 bp were observed at later stages when the enzyme binds more and more DNA/DNA. This 1-base pair variance reflects structural differences between these complexes and further supports the idea that the number of base pairs that fit into the substrate binding groove between the two active sites of RT is an important criterion in determining differences in the conformation of complexed substrates (15).
Free in solution, RNA/DNA hybrids differ in local structural features from the classical A-form, but helical parameters that characterize the overall geometry of the helix, e.g. rise and twist, indicate an A-like conformation (33, 34). The pitch of an A-type helix is lower than that of a standard DNA/DNA B-form, and differences in twist angles between consecutive base pairs result in a 11-fold helix symmetry for the A-form, whereas a B-form helix contains 10 bp per turn (35). Therefore, a stretch representing the distance between the two enzymatic sites should accommodate about 3 more base pairs within a pure A-type helix than within a pure B-type helix. In accord with the crystal structure of the RT-dsDNA complex (12), we therefore suggest that the relatively small difference of 17 versus 18 bp accounts for the partial A-form of the complexed DNA/DNA duplex, whereas the RNA/DNA substrate is expected to retain the preferred A-type conformation.
Although we cannot exclude that subtle differences in the protein
conformation or the kink of the bound dsDNA substrate may also affect
our measurements, a further detailed comparison of our biochemical data
in solution and the crystal structure of the RT-dsDNA complex show
remarkable consistencies. According to the crystallographic data one
would only expect differences in the number of base pairs in the
upstream part of the complexed nucleic acids, since the DNA/DNA
substrate adopts an A-conformation in the vicinity of the polymerase
active site (12). This is supported by the finding that a specific cut
on the DNA template, induced by ONOOK, is located exactly 7 bp upstream
of the primer 3'-end, regardless whether DNA/DNA or RNA/DNA is used as
a substrate. Thus, the 10 DNA/DNA bp versus the 11 RNA/DNA
bp that are measured between the ONOOK and Fe2+ cuts may
represent precise differences of helical turns in B- versus
A-forms (Fig. 11). These findings
suggest that the initiation of (+)-strand DNA synthesis is accompanied
by a conformational change of the bound nucleic acid substrate,
i.e. the enzyme translocates from an initially bound A-type
RNA/DNA substrate into the mixed A/B-conformation of the newly
synthesized DNA/DNA.
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Taken together, our data show that the PPT primer is cleaved immediately after addition of the 12th DNA residue and the ensuing pause event. Were the pausing of RT to be irrelevant in primer removal, and were the latter event to be determined only by the length of the growing strand, RNase H cleavage should be detected even after incorporation of the 12th nucleotide. However, DNA fragments consisting of 13 or more residues were not observed, and we instead detected a single specific band corresponding to the 12-mer product (Fig. 4). Thus, the RT pausing event at position +12 is likely one that facilitates primer removal at this stage of DNA synthesis. Additionally, as stated above, the 12 newly synthesized residues may represent the critical length of the growing chain that permits binding of the competing enzyme to the 5'-end of the primer. We further note that the pausing of the synthesizing RT involved in synthesis and the binding of the competing RT involved in primer removal are not necessarily independent events.
Finally, the time course data obtained with the RNase H-deficient
enzyme show that the absence of the primer removal reaction does not
prevent full-length synthesis of DNA. Regardless, the primer removal
reaction is an important step to prevent reverse transcription of the
PPT fragment at the very last steps of replication, when ()-strand
DNA synthesis resumes after the second strand transfer. Transcription
of the primer at this stage would modify the retroviral U3 ends of the
preintegrative DNA, which in turn would affect the entire integration
process, since the activities of the viral integrase depend on the
proper sequence and structure of both ends of the dsDNA (36).
Therefore, the early removal of the (+)-strand primer, after addition
of the 12th DNA residue, might be of importance to ensure correct
reverse transcription and integration.
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ACKNOWLEDGEMENTS |
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We thank V. E. Anderson (Cleveland) for providing potassium peroxonitrite; S. F. J. Le Grice (Cleveland) for E. coli strains that express wild-type HIV-1 RT and the RNase-deficient mutant enzyme; and M. A. Parniak (Montréal, Quebec, Canada) for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft, the European Community Grant BMH4-CT 97-2641, and the Medical Research Council of Canada.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: McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755, Chemin Côte-Ste-Catherine, Montréal, Québec H3T 1E2, Canada. Tel.: 514-340-7536 or 3299; Fax: 514-340-7537; E-mail: mgoette{at}ldi.jgh.mcgill.ca.
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ABBREVIATIONS |
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The abbreviations used are: RT, reverse transcriptase; HIV-1, human immunodeficiency virus type 1; PBS, primer-binding site; PPT, polypurine tract; nt, nucleotides; bp, base pairs; dNTP, 3'-deoxynucleosidetriphosphate; ddNTP, 2',3'-dideoxynucleosidetriphosphate; ds, double-stranded.
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REFERENCES |
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