Originally published In Press as doi:10.1074/jbc.M102974200 on June 11, 2001
J. Biol. Chem., Vol. 276, Issue 33, 31429-31438, August 17, 2001
DNA Synthesis by HIV-1 Reverse Transcriptase at the
Central Termination Site
A KINETIC STUDY*
Marc
Lavigne
,
Lucette
Polomack§, and
Henri
Buc¶
From the Unité de Physicochimie des Macromolécules
Biologiques, Institut Pasteur, URA1773 du CNRS,
75724 Paris Cedex 15, France
Received for publication, April 4, 2001, and in revised form, June 5, 2001
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ABSTRACT |
Human immunodeficiency virus, type 1 (HIV-1)
reverse transcriptase (RT) terminates plus-strand DNA synthesis at the
center of the HIV-1 genome, a process important for HIV-1 infectivity. The central termination sequence contains two termination sites (Ter1 and Ter2) located at the 3'-end of AnTm motifs,
and the narrowing of the DNA minor groove generated by these motifs is
responsible for termination. Kinetic data associated with the
binding of RT and its ability to elongate in vitro various DNA duplexes and triplexes surrounding the Ter2 terminator were analyzed using a simple kinetic scheme. At Ter2, RT still displays a
reasonable affinity for the corresponding DNA, but the binding of the
next nucleotide and above all its incorporation rate are markedly
hampered. Features affecting the width of the minor groove act directly
at this last step. The constraint exerted against elongation by the
AnTm tract persists at two positions downstream of the terminator.
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INTRODUCTION |
Termination of (+)-strand synthesis on a central termination
sequence (CTS)1 can be
proposed as a new specific target against HIV-1 replication. Mutations
of the CTS that abolish termination are responsible for a large
decrease of HIV-1 infectivity (1). An explanation of this effect has
recently been proposed at the molecular and cellular levels (2). One
characteristic of replication of lentiviruses is that their (+)-strand
is synthesized in two segments, starting at two polypurine tracts,
located in the center and at the 3'-end of the genome and called cPPT
and 3'-PPT, respectively. One of them, the cPPT, is located about 100 base pairs upstream of the CTS. In consequence, between the cPPT and
the CTS, (+)-strand synthesis creates a plus-strand overlap, the
central flap, that acts as a cis-determinant of HIV-1 DNA nuclear
import (2). This event may confer to lentiviruses the property of
replicating in nondividing cells.
It is then essential to determine parameters important for polymerase
activity of HIV-1 reverse transcriptase (RT) at this step and to
compare them rigorously with more classical elongation processes.
In vivo and in vitro, HIV-1 reverse transcriptase
terminates DNA synthesis at two precise termination sites of the CTS,
called Ter1 and Ter2 (1). In vitro, termination is observed
at these sites when elongation is performed on a single-stranded DNA
template and is enhanced in conditions of strand displacement, a result that is consistent with the natural conditions of termination. Furthermore, Ter1 and Ter2 are located at the 3'-end of
AnTm motifs, and the structure generated by these
motifs is responsible for termination (3). This structure consists in a
global curvature of the DNA helix, associated with a narrowing of the
minor groove. This compression, maximal between 5 and 2 base pairs
upstream of the termination site, has been shown to be the structural
element responsible for the termination event (4). Therefore, the width of the minor groove of the synthesized DNA is an important parameter for the polymerase activity of HIV-1 RT.
The role of the DNA minor groove has been confirmed by numerous
biochemical and structural studies. Processivity of the enzyme is
reduced when amino acid side chains entering the DNA minor groove are
mutated (5-7). These amino acids are conserved within lentiviruses,
and the cluster has been defined as the minor groove binding track
(MGBT) (8). Modifications of bases have also been informative.
Replacement of adenines by diaminopurines in the AnTm
motifs both enlarge the DNA minor groove within these motifs and
abolish termination at their 3'-end (4, 9). Styrene oxide
N2-guanine or N6-adenine adducts introduced in
the template strand and pointing toward the minor or major groove,
respectively, have been shown to block HIV-1 RT downstream of these
modifications (10, 11). With both adducts, the profile and efficiency
of termination are different between the WT enzyme and enzymes mutated
in residues of the MGBT (Trp266 and Gly262)
(12). The location of the termination sites and the difference observed
between the WT and mutated enzymes both indicate that the residues of
the MGBT of HIV-1 RT are sensing the DNA minor groove of the
synthesized DNA and that this sensing is important for HIV-1
processivity. Two structures of complexes formed between RT and a
DNA/DNA duplex have been obtained (13, 14). Both structures clearly
show a bend of the DNA molecule 4-5 nucleotides upstream of the 3'-end
of the primer. This bend is associated with an opening of the minor
groove that faces the thumb of the catalytic subunit of the enzyme and
more precisely helix 
H, which contains four of the five
residues of the MGBT. Furthermore, during these studies, efficient
cross-links have been obtained between RTs mutated in a residue of the
MGBT (Q258C, G262C, or W266C) and a duplex that contains a guanine
modified at position N-2 (facing the minor groove) in the
template strand (15). A catalytic complex of HIV-1 RT has been trapped
at various positions by these cross-links, stressing the role of the
DNA minor groove in the interaction between HIV-1 RT and the elongated
DNA duplex.
Kinetic studies have shown that the mechanism of ordinary elongation by
HIV-1 RT is similar to the one proposed for other DNA polymerases
(16-19). Using quench-flow techniques and working in excess of DNA
over active enzyme, these studies have suggested a mechanism where the
catalytic step is preceded by a conformational change of the enzyme.
This isomerization is usually rate-limiting for the incorporation of
the nucleotide at saturation of this substrate (corresponding rate
constant kc) (Scheme
I).
Classical kinetic assays, measuring the extension of a primer by one
nucleotide and initiated by the addition of the corresponding dNTP,
generally display a biphasic profile. The first phase, corresponding to
the pre-steady state, can generally be fitted with a monoexponential function. Two parameters are obtained from this phase at saturation in
dNTP: 1) the burst amplitude, which indicates the quantity of enzyme
that has formed a complex ready to elongate the primer before any event
of dissociation (this complex is called a "productive" complex) and
2) the exponential constant, corresponding to the polymerization rate
equal to kc, at saturating concentrations of dNTP.
This burst is followed by a steady state phase, initially linear with
respect to time. The elongation rate measured during this recycling
process divided by the concentration of active enzyme gives the value
of kobs, which is the constant for the rate-limiting step of the recycling reaction. On DNA duplexes where
synthesis is processive, this step corresponds to the dissociation of
the enzyme from the elongated duplex, and therefore
kobs = koff (+1).
However, this simple sequential pathway is not always sufficient to
account for some specific cases of elongation. Additional complexes
have been postulated to describe RNA-primed initiation of reverse
transcription (20-22) or pauses of DNA synthesis induced by secondary
structures of the template or by modified nucleotides introduced in the
DNA template (23-26).
In the present kinetic study, we examined how the sequential scheme
(Scheme I) should be modified to explain the kinetic behavior at Ter2
and at vicinal positions. Minimal modifications in this scheme were
introduced to take into account the following observations. At Ter2, in
conditions that allow a recycling of the enzyme, elongation by one
nucleotide is not limited by the dissociation of the enzyme from the
elongated duplex (as observed at processive positions) but by a new
rate-limiting step that occurs before this dissociation event and after
binding of nucleotide. Assays performed in the presence of a trap show
that the elongation rate is also slow with respect to the dissociation
rate of the enzyme from the nonelongated duplex. Finally, we examined
how these kinetic features depend on the width of the minor groove that
precedes the Ter2 termination site and on the position of the
elongation complex with respect to this site.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides, Proteins, and
Buffers--
Oligodeoxyribonucleotides (called oligonucleotides) were
purchased from Genset and purified by preparative electrophoresis to
more than 95% homogeneity. dNTP and ddNTP are from Amersham Pharmacia
Biotech.
HIV-1 RT was generously given by T. Unge (27). Two different
batches of RT were used in this study. For each batch, the percentage
of active enzyme was measured on an extended PG5/D22 duplex and
following an active site method described in Ref. 16.
Duplexes were prepared according to the same protocol. One of the two
oligonucleotides (primer or template) is labeled at its 5'-end with
[
-32P]ATP and hybridized to the complementary strand,
at a 2:1 ratio (cold/labeled). Hybridization is performed by
incubating the duplex for 5 min at 75 °C, followed by a slow
decrease in temperature, in a 100 mM Tris-HCl (pH 7.8), 400 mM NaCl, 8% (v/v) polyethylene glycol 6000 solution.
Duplexes were always made at concentrations of oligonucleotide at least
20 times higher than their concentrations in the final assay.
Assays were performed at 37 °C and in 50 mM Tris-HCl (pH
7.8), 6 mM MgCl2, 50 mM KCl, and 2 mM dithiothreitol.
Enzymatic Assays--
Both rapid and slow kinetic assays are
based on the quantification, at different times of the reaction, of
elongated (n + 1) and nonelongated (n) primers.
These two forms were separated on a 16% polyacrylamide sequencing gel
and quantified using the PhosphorImaging technique and ImageQuant software.
Rapid quench experiments were carried out in a Kintek apparatus
(Austin, TX). Experiments were performed by loading the enzyme plus
duplex solution in one loop and the dNTP substrate in the second loop.
Each reaction was started by rapidly mixing both solutions and quenched
with 0.3 M EDTA after time intervals ranging from 11 ms to
several minutes. Before being loaded on the separating gel, the
reaction products were ethanol-precipitated in order to overcome
problems of migration due to the presence of EDTA.
Slow kinetic assays were performed after a manual mixing of two
solutions, preincubated during 5 min at 37 °C. In the measurements of the kobs of steady state phase (Table
I), heparin effect (Fig. 2c),
or dGTP effect (Fig. 3), reaction was started by the addition of
substrate (dNTP or ddNTP), with or without effector, to a premix of DNA
(duplex or triplex) and RT. In the kinetic assays performed in an
excess of enzyme, the reaction is started by the addition of enzyme to
a premix of DNA and nucleotide.
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Table I
Dependence of the kobs (s 1) value measured during the
steady state phase of elongation on the position of elongation and on
the structure of the DNA substrate
Values of kobs were obtained under conditions of
excess of DNA (150 nM) over RT (3 nM),
according to the method described under "Experimental Procedures."
DNA substrates are described in Fig. 1. They are different WT duplexes
corresponding to different positions of elongation around Ter2
(positions 4929-4941), m-C2 and m-C12 duplexes, and a WT triplex.
Elongation is started by the addition of the corresponding incorporable
nucleotide (250 or 900 µM ddNTP or 250 µM
dNTP). At most positions, elongation performed in the presence of dNTP
is restricted to a single incorporation of this substrate. Asterisks
indicate these positions. On the WT duplexes, there are three positions
indicated in boldface type (4934, 4935, and 4936) where the
kobs is significantly lower than the
kobs measured at processive positions. ND, not
determined.
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In both rapid and slow kinetic experiments, performed in an excess of
DNA, the rate constant kobs of the recycling
process was always calculated by dividing the velocity of elongation
during the linear steady state phase by the concentration of active
enzyme (defined on a standard processive hybrid; see above). This
calculation assumes that the percentage of active enzyme does not
depend on the position at which elongation is assayed.
Finally, all time-dependent profiles were fitted to their
analytical expressions using the Kaleidagraph software.
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RESULTS |
Oligonucleotides Designed for This Study--
In our
previous studies, termination at the CTS has been analyzed by
polymerization assays performed on large single-stranded DNA templates
and using a mixture of the four dNTPs (3, 4). These "run-off"
polymerization assays have shown that termination at Ter2, on a WT
template, occurs precisely at position 4934 (position of the last
nucleotide incorporated). However, these assays do not allow to measure
the kinetic constants characterizing the polymerization process at and
around this site.
In order to study the kinetic of incorporation of one single
nucleotide at various positions around Ter2, various small DNA/DNA duplexes were designed. A 60-mer template centered at Ter2 is hybridized to various primers having the same 5'-end but different 3'-ends. Primers are named according to the position of their 3'-end
(4929-4941; Fig. 1). As in the case of
"run-off" assays, these +1 extension assays were performed on small
DNA duplexes having WT or mutated sequences that have been previously
shown to abolish termination (m-C2 and m-C12). These +1 extension
assays were also performed on small DNA substrates, which better mimic the conditions of strand displacement. In these substrates, called triplexes, the single-stranded part of the duplex is hybridized with a
60-mer oligonucleotide that mimics the strand to be displaced by the
enzyme (see Fig. 1).
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Fig. 1.
Oligonucleotides used in the kinetic
study. The sequence surrounding the termination sites Ter1
(position 4925) and Ter2 (position 4934) is presented at the
top. Domains C1 and C2 located upstream of these sites and
the corresponding AnTm motifs are indicated by
brackets and boxes. This sequence has been
mutagenized in domain C2 (mutant m-C2) or in both domains C1 and C2
(mutant m-C12) (3) (mutated nucleotides indicated in
boldface type in the corresponding duplex).
Below this sequence are presented the oligonucleotides used
in the kinetic assays. Primer strands have a common 5'-end (position
4910) and differ by their 3'-end (positions 4929-4941 for WT sequence
and 4934 for m-C2 and m-C12 sequences). Template strands are 60-mer
oligonucleotides of WT, m-C2, or m-C12 sequences between positions 4905 and 4964. Strands to be displaced are also 60-mer oligonucleotides.
They contain an upstream part (between positions 4905 and 4934) that
does not hybridize to the template and a downstream part (between
positions 4934 and 4974) complementary to the WT and m-C12
templates.
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No Burst Is Observed at Ter2 on a WT Duplex--
We first
studied the kinetics of incorporation of dCTP at Ter2 on duplexes of
WT, m-C2, and m-C12 sequences. The corresponding primers are 5'-labeled
and terminate at position 4934. Their extension by one nucleotide (to
position 4935) can be followed by running the elongation products on a
16% sequencing gel and quantifying the nonelongated and elongated primers.
This +1 extension assay was first performed in conditions of large
excess of duplexes (130 nM) relative to RT (6 nM). For each time point, the reaction was started by
mixing a solution containing RT and the duplex with a concentrated
solution of dCTP (250 µM final concentration) and stopped
by the addition of EDTA. Reaction times ranged between 11 ms and
180 s. Fig. 2, a and
b, shows the kinetics of these extensions. RT clearly
displays a different kinetic profile on the WT and mutant duplexes.
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Fig. 2.
Elongation of WT, m-C2, and m-C12 duplexes at
Ter2 by HIV-1 RT and dCTP, using an excess of DNA over RT.
a and b present the kinetic profiles of
elongation by one nucleotide (dCTP) of the primer strand of the WT,
m-C2, and m-C12 k4934/k60 duplexes. Reactions were performed at
37 °C using a Kintek apparatus, and each point of the kinetic
profiles corresponds to one reaction (see "Experimental
Procedures"). Each reaction is started by mixing a preincubated
solution of hybrid (130 nM, labeled primer) and RT (6 nM) with a solution of dCTP (250 µM). It is
terminated by the addition of EDTA (0.3 M) and ethanol
precipitation. Nonelongated (n) and elongated (n + 1) primer oligonucleotides are separated on a sequencing gel and
quantified on PhosphorImager. The concentration (in nM) of
elongated primer is plotted versus the reaction time, with
two different time scales (0-180 s in a and 0-10 s in
b). Filled circles, open
triangles, and filled triangles
correspond to values measured on WT, m-C2, and m-C12 duplexes,
respectively. Values are fitted to a burst equation (see
"Results"). Respectively, on these three duplexes, values of burst
amplitude are 0 ± 0.92, 3.13 ± 0.69, and 4.51 ± 0.36 nM; values of slope are 0.157 ± 0.002, 3.22 ± 0.16, and 2.63 ± 0.1 nM s1; and values
of kpol are 0, 12.4 ± 10, and 23.2 ± 7.1 s1.
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The kinetics of primer extension of the m-C2 and m-C12 duplexes
conform to the behavior expected for a processive synthesis, as
summarized in the introduction (16). They are biphasic and fit well to
the following equation: [Dn + 1] = A(1 
expkpol t) + Bt, where [Dn + 1] represents the
concentration of elongated primer, A is the amplitude of the
burst, kpol is the constant of the pre-steady
state exponential phase, and B is the slope of the steady
state linear phase. Values of burst amplitude, exponential constant,
and slope of the linear phase measured in these assays are given in the
legend of Fig. 2a. The amplitudes of the burst indicate that
50 and 75% of the present enzyme can form a productive complex at the
positions tested, on the m-C2 and m-C12 duplexes, respectively. The
constants of the exponential phase are not significantly different
between the two mutants. They are very close to the values of
kpol measured at processive sites at saturation
of dNTP (kc = 20 s1 in Ref. 16). Last,
the rate constant kobs of the recycling reaction
is equal to the slope of the linear steady state phase divided by the
concentration of active enzyme (defined on a standard processive
hybrid). At processive sites, the recycling process is limited by the
dissociation of the enzyme from the elongated duplex. In consequence,
on the m-C2 and m-C12 duplexes, kobs = koff (+1). These values (0.4-0.5
s1) suggest that RT dissociates slightly faster from
these elongated duplexes than from others (0.2 s1).
On the WT duplex, this biphasic behavior is abolished and a
linear release of products occurs immediately after nucleotide addition. The rate of this process is 0.16 nM
s1 and corresponds to a kobs of
0.027 s1. This value is 7 times lower than a classical
dissociation rate. The simplest explanation for this behavior is that
there is a new rate-limiting step that affects the first round of
incorporation as well as the subsequent ones. The rate of this limiting
step can be compared with the kinetic constant characteristic of a usual burst, kpol, which is on the order of 20 s1 (16). In consequence, a 700-800-fold decrease of the
corresponding constant kc would account for this
atypical behavior .
At Ter2, Recycling Elongation Is Not Limited by Dissociation of
RT--
If the recycling elongation at WT Ter2 is no longer limited by
the dissociation of the enzyme from the elongated duplex, one can then
specifically perturb this last step and study its consequences on the
kobs constants. This study can be performed
at Ter2 on both WT and m-C12 duplexes.
Dissociation of RT after elongation is usually disfavored by the
addition of the next incorporable deoxynucleotide to the reaction
mixture. This effect has been observed when following the incorporation
of a dideoxynucleotide in the presence of the deoxynucleotide
complementary to the next position and when elongation under recycling
conditions is limited by koff (+1) (16, 28).
The corresponding complex has a longer residence time and is thought to
mimic the E'-Dn +1-dNTP ternary complex
formed in the sequential reaction pathway.
We analyzed this effect on WT and m-C12 DNA duplexes made with
primers that terminate at Ter2. Their elongation was performed with
ddCTP in the presence and absence of dGTP (Fig.
3). On the m-C12 duplex, extension is
indeed limited by the dissociation of the enzyme at position 4935, since the corresponding value of kobs is
decreased 14 times in the presence of dGTP. By contrast, a less than
2-fold decrease is observed on the WT duplex. Therefore, during a
recycling phase, elongation at WT Ter2 is not limited by the
dissociation of the enzyme from the elongated duplex but by a step
occurring during the polymerization pathway.
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Fig. 3.
Effect of dGTP on the elongation at Ter2 of
WT and m-C12 duplexes by HIV-1 RT and ddCTP. Duplexes made with a
labeled WT or m-C12 k4934 primer and the corresponding k60 template
were elongated by incorporation of one ddCTP in the absence and
presence of the next incorporable dGTP. These elongation assays were
performed at 37 °C, by mixing manually a premix solution of duplex
(150 nM) and RT (3 nM) with a solution of ddTTP
(250 µM), which either contains or does not include dCTP
(250 µM). The quantity of elongated primer is plotted
versus the time of reaction (in seconds). Values located
within the steady state phase were fitted to a linear function.
Circles and triangles, values obtained on the WT
and m-C12 duplexes, respectively; filled symbols
and thick lines, values and linear fits obtained when the
assays are performed in the absence of stabilizing dGTP;
open symbols and thin
lines, dGTP is present. Values of
kobs (s1) obtained from the linear
fits and defined under "Experimental Procedures" are 0.021 and
0.012 s1 on the WT duplex and 0.24 and 0.017 s1 on the m-C12 duplex, in the absence and presence of
dGTP, respectively.
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Heparin Challenge--
Heparin, a trap of unbound
polymerases, was used to challenge primer extension performed on the WT
and m-C12 duplexes ending at Ter2. After formation of the enzyme-duplex
complexes, in conditions of excess of duplex (200 nM) over
RT (5 nM), heparin was added at the same time as the
nucleotide substrate dCTP. The effect of the competitor on the
elongation of the two duplexes is clearly different (Fig.
4). For the m-C12 duplex (and for m-C2 as
well; data not shown), the amplitude of the burst is not affected by the presence of heparin in the assay. The enzyme-DNA binary complexes formed at m-C12 Ter2 are therefore resistant to heparin and fully committed toward synthesis during the first turnover. This result also
implies that the steps that engage these binary complexes into the
elongation process are faster than the dissociation of this complex
(koff (0) in Scheme I). On the other hand, all
complexes formed at WT Ter2 between RT and the DNA duplex are
heparin-sensitive. If Scheme I still applies,
koff (0) must be faster than the rate-limiting
step kobs, and all of the binary complexes
formed at WT Ter2 can be considered as "slow isomerizing"
complexes.
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Fig. 4.
Effect of heparin on the elongation at Ter2
of WT and m-C12 duplexes by HIV-1 RT and dCTP. This experiment was
performed at 37 °C by mixing manually a premix solution of duplex
(WT and m-C12 k4934/k60, 200 nM, labeled primer) and RT (5 nM) with a solution of dCTP (250 µM) that
either contains or does not include heparin (120 µg/ml). At different
times, the reaction was terminated by the addition of formamide 95%,
EDTA 50 mM, and the n and n + 1 forms
of the primers were separated and quantified. The quantity of elongated
(n + 1) primer is plotted versus the time of the
reaction (in seconds). Triangles and circles
correspond to values obtained on WT and m-C12 duplexes, respectively.
Open symbols and dashed
lines, heparin is added at the same time as dCTP;
filled symbols and continuous
lines, no heparin.
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Substrate Effect--
The dependence of the rate constant
kobs on the dCTP concentration was analyzed at
WT Ter2. It follows a simple binding law as shown in Fig.
5. At the plateau,
kobs is equal to 0.05 s1, and the
corresponding dissociation constant
KD(dCTP) is about 150 µM.
This value is much higher than the value
KD(dNTP), which characterizes, at
positions of processive synthesis, the dependence of the burst rate
kpol on nucleotide concentration. This constant
corresponds to the binding of dNTP on E-Dn in Scheme
I and is reported to be on the order of 4-10 µM (16). Therefore, at Ter2, the rate-limiting step occurs after substrate binding (which is poor) and before dissociation of the enzyme from the
elongated duplex.
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Fig. 5.
Dependence on the dCTP concentration of the
kobs measured during the steady state phase of elongation
at WT Ter2. Elongation of the k4934/k60 WT duplex by HIV-1 RT was
performed at various concentrations of dCTP, and in conditions of
excess of duplex (150 nM) over enzyme (3 nM).
The values of kobs calculated as described under
"Experimental Procedures" are plotted versus the
concentration of dCTP. The dependence on dCTP of the
kobs fits to a hyperbola with a
KD of 153.7 ± 16.9 µM and a
plateau of 0.05 ± 0.01 s1.
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Kinetic Analysis at WT Ter2 Using an Excess of Enzyme over
Duplex--
All experiments reported up to now can be interpreted
though a simple kinetic scheme, requiring only the sequential binding to the enzyme of the duplex DNA and of the deoxynucleotide substrate. The rate-limiting step occurs after this last binding step and before
dissociation of the enzyme from the elongated duplex (Scheme II).
In this model, the role of the enzyme and the duplex are
perfectly symmetrical. We can therefore check its relevance by working in excess of enzyme over duplex. Indeed, at WT Ter2, there is no longer
any rationale to use the converse situation (excess of duplex), since
the information usually provided by a biphasic profile is lost at this
position. Scheme II represents an ordinary pathway where product
formation (corresponding to the last two species in this scheme) is
limited by a step between the ternary complex E-Dn
dNTP and the binary complex E-Dn + 1. The rate of this limiting step is called kls.
Furthermore, we assume that the binding steps leading to the binary
complex (dissociation constant
KD(E)) and to the
ternary complex E-Dn dNTP are fast with respect to
this polymerization step. Formation of the elongation products (P)
occurs in a single turn over and is given by the following.
![V=<FR><NU><UP>d</UP>[<UP>P</UP>]</NU><DE><UP>d</UP>t</DE></FR>=<FR><NU><UP>d</UP>(E′<UP>D</UP><SUB>n</SUB><UP>dNTP</UP>)</NU><DE><UP>d</UP>t</DE></FR>=k<SUB><UP>ls</UP></SUB>[E <UP>D</UP><SUB>n</SUB><UP>dNTP</UP>]](/content/vol276/issue33/fulltext/31429/img001.gif)
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(Eq. 1)
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The concentration of this species is obtained via the two fast
preequilibria.
![[E <UP>D</UP><SUB>n</SUB> <UP>dNTP</UP>]=<FR><NU>[E <UP>D</UP><SUB>n</SUB>][<UP>dNTP</UP>]</NU><DE>K<SUB>D(<UP>dNTP</UP>)</SUB></DE></FR>=<FR><NU>[E][<UP>D</UP><SUB>n</SUB>][<UP>dNTP</UP>]</NU><DE>K<SUB>D(<UP>dNTP</UP>)</SUB>K<SUB>D(E)</SUB></DE></FR>](/content/vol276/issue33/fulltext/31429/img002.gif)
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(Eq. 2)
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In consequence, it is related to [D]T, the total
concentration of DNA, by the following.
![[E <UP>D</UP><SUB>n</SUB> <UP>dNTP</UP>]=<FR><NU>[<UP>D</UP>]<SUB><UP>T</UP></SUB>−[<UP>P</UP>]</NU><DE>1+<FR><NU>K<SUB>D(<UP>dNTP</UP>)</SUB></NU><DE>[<UP>dNTP</UP></DE></FR>+<FR><NU>K<SUB>D(<UP>dNTP</UP>)</SUB>K<SUB>D(E)</SUB></NU><DE>[E][<UP>dNTP</UP>]</DE></FR></DE></FR>](/content/vol276/issue33/fulltext/31429/img003.gif)
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(Eq. 3)
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When this value is incorporated in Equation 1, product
accumulation is shown to proceed via an exponential law characterized by a single time constant, 
[E, dNTP].
=[<UP>D</UP>]<SUB><UP>T</UP></SUB>(1−e<SUP><UP>−</UP>t/&tgr;</SUP>)](/content/vol276/issue33/fulltext/31429/img004.gif)
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(Eq. 4)
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with
![&tgr;<SUP><UP>−</UP>1</SUP>=<FR><NU>k<SUB><UP>ls</UP></SUB>[E][<UP>dNTP</UP>]</NU><DE>K<SUB>D(<UP>dNTP</UP>)</SUB>K<SUB>D(E)</SUB>+[E]([<UP>dNTP</UP>]+K<SUB>D(<UP>dNTP</UP>)</SUB>)</DE></FR>](/content/vol276/issue33/fulltext/31429/img005.gif)
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(Eq. 5)
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At constant substrate concentration, the rate 1
should then follow a Michaelis-Menten behavior as the enzyme
concentration [E] is increased. The maximal rate
kmax is related to the thermodynamic constant
kls through the following equation.
![k<SUB><UP>max</UP></SUB>=k<SUB><UP>ls</UP></SUB><FR><NU>[<UP>dNTP</UP>]</NU><DE>K<SUB>D(<UP>dNTP</UP>)</SUB>+[<UP>dNTP</UP>]</DE></FR>](/content/vol276/issue33/fulltext/31429/img006.gif)
|
(Eq. 6)
|
The apparent Michaelis constant
Km(E) is related to
the dissociation constant
KD(dCTP)(E) through
the following equation.
![K<SUB>m(E)</SUB>=K<SUB>D(E)</SUB> <FR><NU>K<SUB>D(<UP>dNTP</UP>)</SUB></NU><DE>K<SUB>D(<UP>dNTP</UP>)</SUB>+[<UP>dNTP</UP>]</DE></FR>](/content/vol276/issue33/fulltext/31429/img007.gif)
|
(Eq. 7)
|
This last constant characterizes the dissociation of the pooled
complexes E-Dn and E-Dn-dNTP into
the free components E, Dn, and dNTP.
Elongation assays were started by the addition of an excess of RT to a
premix of duplex Dn and dCTP. As predicted from Equations 4 and
5, the formation of elongated primer
Dn + 1 follows an exponential function with a
plateau and a time constant . These assays are performed at
different concentrations of RT, and the values are plotted
versus the inverse of concentration of RT. These plots are
called " plots." This methodology is very close to the one
developed by W. McClure in order to study the kinetics of abortive
initiation by the Escherichia coli RNA polymerase (29,
30).
Data were obtained by this methodology at
WT Ter2. They are displayed in Fig. 6,
a and b, and in Table
II. Three conclusions emerge. First, at
all enzyme concentrations tested, a single exponential suffices to fit
the time course of product formation (Fig. 6a). This is a
test of the homogeneity of population of the kinetically competent
complexes bound at Ter2. They all have to go over similar activation
barriers (probably the same ones) during the process of primer
extension. Second, at the substrate concentration tested, the rate
constant kmax (0.017 s1) is not
very different from the previous rate, kobs,
measured in excess of DNA duplex (0.027 s1). Using
Equation (6) and the value previously determined for KD(dCTP), the
kmax value yields a value for
kls of 0.027 s1. Finally, the
apparent Michaelis constant related to enzyme binding is in the
nanomolar range
(Km(E) = 1.33 ± 0.23 nM). Using Equation 7, this leads to a dissociation
constant for the initial binary complex E-Dn of 3.6 nM, well in the range of ordinary dissociation constants
for the binding of RT on DNA-DNA duplexes.
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Fig. 6.
Elongation of a WT duplex, at position Ter2
by HIV-1 RT and dCTP in excess of RT over DNA. a,
kinetic profiles of extension by one nucleotide (dCTP) of the primer
strand of the WT k4934/k60 duplex at various concentrations of RT.
Reactions were performed at 37 °C by mixing manually a premix
solution of duplex (0.1 nM, labeled primer) and dCTP (250 µM) with a solution of RT (1-5 nM). The
reaction was terminated as indicated in the preceding figure legends.
The quantity of elongated primer (n + 1) is plotted
versus the time of reaction (in seconds). These kinetic
profiles fit to monoexponential functions, and the corresponding fits
are represented on the plot. The values of exponential constants
( constants) measured at different concentrations of RT
(ranging between 1 and 50 nM) on the WT duplex (position
Ter2) were plotted versus the inverse of RT concentration
(Fig. 6b). These values fit to a linear function, and
the fit gives an estimate of kmax (0.017 ± 0.001 s1), Km (1.33 ± 0.23 nM), and kmax/Km
(1.24 ± 0.13 10+7
s1·M1).
|
|
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|
Table II
Values of kmax, Km and kmax/Km measured
at position Ter2 on duplex or triplex substrates with a WT or m-C2
sequence
This table presents the values of kmax,
Km, and
kmax/Km, obtained from the plots described in Figs. 6-8 and the ratios calculated between some of
these values (in boldface type). * and #, values and ratios that are
not significant.
|
|
In summary, all complexes engaged into elongation at WT Ter2 appear to
conform to the simple sequential scheme given above, and the main
constants are shown in Scheme III.
At the termination site Ter2, binding of HIV-1 RT to the duplex is
similar to other processive sites, whereas subsequent binding of dCTP
is disfavored. However, the major effect is observed at the
rate-limiting step, which corresponds to a very slow conversion into
product. The corresponding limiting step is 800 times slower than the
conformational change that precedes the chemistry step at a processive position.
Modulation of the Strength of the
Terminator--
Using the same methodology as the one described above,
we analyzed how the kinetics of elongation at Ter2 were affected either by changes of the minor groove in the C2 domain or by the requirement of strand displacement on the kinetics of elongation at Ter2.
First, a widening of the groove width can be obtained by mutating
the corresponding sequence (mutant m-C2). As observed with the WT
duplex, the formation of elongated primer Dn + 1 with the m-C2 duplex fits a monoexponential function (Fig.
7a). The corresponding time
constant follows a Michaelis-Menten behavior as the enzyme
concentration is increased (Fig. 7b). The maximal observed
rate is 0.13 s1, a value 10 times higher than the one
measured on the WT duplex. However, it is also 100 times smaller than
the kpol measured on the same duplex during the
pre-steady state phase in conditions of excess DNA. This large
difference can be explained by the existence of another limiting step
during elongation of the m-C2 duplex. This step would occur during the
formation of the E-Dn complex, and its rate would be
very close to the rate of dissociation from the elongated duplex. In
consequence, on assays performed with an excess of DNA over enzyme and
started by the addition of substrate, this other limiting step would
not prevent the existence of a burst and would not affect the rate
constant kobs of the steady state phase. On
assays performed in conditions of an excess of enzyme over DNA,
kmax would correspond to the rate of this rate-limiting step.
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Fig. 7.
Elongation of a m-C2 duplex at position Ter2
by HIV-1 RT and dCTP in excess of RT over DNA. +1 extension assays
were performed on the m-C2 k4934/k60 duplex under similar conditions as
the assays performed on the WT homologous duplex (see legend to Fig.
6). Fig. 7, a and b, represent respectively the
kinetic profiles of +1 extension and the derived plot. Values of
kmax, Km, and
kmax/Km estimated from the plot are
0.132 ± 0.015 s1, 1 ± 0.3 nM, and
13.1 ± 2.4 10+7
s1·M1 respectively.
|
|
Conversely, we looked at the effect of distamycin on the elongation at
WT Ter2. This drug is known to have a good affinity for
AnTm motifs like the one present upstream of Ter2. A +1
extension assay was performed at position 4934 on a WT duplex, in
conditions of an excess of enzyme over DNA and in the presence of
distamycin (25 nM). The corresponding plot, presented
in Fig. 8, fits again a linear relation.
Values of kmax and
Km(E) obtained from
this fit show that the major parameter affected by the presence of
distamycin is again kmax. Its value is about 4 times smaller than the one measured in the absence of the drug.
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Fig. 8.
Effect of distamycin on the elongation of a
WT duplex, at position Ter2, by HIV-1 RT and dCTP. A plot is
shown. The WT k4934/k60 duplex (0.1 nM), incubated with
distamycin (25 nM) was elongated at 37 °C by dCTP (250 µM) using various concentrations of RT (enzyme added at
the beginning of the reaction). Each +1 extension profile was fitted to
a monoexponential function, and the values obtained from these fits
were plotted versus the inverse of RT concentration and
fitted to a linear function. This plot and the values of
kmax, Km, and
kmax/Km estimated from this plot are
0.0043 ± 0.0003 s1, 0.75 ± 0.18 nM, and 5.7 ± 1 10+6
s1·M1, respectively.
|
|
Since we did not measure the values of
KD(dNTP) at m-C2 Ter2 and at WT Ter2 in
the presence of distamycin, we cannot estimate the corresponding values
of kc in these conditions. However, at a
constant substrate concentration, the overall activation barrier is
reflected by the following.
![<FR><NU>k<SUB><UP>max</UP></SUB></NU><DE>K<SUB>m(E)</SUB></DE></FR>=<FR><NU>k<SUB><UP>ls</UP></SUB>[<UP>dNTP</UP>]</NU><DE>K<SUB>D(E)</SUB> K<SUB>D(<UP>dNTP</UP>)</SUB></DE></FR>](/content/vol276/issue33/fulltext/31429/img008.gif)
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(Eq. 8)
|
This ratio is increased by a factor of 10 when the minor groove in
C2 is enlarged (as observed on the m-C2) and decreased by a factor of 2 when this groove is occupied by distamycin.
Finally, because strand displacement conditions increase the efficiency
of termination, we also performed +1 extension assays on a WT Ter2
triplex, in conditions of excess of enzyme over DNA. Fig.
9 shows the corresponding plot. This
plot allows a reasonable estimate of kmax and of
kmax/Km(E)
(decreased, respectively, by a factor of 9 and 5 with regard to the WT
duplex). Control experiments were performed on the m-C2 triplex (Fig.
9). As already observed by comparison between the WT and mutant
duplexes, the major parameter affected by the width of the DNA minor
groove on the triplexes is kmax (decreased by a
factor of 19). Furthermore, on both WT and m-C2 sequences, converting
the duplex into a triplex results in a similar decrease of the
ratio
kmax/Km(E) (Table II).
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Fig. 9.
Elongation of WT and m-C2 triplexes, at
position Ter2, by HIV-1 RT and dCTP, in excess of RT over DNA. A
plot is shown. Triplexes were obtained after annealing of the
duplexes (WT and m-C2 k4934/k60) with a 60-mer oligonucleotide
complementary to the single-stranded part of the duplex (see Fig. 1).
The WT and m-C2 triplexes (0.1 nM) were elongated at
37 °C by dCTP (250 µM) and various concentrations of
RT (enzyme added at the beginning of the reaction). Each +1 extension
profile was fitted to a monoexponential function, and the values
obtained from these fits were plotted versus the inverse of
RT concentration and fitted to a linear function. The fit of the
corresponding plots give values of kmax
(0.0019 ± 0.0002 and 0.035 ± 0.006 s1),
Km (0.75 ± 0.85 and 1.95 ± 1.59 nM) and kmax/Km
(2.6 ± 2.6 and 1.78 ± 1.18 s1·M1) for the WT and m-C2
triplexes, respectively.
|
|
Therefore, termination at Ter2 depends on two inhibitory effects. The
major one is the compression of the minor groove due to the
AnTm motif. The minor one is the necessity to unwind a
preexisting double helix placed downstream of the terminator. The two
effects appear to be independent and additive.
Values of kobs Measured during the Steady State Phase
at Various Positions around WT Ter2--
The kinetic studies presented
above revealed that the polymerization rate kls
measured at Ter2 is 740 times lower than polymerization rates measured
at processive positions. We wondered whether this very large effect is
focused on the Ter2 site or is also observed at vicinal positions. For
this purpose, +1 extension assays were performed at positions
4929-4941, with an excess of duplex over enzyme, and the corresponding
values of kobs were calculated. Assays were
performed using ddNTP instead of dNTP as incorporated substrate, in
order to avoid extensions of more than one nucleotide. Furthermore, two
different concentrations of ddNTP (250 and 900 µM) were
used to investigate the possible stabilizing effect of this nucleotide
in case it is complementary to the next position of the template.
Finally, whenever possible (positions 4929, 4933, 4934, 4937, 4940, and
4941), the values of kobs corresponding to the
incorporation of dNTP and ddNTP (at the same concentration of 250 µM) were compared.
The values of kobs measured at these different
positions on the WT sequence as well as at position 4934 on the m-C2
and m-C12 sequences are presented in Table I. A significant decrease
(about 10-fold) of these values is observed at positions 4934, 4935, and 4936 only. This change is independent of the concentration of
substrate used in the assay. At Ter2, it does not depend on the nature
of the incorporated nucleotide (dNTP or ddNTP). Therefore, extension at
the two positions immediately downstream of Ter2 is probably limited,
as at Ter2, by a step distinct from the dissociation of the enzyme from
the elongated duplex. We propose that this limiting step is similar to
the one defined at Ter2.
The self-consistency of our data was furthermore checked by measuring,
under the present conditions of excess DNA, the turnover number on a WT
triplex at Ter2. This value was found to be 30 times lower than on the
corresponding duplex. It is very close to the
kmax value measured in assays performed with an
excess of enzyme over DNA (0.001 s1 instead of 0.0019 s1).
| |
DISCUSSION |
The main results arising from these kinetic studies are quite
simple. At the termination site Ter2, the elongation of the primer
strand by one nucleotide is not prevented by a poor binding of the
enzyme to the relevant DNA substrate, but by a more difficult association of the nucleotide substrate to the binary complex and,
above all, by a very slow elongation rate by the ternary complex
E-Dn dNTP. This effect is lost when the DNA minor groove located upstream of the termination site is enlarged by mutagenesis of the corresponding AnTm tract. On the
other hand, this effect is enhanced when the minor groove is occupied
by distamycin or when the enzyme must also unwind a double-stranded DNA
template, downstream of the polymerization site.
Termination Can Be Described by a Simple Sequential Elongation
Scheme--
The first remarkable aspect of these results is that the
simple sequential scheme initially proposed to describe elongation by
different DNA polymerases is sufficient to explain the kinetics of
elongation by HIV-1 RT at Ter2. Results obtained in excess of enzyme
over DNA are consistent with the ones obtained in the converse
situation. The kinetics of +1 extension at Ter2 performed with an
excess of enzyme over DNA can always be interpreted by one single
exponential function, a result that argues in favor of a unique kinetic
pathway for all bound enzyme species.
This uniqueness contrasts with the branched pathways proposed to
explain elongation at other nonprocessive sites, like the ones induced
by secondary structures of the template or by modified nucleotides
introduced in the DNA template (23-26). At these sites, a double
exponential function is necessary to fit the initial phase of
elongation, in +1 extension assays performed under conditions of excess
of DNA over enzyme. This double exponential fit of the initial burst
has also been observed during studies on the RNA-primed initiation of
reverse transcription (20-22) and on the extension of a DNA/DNA duplex
(31). These different studies have proposed the existence of different
binding modes of RT to the duplex. One binding mode would lead to the
formation of a kinetically competent or productive complex which is
characterized by a fast polymerization rate. Another binding mode would
lead to the formation of a slow complex, which has to isomerize to the
productive complex. This isomerization is attributed to the slow
polymerization rate and the amplitudes associated with each
polymerization rate (fast and slow) yielding the percentage of
productive and slow complexes.
The simplicity of the formalism accounting for our results at WT Ter2
is due to the very slow maximal rate of the limiting step
kls. Even if several conformers exist in the
populations of binary (E-Dn) and ternary
(E-Dn-dNTP) complexes, reequilibration between these
populations is rapid with respect to kls. It is
then interesting to estimate the overall effect exerted by the
AnTm tract on the processivity of HIV-1 RT. This can be
assessed by comparing at WT Ter2 and at one given concentration of dNTP
(250 µM) the probability to elongate
kmax/ (kmax + koff) versus the probability to
dissociate koff/(koff + kmax). As a result, more than 10 encounters
(inverse of the probability to elongate) are necessary for synthesis to proceed. If one assumes a constant koff, then
the presence of distamycin increases this number to more than 50 encounters, and the presence of a strand to be displaced downstream of
the elongation site increases this value to more than 100 encounters.
Together with the sensitivity of the binary complexes to heparin
challenge, these calculations indicate that most encounters between the
enzyme and the template at Ter2 abort before primer extension.
It is also possible to quantify the negative effect exerted by the
AnTm tract located upstream of Ter2, by comparing the
thermodynamic constants measured here at each step of the sequential
pathway with similar constants determined at a position where synthesis
is processive (e.g. in Ref. 16). At Ter2, the value of
KD(E) is around 3.6 nM, which is very close to the value measured at a
processive position (5 nM). Binding of HIV-1 RT to the Ter2
duplex is therefore only slightly favored over binding to a processive
duplex (
G1 
0.2 kcal/mol). The value of
KD(dNTP) at Ter2 is equal to 150 µM, indicating that the second step of the sequential
scheme is disfavored by 2.17 kcal/mol. Finally,
kls is equal to 0.027 s1, which is
740 times lower than kc at a processive
site (20 s1) and corresponds to a major penalty of this
process (4 kcal/mol). On the whole, the penalty is on the order of 6 kcal/mol.
Same Kinetic Steps Affected at Ter2 by the Compression of the Minor
Groove and by the Constraint of DNA Unwinding--
The second
important result obtained in this study is that the main kinetic steps
affected during the process of termination at WT Ter2 are always
located after the formation of the ternary complex and before the
dissociation of the enzyme from the elongated product. Indeed, both
modifications in the access of the minor groove of the synthesized DNA
(by mutagenesis or binding of distamycin) and the constraint of
unwinding the DNA template downstream of the elongation site affect
mainly these steps of the sequential scheme. Similarly, pauses observed
on RNA and due to the formation of RNA secondary structures downstream
of the catalytic site are generally associated with a large decrease of
the burst amplitude and with the formation of complexes characterized
by good binding affinities and by a slower isomerization into
kinetically competent species (corresponding
kpol is 1000 times lower) (23). The effect of
distamycin on elongation at Ter2 can also be compared with the effects
observed during polymerization on a DNA duplex containing cisplatin
modifications (25). Again, the major parameter affected by cisplatin
modifications is a polymerization rate ("slow" rate = 0.06 s1). On the other hand, pauses of HIV-1 RT (WT or mutated
forms affected in their processivity) are not always associated with modifications of the same steps of the polymerization process. In
particular, mutations in the MGBT domain, responsible for a decrease of
processivity of HIV-1 RT, lead to a decrease of the burst amplitude and
to an increase of the dissociation rates of the mutated enzyme from DNA
duplexes (5-8, 32). In the case of Ter2, we did not directly measured
the rate of dissociation of the enzyme at the termination site.
However, the kobs of the rate-limiting step
measured during the +1 extension assay performed at the previous
position (4933) can be used to estimate the koff at Ter2 (position 4934). As shown in Table I, this value is very close
to the value of koff measured at a processive
site (0.2 s1 in Ref. 16). We conclude that the
introduction of mutations in the MGBT motif has a more drastic effect
than the narrowing of the minor groove where its side chains interact.
They prevent the formation of stable initial complexes, while a
decreased accessibility of the same groove to the WT thumb essentially
prevents the formation of complexes that can quickly isomerize into a
productive species.
Finally, we observed that the AnTm tract, which
disfavors so strongly the elongation rate at Ter2 (position 4934),
continues to exert its negative effect at the two following positions
(4935 and 4936). This phenomenon is indicated by the values of
kobs measured at these positions, which are
strikingly lower than the expected values of dissociation rates. At
these positions and because of the sequence, the incorporated ddGTP could also play the role of stabilizing nucleotide. However, the low
values of kobs measured at positions 4935 and
4936 do not change significantly when the concentration of ddGTP is
increased. Therefore, at these positions, elongation in recycling
conditions is also limited by a step different than dissociation from
the elongated duplex. Surprisingly, no decrease of the
kobs is observed at the positions located
immediately upstream of Ter2. Even if there is a decrease of
kc at these positions, its values are
still larger than (or on the order of) the local dissociation rates. The restriction imposed at Ter2 and at the two vicinal positions on
further elongation is therefore extremely severe.
Open Questions--
These kinetic studies did not allow to define
which step is precisely slowed down at Ter2. If the kinetic pathway
between the intermediates E-Dn dNTP and
E-Dn + 1 is the same at Ter2 as at
processive positions, then this slower step could be the isomerization
of the ternary complex into an activated ternary complex and/or the
chemical step that follows this isomerization. The high value of the
dissociation constant associated with dCTP binding already suggests
that the substrate binding site is not correctly folded in the
E-Dn dNTP ternary complex. In consequence, an isomerization of
this complex would be required to reach the kinetically competent
complex and would correspond to the rate-limiting step. However, it is
also possible that the ternary complex E-Dn-dNTP
formed at Ter2 follows a different kinetic path to generate the
elongation product. Furthermore, although the same simplified
sequential pathway can account for the elongation mechanism at
positions where synthesis is processive or distributive (Ter2), it
would be misleading to assume that the structure of the postulated
intermediates will be roughly identical. The use of footprinting
techniques, reported in the companion paper (33), helps to specify the
nature of the interactions involved in these transient intermediates.
| |
ACKNOWLEDGEMENTS |
We are very grateful to Dr. Torsten Unge
(Uppsala, Sweden) for the steady supply of HIV-1 RT and to Dr. Bianca
Sclavi (Institut Gustave Roussy, France) for precious help in
experiments with the Kintek and for a critical reading of the
manuscript. We also thank Geneviève Legat for technical assistance.
| |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Wellman 10, Massachusetts General Hospital, Fruit St., Boston,
MA 02114. E-mail: lavigne@molbio.mgh.harvard.edu.
§
Present address: Dept. of Molecular Biology, Institut Pasteur,
75724 Paris Cedex 15, France.
¶
Present address: URA 1960 CNRS, Institut Pasteur, 75724 Paris
Cedex 15, France.
Published, JBC Papers in Press, June 11, 2001, DOI 10.1074/jbc.M102974200
| |
ABBREVIATIONS |
The abbreviations used are:
CTS, central
termination sequence;
RT, reverse transcriptase;
WT, wild type;
MGBT, minor groove binding track;
HIV-1, human immunodeficiency virus,
type 1.
| |
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TOP
ABSTRACT
INTRODUCTION
