RNA Dependent DNA Replication Fidelity of HIV-1 Reverse Transcriptase: Evidence of Discrimination between DNA and RNA Substrates†
Identifieur interne : 000B19 ( Istex/Corpus ); précédent : 000B18; suivant : 000B20RNA Dependent DNA Replication Fidelity of HIV-1 Reverse Transcriptase: Evidence of Discrimination between DNA and RNA Substrates†
Auteurs : Stephen G. Kerr ; Karen S. AndersonSource :
- Biochemistry [ 0006-2960 ] ; 1997.
Abstract
The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template−primer is employed as compared with the corresponding DNA/DNA template−primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 × 106 for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template−primer substrates, HIV-1 RT exhibits approximately a 10−60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.
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DOI: 10.1021/bi971385+
Links to Exploration step
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<front><div type="abstract">The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template−primer is employed as compared with the corresponding DNA/DNA template−primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 × 106 for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template−primer substrates, HIV-1 RT exhibits approximately a 10−60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.</div>
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<profileDesc><abstract><p>The RNA dependent DNA replication fidelity of HIV-1 reverse
transcriptase has been
investigated using pre-steady-state kinetics under single turnover
conditions. In contrast to previous
estimates of low replication fidelity of HIV-1 reverse transcriptase,
the present study finds the enzyme to
be more highly discriminating when an RNA/DNA template−primer is
employed as compared with the
corresponding DNA/DNA template−primer. The basis of this
selectivity is due to extremely slow
polymerization kinetics for incorporation of an incorrect
deoxynucleotide. The maximum rates for
misincorporation (<hi rend="italic">k</hi>
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dGTP, 176 000 for dCTP, and 1 ×
10<hi rend="superscript">6</hi>
for dTTP misincorporation at this position.
Similar observations were obtained when incorrect
nucleotide misincorporation was examined opposite a template adenine.
Thus in a direct comparison of
RNA/DNA and DNA/DNA template−primer substrates, HIV-1 RT exhibits
approximately a 10−60-fold
increase in fidelity. This study augments our current
understanding of the similarities and differences of
catalytic activity of HIV-1 reverse transcriptase using RNA and DNA
substrates. Moreover, these studies
lend further support for a model for nucleotide incorporation by HIV-1
reverse transcriptase involving a
two-step binding mechanism governed by a rate-limiting conformational
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Present address: Massachusetts College of
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Health Sciences, 179 Longwood Ave., Boston, MA 02115.</p>
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Author to whom correspondence should be addressed.
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(203)-785-4526. Fax: (203)-785-7670. email:
karen.anderson@yale.
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<abstract><p>The RNA dependent DNA replication fidelity of HIV-1 reverse
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conditions. In contrast to previous
estimates of low replication fidelity of HIV-1 reverse transcriptase,
the present study finds the enzyme to
be more highly discriminating when an RNA/DNA template−primer is
employed as compared with the
corresponding DNA/DNA template−primer. The basis of this
selectivity is due to extremely slow
polymerization kinetics for incorporation of an incorrect
deoxynucleotide. The maximum rates for
misincorporation (<italic toggle="yes">k</italic>
<sub>pol</sub>
) of dGTP, dCTP, and dTTP
opposite a template uridine were 0.2, 0.03, and 0.003
s<sup>-1</sup>
, respectively. The equilibrium
dissociation constants (<italic toggle="yes">K</italic>
<sub>d</sub>
) for the incorrect
nucleotide opposite a
template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP,
respectively. These kinetic
values provide fidelity estimates of 26 000 for discrimination against
dGTP, 176 000 for dCTP, and 1 ×
10<sup>6</sup>
for dTTP misincorporation at this position.
Similar observations were obtained when incorrect
nucleotide misincorporation was examined opposite a template adenine.
Thus in a direct comparison of
RNA/DNA and DNA/DNA template−primer substrates, HIV-1 RT exhibits
approximately a 10−60-fold
increase in fidelity. This study augments our current
understanding of the similarities and differences of
catalytic activity of HIV-1 reverse transcriptase using RNA and DNA
substrates. Moreover, these studies
lend further support for a model for nucleotide incorporation by HIV-1
reverse transcriptase involving a
two-step binding mechanism governed by a rate-limiting conformational
change for correct incorporation.
</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>bi971385+</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes id="bi9713851AF2"><label>†</label>
<p>
This work was supported by NIH Grant GM 49551 to
K.S.A.</p>
</notes>
<notes id="bi9713851AF7"><label>✗</label>
<p>
Abstract published in <italic toggle="yes">Advance ACS
Abstracts,</italic>
November 1, 1997.</p>
</notes>
</front>
<body><sec id="d7e134"><title></title>
<p>HIV-1 reverse transcriptase (RT<xref rid="atyp_ref1" ref-type="bibr"></xref>
) is
a virally encoded
enzyme essential for viral replication and the target of
clinically useful drugs for treatment of aquired immunodeficiency syndrome (AIDS). An in-depth understanding of
the catalytic mechanism of HIV-1 RT may ultimately aid in
the development of better therapeutics.
</p>
<p>Pre-steady-state kinetic techniques have been previously
employed to determine the mechanism for polymerases and
to provide estimates of replication fidelity (Kuchta et
al.,
1987; Kuchta et al., 1988). Such transient kinetic
model
systems have also been used to provide mechanistic
information for HIV-1 RT (Kati et al., 1992; Reardon, 1992;
Reardon, 1993; Hsieh et al., 1993). Earlier
mechanistic
studies on HIV-1 reverse transcriptase (RT) using rapid
transient kinetic analysis suggest that catalysis at the
active
site is similar to other polymerases such as T7 DNA
polymerase (Patel et al., 1991; Wong et al., 1991)
involves
an “induced fit” model for polymerization (Kati et al.,
1992,
Hsieh et al., 1993). Although the proposal of a
two-step
binding model involving a rate-limiting conformational
change (Kati et al., 1992) has been challenged (Reardon,
1993), more recent transient kinetic studies on HIV-1 RT
substantiate the validity of this model (Spence et al.,
1995;
Rittinger et al., 1995). This model describes the
reaction
pathway for deoxynucleotide triphosphate (dNTP) incorporation for HIV-1 RT in terms of a two-step binding process
(Kati et al., 1992; Hsieh et al., 1993; Spence et al.,
1995;
Rittinger et al., 1995). The first step in dNTP
binding
comprises an initial binding complex in which the dNTP is
base paired with the requisite base in the template strand.
In
the second step the enzyme checks for proper base pairing
geometry in which only correct base pairs may induce the
formation of a tight ternary complex leading to catalysis.
The maximal rate of polymerization is governed by a
rate-limiting conformational change which precedes the chemical
catalytic step and is observed with both DNA/DNA
template−primer substrates as well as RNA/DNA template−primer
substrates.
</p>
<p>This two-step mechanism controls the selectivity for
correct versus incorrect dNTP incorporation. Whereas
T7
DNA polymerase is considered to exhibit high DNA replication fidelity (Johnson, 1993), HIV-1 RT has been reported
to have a low DNA replication fidelity (Preston et al.,
1988;
Roberts et al., 1988; Weber et al., 1989; Bebenek et al.,
1989;
Ji and Loeb, 1992, 1994; Perrino et al., 1989; Mendelman
et al., 1989; Mendelman et al., 1990; Yu et al., 1992;
Preston
et al., 1997). The low estimates for DNA fidelity are
based
upon analysis of mutation frequency using assays involving
transfection (Preston et al., 1988; Roberts et al., 1988;
Weber
et al., 1989; Bebenek et al., 1989; Ji and Loeb, 1992;
Perrino
et al., 1989) or steady-state kinetic gel shift systems
(Preston
et al., 1988; Mendelman et al., 1989; Mendelman et al.,
1990;
Ricchetti and Buc, 1990; Yu et al., 1992).
Error-prone
polymerization of RT has also been
described in terms of
errors initiated by template−primer misalignments
(Roberts
et al., 1988; Bebenek et al., 1989; Bebenek et al., 1993).
These studies have provided insight toward our
understanding
of the biologically important reactions contributing to
observed mutation frequencies. However, these studies
do
not provide a detailed mechanistic picture concerning how
HIV-1 RT accommodates both RNA/DNA and DNA/DNA
duplexes and controls DNA replication fidelity.
</p>
<p>A rapid transient kinetic approach will provide an
in-depth
mechanistic understanding of factors governing replication
fidelity for DNA dependent and RNA dependent polymerization. The DNA dependent replication fidelity of
HIV-1
RT has been examined using rapid transient kinetic
analysis
(Kati et al., 1992; Zinnen et al., 1994). In an effort to
further
understand the similarities and differences for the
catalytic
activity of HIV-1 RT in utilizing DNA and RNA substrates,
the present study was undertaken to examine the RNA
dependent DNA replication fidelity of HIV-1 RT. The
biological implications are important since it is likely
that
the RNA dependent DNA replication fidelity may play an
essential role in successful viral survival.
</p>
<p>In this report we provide a comparison of replication
fidelity using DNA/DNA and RNA/DNA template−primer
substrates. The fidelity estimates provided in this
study
reveal differences in DNA and RNA substrates and lend
further support for an induced fit mechanism in which
conformational coupling plays an important role in polymerase fidelity (Johnson, 1993).
</p>
</sec>
<sec id="d7e150"><title>Experimental Procedures</title>
<p><italic toggle="yes">Overexpression and Purification of Recombinant HIV-1
RT</italic>
. In these experiments, RT was purified from a
clone
generously provided by Barbara Müller and Roger
Goody
which expresses both subunits (66 and 51 kDa) of the
protein
(Müller et al., 1989). The <italic toggle="yes">Escherichia coli </italic>
cells
were grown
up in 2× YT broth containing ampicillin (100 mg/L) at 37
°C for 4 h to an optical density of 0.5 unit at 595 nm.
The
growth was then induced by the addition of IPTG (1 mM),
and the cells were allowed to grow for an additional 5−6
h.
All purification steps were done in the cold, 0−4 °C.
Cells
were harvested by centrifugation at 4 °C and resuspended
in buffer A (50 mM Tris-Cl, pH 8.0, 2 mM EDTA, 2 mM
dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 10%
glycerol) containing 0.5 M NaCl and 0.1% Triton X-100.
The cells were lysed using a french pressure cell press
(SLM
Instruments) at 20 000 psi. The lysed cells were
centrifuged
at 15 000 rpm. The supernatant was decanted and the
pellet
extracted once more with the lysis buffer followed by
centrifugation. The two supernatants were combined
and
nucleic acids precipitated by addition of 5%
poly(ethyleneimine) to a final concentration of 0.3%. The
precipitated
nucleic acids were removed by centrifugation at 10 000
rpm.
To the supernatant was then added ammonium sulfate
(60%).
Crude precipitated protein was centrifuged, resuspended,
and
dialyzed against buffer A. Crude RT was purified by
column
chromatography over a phosphocellulose column (Waters)
using a linear gradient of 0.05−1 M NaCl in buffer A.
Fractions containing RT were pooled and dialyzed
against
buffer A and subsequently loaded onto a Q-Sepharose
(Pharmacia) column. RT was eluted using a linear
gradient
from 10 to 250 mM NaCl. The pooled RT fractions were
combined, concentrated, and further purified by strong
cation
exchange FPLC using a Mono S 5/5 column (Pharmacia) as
previously described (Stahlhut et al., 1994). This step
has
been shown to separate homodimeric p66 and p51 species
from p66/p51 heterodimer and provide the desired heterodimer in a 1:1 ratio. The RT was dialyzed against
buffer
A containing 50 mM NaCl, concentrated, aliquoted, and
stored at −70 °C. Enzyme concentration was estimated
by
UV at 280 nm using an extinction coefficient of 260 450
M<sup>-1</sup>
cm <sup>-1</sup>
as
previously described (Kati et al., 1992).
Concentrations of RT used in subsequent experiments
were
determined by an active site titration method as
previously
described (Kati et al., 1992). The preparation of RT
with
this system and purification procedure gave burst
amplitudes
of 30−40%, and the enzyme concentrations for all
experiments were performed using the corrected active concentration. The RT obtained after this purification procedure
was
highly pure and free of contaminating exonuclease or RNAse
activities. The exonuclease activity was judged by
incubation
of enzyme with radiolabeled DNA/DNA 45−25-mer for 30
min at 37 °C and the RNase activity was evaluated by
incubation of enzyme with radiolabeled RNA 45-mer for 30
min at 37 °C. In each case <5% of the substrate
was
degraded to cleavage products.
</p>
<p><italic toggle="yes">Nucleotide Triphosphates and Other Materials.</italic>
All
dNTPs were obtained from Pharmacia LKB Biotechnology
Inc. and determined to be >99% pure by anion-exchange
HPLC. [γ-<sup>32</sup>
P]ATP was obtained from Amersham
Co.
Biospin columns for purification of labeled oligomers were
obtained from BioRad.
</p>
<p><italic toggle="yes">Synthetic Oligonucleotides. </italic>
The DNA 25-mer and
22-mers (Table <xref rid="bi9713851t00001"></xref>
) were synthesized on an
Applied Biosystems
380A DNA synthesizer from the Yale DNA Synthesis
Facility and purified by denaturing polyacrylamide gel
electrophoresis (16%) as previously described (Kati et
al.,
1992). RNA 45-mer (Table <xref rid="bi9713851t00001"></xref>
) was obtained from the
Yale
Synthesis Facility and purified as described (Kati et al.,
1992)
or obtained as the deprotected gel and HPLC purified
material from New England Biolabs, Inc.
<table-wrap id="bi9713851t00001" position="float" orientation="portrait"><label>1</label>
<caption><p>RNA and DNA Oligonucleotides Substrates</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="bi9713851u00001a.tif" position="float" orientation="portrait"></graphic>
</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
</table-wrap>
</p>
<p>The heteroduplex RNA/DNA 45/25-mer template−primer
strands were annealed using equimolar ratios of pure
template−primer at 80 °C for 4 min and 50 °C for 30
min
as previously described (Kati et al.,
1992). Concentrations
of the oligomers were estimated from UV absorbances at
260 nm using extinction coefficients of 507 960 and
249 040
M<sup>-1</sup>
cm<sup>-1</sup>
for the
RNA 45-mer, and DNA 25-mer, respectively. The corresponding extinction coefficient for the
DNA
22-mer was 218 390 M<sup>-1</sup>
cm<sup>-1</sup>
.
</p>
<p><italic toggle="yes">Buffers. </italic>
All experimental procedures involving
kinetic
experiments of RT were done in 50 mM Tris-Cl, 50 mM
NaCl, pH 7.5 at 37 °C, and used sterile buffers,
reagents,
and laboratory ware wherever possible.
</p>
<p><italic toggle="yes">5</italic>
‘<italic toggle="yes">-[</italic>
<italic toggle="yes"><sup>32</sup>
</italic>
<sup></sup>
<italic toggle="yes">P]-Labeling
the 45/25-mers and 45/22-mers.</italic>
The
RNA 45-mer, DNA 25-mer, and DNA 22-mer strands were
5‘-labeled with [γ-<sup>32</sup>
P]ATP using T4 polynucleotide
kinase
(New England Biolabs, Inc.) according to previously described procedures (Kati et al., 1992).
</p>
<p><italic toggle="yes">Rapid Quench Experiments.</italic>
Rapid quench
experiments
were carried out in an apparatus designed by Johnson
(Johnson, 1986; Johnson, 1992) and built by Kintek Instruments (State College, PA). The apparatus was modified
to
allow small reaction volumes of 15 μL. Experiments
were
carried out as described (Kati et al., 1992). Briefly, 15
μL
of substrate (RNA or DNA template−primer) preincubated
with enzyme (RT) was loaded in one sample loop while the
other loop contained an equal amount of the nucleotide to
be incorporated, preincubated with Mg<sup>2+</sup>
. Enzyme
catalysis
was initiated by rapidly mixing the two reactants together
and was terminated by quenching with 0.3 M EDTA (final
concentration) after time points ranging from milliseconds
to several seconds. In the cases where long reaction
times
were required (as in the case for dTMP incorporation),
manual quench experiments were undertaken in which the
two solutions (substrate preincubated with RT and dTTP-Mg<sup>2+</sup>
) were incubated at 37 °C, and at regular time
intervals
30 μL of the reaction solution was removed and quenched
with EDTA (0.3 M final concentration). All
concentrations
reported in the text are final concentrations after
mixing.
</p>
<p><italic toggle="yes">Product Analysis.</italic>
The products were analyzed by
sequencing gel analyses (16% polyacrylamide gel in 8 M urea)
where elongation products from the primer strand and the
template strand and its degradation products could be
resolved and quantitated. The products and substrates
were
quantitated by scanning the dried gel using a either a GS-250 Molecular Imager System (BioRad) or Betascope
(Betagen).
</p>
<p><italic toggle="yes">Data Analysis.</italic>
Data were fit by nonlinear
regression
analysis using the commercially available program KaleidaGraph (Synergy Software, Reading, PA) for the Macintosh
computer. The data were fit to a burst equation, product
=
<italic toggle="yes">A</italic>
(1 − exp(−<italic toggle="yes">kt</italic>
)) + <italic toggle="yes">mt</italic>
, where
<italic toggle="yes">A</italic>
is the amplitude of the burst,
<italic toggle="yes">k</italic>
is the observed first-order burst rate constant, and
<italic toggle="yes">m</italic>
is the
linear steady-state rate constant; for pre-steady-state
bursts
experiments or, to a single exponential, product = <italic toggle="yes">A</italic>
(1
−
exp(−<italic toggle="yes">kt</italic>
)) according to the mechanism previously
described
(Kati et al., 1992). The concentration dependence of
the
burst rate were fit to a hyperbolic function in which <italic toggle="yes">k</italic>
=
<italic toggle="yes">k</italic>
<sub>pol</sub>
[dNTP]/(<italic toggle="yes">K</italic>
<sub>d</sub>
+
[dNTP]), where <italic toggle="yes">k</italic>
is the observed rate,
<italic toggle="yes">k</italic>
<sub>pol</sub>
is the maximum rate of incorporation and <italic toggle="yes">K</italic>
<sub>d</sub>
is
the equilibrium dissociation constant for the dNTP. The term
<italic toggle="yes">k</italic>
<sub>pol</sub>
is
used even in cases where the rates are extremely slow
since
under single turnover conditions the observed rate is still
a
function of the maximum rate of nucleotide incorporation.
Since <italic toggle="yes">k</italic>
<sub>pol</sub>
is most likely defined by a slow
conformational
or chemical step, and not product release, it would be
equivalent to <italic toggle="yes">k</italic>
<sub>cat</sub>
.
</p>
</sec>
<sec id="d7e312"><title>Results</title>
<p>In this report we contrast the RNA dependent and DNA
dependent replication fidelity of recombinant HIV-1
reverse
transcriptase by examining the kinetics of incorrect
nucleotide
incorporation into a RNA/DNA template−primer as compared with the corresponding DNA/DNA template−primer
substrate. Studies were conducted with two synthetic
oligonucleotide substrates: a 45/25 template−primer and
a
45/22 template−primer (shown in Table <xref rid="bi9713851t00001"></xref>
) . For the
45/25
template−primer, the mismatch involved incorporation of
an incorrect dNTP (dGTP, dCTP, and dTTP) opposite a
template uridine (U). This analysis was extended to a
45/22 template−primer to examine the incorporation of an
incorrect dNTP (dGTP, dCTP, and dATP) opposite a
template adenosine (A). The polymerization rate
constants
(<italic toggle="yes">k</italic>
<sub>pol</sub>
) and equilibrium dissociation constants
(<italic toggle="yes">K</italic>
<sub>d</sub>
) of the
incorrect dNTPs have been determined. From these
kinetic
parameters, fidelity estimates for the RNA dependent DNA
polymerization have been calculated. The results are
compared and contrasted with the fidelity estimates of DNA
dependent DNA polymerization (Kati et al., 1992).
</p>
</sec>
<sec id="d7e327"><title></title>
<sec id="d7e329"><title>Pre-Steady-State Kinetic Studies Using a 45/25
Template−Primer</title>
<p><italic toggle="yes">Correct Incorporation of dATP into Heteroduplex RNA/DNA 45/25 </italic>
<italic toggle="yes">Template</italic>
<italic toggle="yes">−</italic>
<italic toggle="yes">Primer.</italic>
We began our studies
examining RNA dependent replication fidelity by determining the kinetic parameters for correct dNTP incorporation
to provide a framework for comparison of incorrect incorporation. A representative pre-steady-state burst
experiment
for the correct incorporation of dATP is shown in Figure
<xref rid="bi9713851f00001"></xref>
.
The experiment was performed by mixing a preincubated
solution of RT (175 nM active site concentration) and 5‘-[<sup>32</sup>
P]-labeled RNA/DNA 45/25-mer (300 nM) with
Mg<sup>2+</sup>
(10
mM) and dATP (200 μM), the nucleotide for correct
incorporation. The resulting time
course of dATP incorporation (Figure <xref rid="bi9713851f00001"></xref>
) showed biphasic kinetics with the first
turnover occurring at a rate (<italic toggle="yes">k</italic>
<sub>pol</sub>
) of 68
s<sup>-1</sup>
and subsequent
turnovers at a rate (steady-state rate) of 0.13
s<sup>-1</sup>
similar to
our previous results (Kati et al., 1992). Likewise,
biphasic
kinetics were also observed for the correct incorporation
of
dTTP into an RNA/DNA or DNA/DNA 45/22 template−primer (see below).
<fig id="bi9713851f00001" position="float" orientation="portrait"><label>1</label>
<caption><p>Pre-steady-state kinetics of correct incorporation of dATP, U·dA base pair. Pre-steady-state kinetics of correct incorporation of dATP into heteroduplex RNA/DNA 45/25-mer (template−primer) were measured by mixing a preincubated solution of RT (175 nM active site concentration) and 5‘-[<sup>32</sup>
P]-labeled RNA/DNA (300 nM) with Mg<sup>2+</sup>
(10 mM) and dATP (200 μM)
under
rapid quench conditions. The reactions were quenched with 0.3
M
EDTA at the indicated times, and the product 26-mer primer
was
separated and quantitated by sequencing gel analysis (16%
acrylamide, 8 M urea). The solid line represents the best fit of the
data
(·) to a burst equation with rate constants equal to 68 and
0.13
s<sup>-1</sup>
for the exponential and linear phases,
respectively.</p>
</caption>
<graphic xlink:href="bi9713851f00001.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p><italic toggle="yes">Misincorporation of dNTP into RNA/DNA 45/25-mer.
</italic>
The
kinetics of misincorporation of the three deoxynucleoside
triphosphates, dGTP, dCTP, and dTTP, into the heteroduplex
RNA/DNA 45/25-mer were determined. The experiments
with dGTP and dCTP required the use of a rapid quench
apparatus while those for dTTP were conducted by manual
quench. The equilibrium dissociation constants,
<italic toggle="yes">K</italic>
<sub>d</sub>
s, and
maximum rates of incorporation, <italic toggle="yes">k</italic>
<sub>pol</sub>
, of the
three incorrect
dNTPs were determined under single turnover conditions
with enzyme (RT 100 nM) in slight excess of substrate
(RNA/DNA, 90 nM) and quantitating all products formed.
Substrate inhibition occurred with dNTP concentrations
in
excess of 2 mM as previously observed (Kati et al., 1992).
</p>
<p><italic toggle="yes">(a) Misincorporation of dGTP: Kinetics of a
U·dG
Mismatch.</italic>
Figure <xref rid="bi9713851f00002"></xref>
A shows the
concentration dependence
on the first-order rates of incorporation of dGTP into
the
primer 25-mer strand opposite a template uridine.
Reactions
were carried out as indicated in the figure legend.
The
misincorporation of dGTP led to the elongation of the 25-mer by one base to form a 26-mer product. The
formation
of the 26-mer product was quantified and plotted versus
time.
The data for each time course was fit to a single
exponential
to provide the rate of polymerization. Figure <xref rid="bi9713851f00002"></xref>
B is a
plot
of the first-order rates, determined above, versus dGTP
concentration (mM) and fit to a hyperbola to determine,
<italic toggle="yes">k</italic>
<sub>pol</sub>
,
the maximum rate of incorporation (0.203 ± 0.03
s<sup>-1</sup>
), and
the dissociation constant, <italic toggle="yes">K</italic>
<sub>d</sub>
, for dGTP (1 ±
0.3 mM) for
the U·dG mismatch.
<fig id="bi9713851f00002" position="float" orientation="portrait"><label>2</label>
<caption><p>dGTP concentration dependence on the first-order rates for misincorporation, U·dG mismatch. (A) A preincubated solution of RT (100 nM) and 5‘-[<sup>32</sup>
P]-labeled 45/25-mer RNA/DNA
(90
nM) was mixed with increasing concentrations of dGTP in
Mg<sup>2+</sup>
buffer to start the reactions. The reactions were quenched with
0.3
M EDTA at the indicated times, and the products were
analyzed
and quantitated as indicated previously. The dGTP
concentrations
were (▴) 0.125 mM; (▪) 0.25 mM; (⧫) 0.5 mM; (○) 0.9 mM;
(·)
2 mM. The solid lines represent the best fit of the data to the
first-order processes. (B) The first-order rates (·) measured above
were
plotted against the dGTP concentrations. The fit to a
hyperbola
yielded a <italic toggle="yes">K</italic>
<sub>d</sub>
value of 1 ± 0.3 mM for dGTP
dissociation and a
maximum rate of misincorporation of 0.203 ± 0.03
s<sup>-1</sup>
.</p>
</caption>
<graphic xlink:href="bi9713851f00002.tif" position="float" orientation="portrait"></graphic>
</fig>
<fig id="bi9713851f00003" position="float" orientation="portrait"><label>3</label>
<caption><p>Gel analysis of misincorporation of dCTP into RNA/DNA 45/25-mer, a U·dC mismatch. The bands indicate the time course (0−384 s) of polymerization of the 45/25-mer RNA/DNA heteroduplex (90 nM) by RT (100 nM) in presence of dCTP (1.25 mM) and Mg<sup>2+</sup>
(10 mM). Polymerization product bands are at
26-,
27-, 28-, 29-, and 30-mer (see text for explanation). The
product
was quantitated by the summation of all product bands
(26−30-mer) for the individual time courses. The RNA template 45-mer
is
also visible along with the RNA cleavage fragments at 39-
and
38-mer and smaller, depicting the simultaneous RNAse H
activity
of HIV-1 RT. These cleavage fragments are similar to those
seen
previously (Kati et al., 1992) and confirm the distance
between
the polymerase and RNAse H sites to be approximately
18−19
nucleotides apart.</p>
</caption>
<graphic xlink:href="bi9713851f00003.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p><italic toggle="yes">(b) Misincorporation of dCTP: Kinetics of a
U·dC
Mismatch.</italic>
Similar experiments to those done above
for
dGTP were conducted to obtain the dissociation constant
and
an estimate of the maximum rate of incorporation of dCTP
opposite a template uridine. The incorporation of dCTP
led
to a series of five bands of sizes 26−30 bases. This
resulted
from the fact that after the initial misincorporation of
dCMP
to form DNA 26-mer, dCTP is the correct nucleotide for
the next two additions (27- and 28-mers). At higher
concentrations and longer time points, a second misincorporation on the 28-mer generates a 29-mer, and since dCTP
is again the correct nucleotide for the next addition, a
30-mer accumulates. The gel analysis of the time course
of
the reaction of a preincubated solution of RT (100 nM) and
5‘-[32P]-doubly-labeled 45/25-mer RNA/DNA (90 nM) with
dCTP (1.25 mM) in Mg<sup>2+</sup>
buffer is shown in Figure <xref rid="bi9713851f00003"></xref>
. The
major polymerization products (dark bands) of 28-mer and
30-mer, where the reaction has stalled, are clearly
visible,
while faint bands of the 26-mer, 27-mer, and 29-mer can
also been seen. Other bands seen are those from the
RNAse
H cleavage reaction (see Discussion below). All five
bands
were summed and quantified to yield total products, thus
defining the kinetics of the first misincorporation (Wong
et
al., 1991; Kati et al., 1992). Thus, from a plot of
total
products (26−30-mer) formed versus time and fitting the
plot to a first-order (single turnover) equation, one can
obtain
a lower limit for the rate of dCTP incorporation, Figure
<xref rid="bi9713851f00004"></xref>
A.
From a plot of the rate constants of dCTP
incorporation
versus dCTP concentration and fitting the data to a
hyperbola,
the lower limit for maximum rate of incorporation of the
U·dC mismatch was determined to be 0.033 ± 0.002
s<sup>-1</sup>
and <italic toggle="yes">K</italic>
<sub>d</sub>
for dCTP was 1.1 ± 0.15 mM, Figure
<xref rid="bi9713851f00004"></xref>
B.
<fig id="bi9713851f00004" position="float" orientation="portrait"><label>4</label>
<caption><p>Misincorporation of dCTP into RNA/DNA 45/25-mer, U·dC mismatch. (A) A preincubated solution of RT (100 nM) and 5‘-[<sup>32</sup>
P]-labeled 45/25-mer RNA/DNA (90 nM) was mixed
with
dCTP (1.25 mM) in Mg<sup>2+</sup>
buffer to start the reaction. The
reaction
was quenched with 0.3 M EDTA at the indicated times, and
the
products were analyzed and quantitated as indicated in the
text.
The solid line represents the best fit of the data (·) under
single
turnover conditions, using a rate constant of 0.018
s<sup>-1</sup>
for the first-order exponential process. (B) The first-order rate
constants
determined analogously to the above experiment, with
varying
concentrations of dCTP, were plotted against the dCTP
concentrations. The data (·) were fit to a hyperbola (solid line) to yield
a
<italic toggle="yes">K</italic>
<sub>d</sub>
value of 1.11 ± 0.15 mM for dCTP
dissociation and a rate of
misincorporation of 0.033 ± 0.002
s<sup>-1</sup>
.</p>
</caption>
<graphic xlink:href="bi9713851f00004.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p><italic toggle="yes">(c) Misincorporation of dTTP: U·dT Mismatch.</italic>
Identical
experiments to those conducted for dCTP and dGTP were
conducted for determining the incorporation rate for a
U·dT
mismatch. The maximum rate of incorporation was determined to be 0.0034 ± 0.0004 s<sup>-1</sup>
and
<italic toggle="yes">K</italic>
<sub>d</sub>
for dTTP
dissociation was 0.7 ± 0.18 mM for the U·dT
mismatch.
Striking differences are noted between the rates of
misincorporation for the RNA template as compared with DNA
for each of the mismatches examined as shown in Figure <xref rid="bi9713851f00005"></xref>
.
This figure visually illustrates these findings by showing
a
gel analysis directly comparing misincorporation of dTMP
using the RNA 45-mer template (right) with the DNA 45-mer template (left). At the 40 s time point, most of the
25-mer has been converted to 26-mer for the DNA while only
a trace of 26-mer is seen with the RNA template. Also
shown in Figure <xref rid="bi9713851f00005"></xref>
are the anticipated RNAase H cleavage
products (RNA 38-mer and 39-mer) as discussed below.
</p>
<p><italic toggle="yes">Fidelity Estimates.</italic>
Table
<xref rid="bi9713851t00002"></xref>
summarizes the kinetic
parameters for correct and incorrect incorporation into
the
heteroduplex RNA/DNA 45/25 template−primer. The RNA
dependent DNA replication fidelity was estimated from the
ratios of incorporation of the second-order rate constants
of
correct to incorrect, as calculated by the expression
shown
in Table <xref rid="bi9713851t00002"></xref>
. Fidelity estimates for the U·dG, U·dC,
and U·
dT mismatches were 26 000, 176 000, and 1 000 000,
respectively. The corresponding estimates for
<italic toggle="yes">k</italic>
<sub>pol</sub>
(shown
in parentheses) and DNA dependent replication fidelity for
the DNA/DNA 45/25 template−primer are also shown for
comparison.
<table-wrap id="bi9713851t00002" position="float" orientation="portrait"><label>2</label>
<caption><p>Fidelity of HIV-1 RT with 45/25 Template−Primer</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1">dNTP</oasis:entry>
<oasis:entry colname="2"><italic toggle="yes">K</italic>
<sub>d</sub>
(μM)</oasis:entry>
<oasis:entry colname="3"><italic toggle="yes">k</italic>
<sub>pol</sub>
(s<sup>-1</sup>
)</oasis:entry>
<oasis:entry colname="4"><italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
(μM<sup>-1</sup>
s<sup>-1</sup>
)</oasis:entry>
<oasis:entry colname="5">fidelity<italic toggle="yes"><sup>a</sup>
</italic>
<sup></sup>
(RNA)</oasis:entry>
<oasis:entry colname="6">fidelity<italic toggle="yes"><sup>b</sup>
</italic>
<sup></sup>
(RNA)
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dATP
</oasis:entry>
<oasis:entry colname="2">14
</oasis:entry>
<oasis:entry colname="3">74 (33)
</oasis:entry>
<oasis:entry colname="4">5.28
</oasis:entry>
<oasis:entry colname="5"></oasis:entry>
<oasis:entry colname="6"></oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dGTP
</oasis:entry>
<oasis:entry colname="2">1000
</oasis:entry>
<oasis:entry colname="3">0.2 (4.8)
</oasis:entry>
<oasis:entry colname="4">2.0 × 10<sup>-4</sup>
</oasis:entry>
<oasis:entry colname="5">26 000
</oasis:entry>
<oasis:entry colname="6">1740
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dCTP
</oasis:entry>
<oasis:entry colname="2">1100
</oasis:entry>
<oasis:entry colname="3">0.03 (0.05)
</oasis:entry>
<oasis:entry colname="4">3.0 × 10<sup>-5</sup>
</oasis:entry>
<oasis:entry colname="5">176 000
</oasis:entry>
<oasis:entry colname="6">19 700
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dTTP
</oasis:entry>
<oasis:entry colname="2">700
</oasis:entry>
<oasis:entry colname="3">0.003 (0.4)
</oasis:entry>
<oasis:entry colname="4">4.9 × 10<sup>-6</sup>
</oasis:entry>
<oasis:entry colname="5">1 100 000
</oasis:entry>
<oasis:entry colname="6">16 900</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic toggle="yes"><sup>a</sup>
</italic>
<sup></sup>
Calculated as
[(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>correct</sub>
+
(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>incorrect</sub>
]/(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>incorrect</sub>
.<italic toggle="yes"><sup>b</sup>
</italic>
<sup></sup>
Kati
et al., 1992; values in parentheses for <italic toggle="yes">k</italic>
<sub>pol</sub>
are
the rates of incorporation
for the DNA template.</p>
</table-wrap-foot>
</table-wrap>
</p>
</sec>
</sec>
<sec id="d7e686"><title></title>
<sec id="d7e688"><title>Pre-Steady-State Kinetic Studies Using a 45/22
Template−Primer</title>
<p>Although the higher fidelity for an RNA/DNA template−primer compared to a DNA/DNA template−primer in the
case of a 45/25 template−primer was interesting, the
generality of this observation was uncertain.
Therefore,
analysis of a 45/22 template−primer was undertaken to
examine the correct and incorrect incorporation of dNTP
opposite a template adenosine (A) using the DNA and RNA
substrates shown in Table <xref rid="bi9713851t00001"></xref>
. The equilibrium
dissociation
constants, <italic toggle="yes">K</italic>
<sub>d</sub>
s, and maximum rates of
incorporation, <italic toggle="yes">k</italic>
<sub>pol</sub>
, for
the correct and three incorrect dNTPs were determined
under
single turnover conditions analogous to that described
above
for the 45/25 template−primer. The kinetic parameters,
K<sub>d</sub>
and <italic toggle="yes">k</italic>
<sub>pol</sub>
, and fidelity estimates for the
A·dC, A·dA, and A·
dG mismatches using a DNA or RNA substrate are shown
in Table <xref rid="bi9713851t00003"></xref>
. The estimates for
DNA dependent replication
fidelity for a dC, dA, and dC mismatch were 800, 150 000,
and 350 000. For the corresponding RNA dependent replication fidelity for dC, dA, and dC mismatches, the
estimates
were 12 000, 3 500 000, and 4 600 000. These
results
suggest that HIV-1 RT is, in general, more highly discriminating toward an RNA/DNA template−primer.
<table-wrap id="bi9713851t00003" position="float" orientation="portrait"><label>3</label>
<caption><p>Fidelity of HIV-1 RT for 45/22 Template−Primer</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="9"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:colspec colnum="9" colname="9"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry>
<oasis:entry namest="2" nameend="3"><italic toggle="yes">k</italic>
<sub>pol</sub>
(s<sup>-1</sup>
)</oasis:entry>
<oasis:entry namest="4" nameend="5"><italic toggle="yes">K</italic>
<sub>d</sub>
(μM)</oasis:entry>
<oasis:entry namest="6" nameend="7"><italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
(μM<sup>-1</sup>
s<sup>-1</sup>
)</oasis:entry>
<oasis:entry namest="8" nameend="9">fidelity<italic toggle="yes"><sup>a</sup>
</italic>
<sup></sup>
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dNTP</oasis:entry>
<oasis:entry colname="2">RNA</oasis:entry>
<oasis:entry colname="3">DNA</oasis:entry>
<oasis:entry colname="4">RNA</oasis:entry>
<oasis:entry colname="5">DNA</oasis:entry>
<oasis:entry colname="6">RNA</oasis:entry>
<oasis:entry colname="7">DNA</oasis:entry>
<oasis:entry colname="8">RNA</oasis:entry>
<oasis:entry colname="9">DNA
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dTTP
</oasis:entry>
<oasis:entry colname="2">72
</oasis:entry>
<oasis:entry colname="3">0.96
</oasis:entry>
<oasis:entry colname="4">17
</oasis:entry>
<oasis:entry colname="5">7
</oasis:entry>
<oasis:entry colname="6">4.2
</oasis:entry>
<oasis:entry colname="7">0.14
</oasis:entry>
<oasis:entry colname="8"></oasis:entry>
<oasis:entry colname="9"></oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dCTP
</oasis:entry>
<oasis:entry colname="2">0.5
</oasis:entry>
<oasis:entry colname="3">0.29
</oasis:entry>
<oasis:entry colname="4">1400
</oasis:entry>
<oasis:entry colname="5">1700
</oasis:entry>
<oasis:entry colname="6">3.5 × 10<sup>-4</sup>
</oasis:entry>
<oasis:entry colname="7">1.7 × 10<sup>-4</sup>
</oasis:entry>
<oasis:entry colname="8">12 000
</oasis:entry>
<oasis:entry colname="9">800
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dATP
</oasis:entry>
<oasis:entry colname="2">1.3 × 10<sup>-3</sup>
</oasis:entry>
<oasis:entry colname="3">9.2 × 10<sup>-4</sup>
</oasis:entry>
<oasis:entry colname="4">1100
</oasis:entry>
<oasis:entry colname="5">970
</oasis:entry>
<oasis:entry colname="6">1.2 × 10<sup>-6</sup>
</oasis:entry>
<oasis:entry colname="7">9.5 × 10<sup>-7</sup>
</oasis:entry>
<oasis:entry colname="8">3 500 000
</oasis:entry>
<oasis:entry colname="9">148 000
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1">dGTP
</oasis:entry>
<oasis:entry colname="2">3.3 × 10<sup>-3</sup>
</oasis:entry>
<oasis:entry colname="3">2.7 × 10<sup>-3</sup>
</oasis:entry>
<oasis:entry colname="4">3700
</oasis:entry>
<oasis:entry colname="5">6800
</oasis:entry>
<oasis:entry colname="6">9.0 × 10<sup>-7</sup>
</oasis:entry>
<oasis:entry colname="7">3.9 × 10<sup>-7</sup>
</oasis:entry>
<oasis:entry colname="8">4 700 000
</oasis:entry>
<oasis:entry colname="9">360 000</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic toggle="yes"><sup>a</sup>
</italic>
<sup></sup>
Calculated as
[(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>correct</sub>
+
(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>incorrect</sub>
]/(<italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
)<sub>incorrect</sub>
.</p>
</table-wrap-foot>
</table-wrap>
</p>
<p><italic toggle="yes">RNase H Activity of RT for Correct versus Incorrect
Nucleotide Incorporation.</italic>
We have previously
investigated
the simultaneous catalytic activities of DNA
polymerization
and RNA cleavage by using the 5‘-doubly-labeled RNA/
DNA 45/25 template−primer shown in
Table <xref rid="bi9713851t00001"></xref>
(Kati et al.,
1992). In the present study we have examined the
RNase
H activity and sites of template cleavage under conditions
of incorrect nucleotide incorporation. The rate of RNase
H
cleavage ranged from 3 to 16 s<sup>-1</sup>
and appeared
to be
independent of the rate of polymerization. The site of
RNase
H cleavage on the template was also independent of the
nucleotide (correct or incorrect) being incorporated.
This
analysis also provided further confirmation of the
distance
between the polymerase and RNase H active sites (Kati et
al., 1992; Gopalakrishnan et al., 1992) by determining the
length of RNA cleavage products produced (see Figure <xref rid="bi9713851f00003"></xref>
and <xref rid="bi9713851f00005"></xref>
). On the basis of the 38- or
39-mer RNA cleavage
products obtained, a distance of 19 RNA/DNA heteroduplex
bases is suggested between the two sites and is consistent
with the three-dimensional structural studies on RT (Kohlstaedt et al., 1992; Jacobo-Molina et al., 1993). As
previously shown with correct incorporation, 41-mer RNA
cleavage product was also observed which may result from
an alternate binding mode. In general, the RNase H
catalytic
activity was similar to that previously determined for
correct
nucleotide incorporation (Kati et al., 1992).
<fig id="bi9713851f00005" position="float" orientation="portrait"><label>5</label>
<caption><p>Comparison of misincorporation of dTTP into DNA/DNA 45/25-mer (T· dT mismatch) or RNA/DNA 45/25-mer (U·dT mismatch). A preincubated solution of RT (100 nM) and doubly-labeled 5‘-[<sup>32</sup>
P]-labeled 45/25-mer DNA/DNA (left) or RNA/DNA
(right) (90 nM)
was mixed with dTTP (2 mM) in Mg<sup>2+</sup>
buffer to initate the
reaction. The time course for the formation of the 26-mer
misincorporation
product is shown over a period of 40 s. In the case of the DNA
substrate , >70% of the product is formed after 40 s; however with
the RNA
substrate less than 15% of the product is observed at the same
reaction time. Thus the catalytic activity of HIV RT for
incorporating
mismatches is substantially different for RNA versus DNA.</p>
</caption>
<graphic xlink:href="bi9713851f00005.tif" position="float" orientation="portrait"></graphic>
</fig>
</p>
</sec>
</sec>
<sec id="d7e964"><title>Discussion</title>
<p>Previous studies have determined the kinetic parameters
of HIV-1 RT for both RNA dependent as well as DNA
dependent polymerization (Kati et al., 1992). It was
shown
that for correct nucleotide incorporation (dATP) into a
predefined template−primer 45/25-mer, the RNA dependent
polymerization rates (<italic toggle="yes">k</italic>
<sub>pol</sub>
) were at least 2-fold
faster than
the DNA dependent polymerization rates (74
s<sup>-1</sup>
compared
to 33 s<sup>-1</sup>
), while the dissociation constants
(<italic toggle="yes">K</italic>
<sub>d</sub>
) for the
incoming nucleotide were essentially similar to that of
the
deoxynucleotide triphosphate, dATP, showing slightly
greater
affinity for the DNA/DNA 45/25-mer template−primer (4
μM) compared with the RNA/DNA 45/25-mer (14 μM) (Kati
et al., 1992). Fidelity estimates from single
turnover
experiments for the DNA dependent polymerization were
in the range of 1/1700 for misincorporation of dGMP
opposite a template thymidine to approximately 1/18000 for
misincorporation of either a dCMP or dTMP opposite a
template thymidine. The results were in general
agreement
with other fidelity studies on HIV-1 RT (Preston et al.,
1988;
Roberts et al., 1988; Weber et al., 1989; Bebenek et al.,
1989;
Ji and Loeb, 1992; Perrino et al., 1989; Mendelman et al.,
1989; Mendelman et al., 1990; Yu et al., 1992; Kati et
al.,
1992, Johnson, 1993).
</p>
<p>The current study provides fidelity estimates for the RNA
dependent DNA replication reaction of HIV-1 RT under the
same conditions as previously described (Kati et al.,
1992).
The RNA-dependent fidelity for RT relies solely on
the
polymerase contribution since there is no exonuclease
activity
associated with RT and is thus a relationship between the
selectivity for correct dNTP discrimination over that of
incorrect dNTP. Fidelity may be estimated therefore as
a
ratio of the <italic toggle="yes">k</italic>
<sub>pol</sub>
/<italic toggle="yes">K</italic>
<sub>d</sub>
values for correct to incorrect nucleotide
incorporation, as given in Tables <xref rid="bi9713851t00002"></xref>
and
<xref rid="bi9713851t00003"></xref>
. In contrast to
previous studies (Kati et al., 1992; Preston et al., 1988;
Roberts et al., 1988; Weber et al., 1989; Bebenek et al.,
1989;
Ji and Loeb, 1992; Perrino et al., 1989; Mendelman et al.,
1989; Mendelman et al., 1990; Yu et al., 1992), our
results
from the present work show HIV-1 RT to be more discriminating to RNA dependent polymerization than previously
observed for either DNA or RNA dependent polymerization.
A forward mutation assay has suggested a higher
fidelity
for RNA versus DNA templates for frameshifts (Boyer al.,
1992) but not for substitutions. In using an RNA/DNA
45/25 template−primer, the fidelity estimate for
misincorporation of a dGMP was 26 000 while those for dCMP and dTMP
were 176 000 and approximately 1 × 10<sup>6</sup>
, respectively,
over
a template uridine. These estimates indicate an
increased
fidelity of HIV-1 RT of 9−64-fold (approximately 1 to 2
orders of magnitude) over that of the corresponding DNA-directed polymerization fidelity of RT, on direct
comparison
with the previous results (Kati et al., 1992).
</p>
<p>The apparent dissociation rate constants (<italic toggle="yes">K</italic>
<sub>d</sub>
)
for the
incorrect nucleotide incorporation for RNA-dependent polymerization were similar to those determined previously
for
the DNA dependent polymerization of RT (Kati et al.,
1992).
The 50−80-fold difference in binding affinities (Table
<xref rid="bi9713851t00002"></xref>
)
suggests that incorrect nucleotides do not act as
competitive
inhibitors of correct nucleotide incorporation. However,
the
polymerization rates (<italic toggle="yes">k</italic>
<sub>pol</sub>
) differed
significantly. In contrast
to what was observed with correct dNTP incorporation, RNA
dependent polymerization being 2-fold faster than DNA
dependent polymerization (Kati et al., 1992), the rates
for
incorrect nucleotide incorporation with the RNA template
were surprisingly slow. Since the DNA dependent
polymerization rates for misincorporation were only 7−90-fold
slower (for dGMP to dCMP or dTMP) than the corresponding rate for correct incorporation (dAMP) (Kati et al.,
1992),
a decrease of similar magnitude (7−90-fold) was expected
for the RNA dependent misincorporation rates. In
contrast
to the expectation, the RNA dependent misincorporation
rates
ranged from 364-fold slower for dGMP to more than 2000-fold slower for dCMP and greater than 21000-fold slower
for a dTMP misincorporation, compared to the rate of
correct
(dAMP) incorporation (Table <xref rid="bi9713851t00002"></xref>
), indicating a highly discriminating enzyme for RNA dependent polymerization.
While the misincorporation rate for dGTP (0.203
s<sup>-1</sup>
) was
only slightly faster than the product dissociation rate
(0.13
s<sup>-1</sup>
), the rates for misincorporation of dCTP
(0.033 s<sup>-1</sup>
) and
dTTP (0.003 s<sup>-1</sup>
) were 1−2 orders of
magnitude slower than
the dissociation rate of the enzyme−RNA/DNA complex.
Since these experiments were carried under single
turnover
conditions in which the substrate is stoichiometric with
enzyme, the maximum rate of polymerization
(<italic toggle="yes">k</italic>
<sub>pol</sub>
) is limited
by either a conformational change or chemical catalysis
but
not product dissociation for the dCTP and dTTP incorporation and is most likely equal to <italic toggle="yes">k</italic>
<sub>cat</sub>
under
steady-state
conditions. To establish if the higher discrimination
with
RNA may be a more general property of HIV-1 RT, this
analysis was extended to a 45/22 template−primer. In
this
case the correct and incorrect incorporation opposite a
template adenine was examined with both a DNA and an
RNA substrate. A 14−23-fold higher fidelity was
observed
for the RNA template in comparision with the DNA
indicating this may be a common feature for HIV-1 RT.
</p>
<p>This high degree of selectivity by RT <italic toggle="yes">in vitro</italic>
has not
been
detected prior to our study although more recent results
from
an <italic toggle="yes">in vivo </italic>
study of HIV fidelity (Mansky and Temin,
1995)
is consistent with our observations. Previous studies
using
steady-state analysis (Yu and Goodman, 1992; Ji and Loeb,
1992; Johnson, 1993) have provided RNA dependent fidelity
similar to that observed for the DNA replication fidelity
estimates. In present investigation to examine RNA
dependent fidelity, a pre-steady-state kinetic analysis was
undertaken to directly observe the events at the active
site
of RT. The relative ease with which some mismatches
occur
with an RNA/DNA template−primer is consistent with
earlier studies examining DNA/DNA template−primers (Kati
et al., 1992; Ji and Loeb, 1992). For instance, the
G/U
mismatch was still observed to be the easiest mismatch to
form. According the the rates presented in Table <xref rid="bi9713851t00002"></xref>
, the
G/U
mismatch formed approximately 6-fold faster than C/U and
about 60-fold faster than T/U, suggesting that
conformational
and other thermodynamic factors (Abbots et al., 1991)
contribute to the inability of the enzyme to sufficiently
discriminate between a dA/T (dA/U) or dG/T (dG/U) base
pair. The difference in <italic toggle="yes">K</italic>
<sub>m</sub>
s for correct
versus incorrect dNTP
insertion were found to be in general agreement to the
differences in <italic toggle="yes">K</italic>
<sub>d</sub>
s seen in this investigation,
as was the ability
of RT to extend a mismatch (Figure <xref rid="bi9713851f00003"></xref>
), and continue
polymerization (Yu and Goodman, 1992) albeit at a slower
rate.
</p>
<p>The high fidelity exhibited by HIV-1 RT for the RNA
dependent DNA polymerization, as shown in this study, may
be anticipated as an essential element for survival of an
RNA
virus. Since the virus inherently lacks a
proofreading
mechanism in its replication, the virus must strive to
make
an efficient and correct first (minus) strand DNA copy.
This
must be done prior to the degradation of its RNA template.
While errors in the second DNA strand synthesis may
involve
error correction mechanisms, by proofreading using host
polymerases (Glazer et al., 1987; Stephenson and Karran,
1989), the first DNA strand synthesis has no such
correction
mechanism. Thus, in the absence of error-free
replication
for the RNA dependent polymerization, the final proviral
DNA may have imperfections which could have further
deleterious consequences for the virus such as production
of a nonviable virus. The high fidelity seen in this
investigation may perhaps be a necessary mechanism for
formation of viable viral particles. The differences in
the
fidelity estimates seen here and by earlier studies may be
due to several factors. By examining the replication
fidelity
using pre-steady-state kinetic analysis under single
turnover
conditions, our primary focus was on mechanism by looking
directly at the events at the active site of RT. Other
studies
showing lower RNA dependent fidelity estimates than those
reported here have been conducted using steady-state
assays
(Yu and Goodman, 1992; Johnson, 1993; Johnson, 1992),
which may not entirely reflect events at the active site
of
the enzyme by masking mechanistic detail. The genomic
hypervariation that has been observed with HIV-1 viral
isolates (Coffin, 1986; Goodenow et al., 1989) has been
attributed to decreased fidelity of the RNA dependent DNA
replication and may in part be due to other factors such
as
viral survival. In light of the high fidelity exhibited by
RT
in this investigation and previous fidelity estimates
suggesting
RT to be no less efficient than other polymerases (Preston
et al., 1988; Mendelman et al., 1989; Takeuchi et al.,
1988),
one may speculate that the high degree of
genomic variation
of HIV-1 may be due more to a defense mechanism of the
virus to escape the surveillance of the host immune system
than to the “sloppiness” on the part of RT.
</p>
<p>The extremely slow reaction rates seen for the RNA
dependent mismatch experiments is consistent with the
model
of a two-step binding mechanism of HIV-1 RT involving a
rate-limiting conformational change as previously proposed
(Kati et al., 1992; Hsieh et al., 1993; Spence et al.,
1995;
Rittinger et al., 1995) and similar to the kinetic model
seen
with T7 DNA polymerase (Patel et al., 1991; Wong et al.,
1991; Spence et al., 1995). This model argues that RT
or
T7 DNA polymerase provides the high fidelity of dNTP
incorporation in a two-step mechanism (Spence et al.,
1995).
In the first step, ground state binding between enzyme in
an
“open” binding state with template−primer and dNTP
is
formed, while the second step involves a conformational
change to a “closed” state whereby the protein can
encompass the template−primer and dNTP so as to form an
extremely tight binding template−primer−dNTP complex
from which catalysis can occur. It is in this step that
fidelity
of the protein is most apparent. Catalysis can only occur
if
the protein can adopt the critical catalytic configuration
which
is dependent on correct Watson−Crick base pairing with
the
incoming nucleotide and template (Johnson, 1993). However, if the protein cannot adopt this critical
configuration,
then the reaction proceeds extremely slowly and this step,
perhaps, may be the rate-limiting step in the overall
pathway.
This extremely slow rate for RNA misincorporation is
precisely what is observed in the present investigation.
Thus,
the high fidelity estimates for the RNA dependent
replication
reaction are primarily due to the extremely slow reaction
rates for incorporation of incorrect dNTPs and is most
likely
the result of either a conformational change or chemical
catalysis being the rate-limiting step.
</p>
<p>An intriguing question concerns the mechanistic basis for
the discrimination of RNA versus DNA mismatches by HIV
RT? The answer, in part, may lie in differences in
thermodynamic stability of RNA/DNA heteroduplexes and
DNA/DNA duplexes (Fedoroff et al., 1993; Wang et al.,
1992; Zhu et al, 1995; Sugimoto, et al., 1995) although
our
data indicate that efficiency of nucleotide incorporation
plays
a significant role. Insight into this question would
be
provided by structural information on HIV RT comparing
ternary complexes with an RNA/DNA substrate or DNA/DNA substrate and dNTP bound in the active site; however,
these structures are not available at present.
</p>
</sec>
</body>
<back><ref-list><title>References</title>
<ref id="atyp_ref1"><mixed-citation><name name-style="western"><surname>Abbotts</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Jaju</surname>
<given-names>M.</given-names>
</name>
, & <name name-style="western"><surname>Wilson</surname>
<given-names>S.</given-names>
</name>
(1991) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">266</italic>
, 3937−3943.</mixed-citation>
</ref>
<ref id="atyp_ref2"><mixed-citation><name name-style="western"><surname>Bebenek</surname>
<given-names>K.</given-names>
</name>
, <name name-style="western"><surname>Abbotts</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Roberts</surname>
<given-names>J. D.</given-names>
</name>
, <name name-style="western"><surname>Wilson</surname>
<given-names>S. H.</given-names>
</name>
, & <name name-style="western"><surname>Kunkel</surname>
<given-names>T. A.</given-names>
</name>
(1989) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">264</italic>
, 16948−16956.</mixed-citation>
</ref>
<ref id="atyp_ref3"><mixed-citation><name name-style="western"><surname>Bebenek</surname>
<given-names>K.</given-names>
</name>
, <name name-style="western"><surname>Abbotts</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Wilson</surname>
<given-names>S. H.</given-names>
</name>
, & <name name-style="western"><surname>Kunkel</surname>
<given-names>T. A.</given-names>
</name>
(1993) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">268</italic>
, 10324−10334</mixed-citation>
</ref>
<ref id="atyp_ref4"><mixed-citation><name name-style="western"><surname>Boyer</surname>
<given-names>J. C.</given-names>
</name>
, <name name-style="western"><surname>Bebenek</surname>
<given-names>K.</given-names>
</name>
, & <name name-style="western"><surname>Kunkel</surname>
<given-names>T. A.</given-names>
</name>
(1992) <italic toggle="yes">Proc.</italic>
<italic toggle="yes">Natl.</italic>
<italic toggle="yes">Acad. Sci.</italic>
<italic toggle="yes">U.S.A</italic>
. <italic toggle="yes"> 89</italic>
, 6919−6923</mixed-citation>
</ref>
<ref id="atyp_ref5"><element-citation publication-type="journal"><name name-style="western"><surname>Coffin</surname>
<given-names>J. M.</given-names>
</name>
<source>Cell</source>
<year>1986</year>
<volume>46</volume>
<fpage>1</fpage>
<lpage>4</lpage>
</element-citation>
</ref>
<ref id="atyp_ref6"><mixed-citation><name name-style="western"><surname>Fedoroff</surname>
<given-names>O. Y.</given-names>
</name>
, <name name-style="western"><surname>Salazar</surname>
<given-names>M.</given-names>
</name>
, & <name name-style="western"><surname>Reid</surname>
<given-names>B. R.</given-names>
</name>
(1993) <italic toggle="yes">J.</italic>
<italic toggle="yes">Mol.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">233</italic>
, 509−523.<pub-id pub-id-type="doi">10.1006/jmbi.1993.1528</pub-id>
</mixed-citation>
</ref>
<ref id="atyp_ref7"><mixed-citation><name name-style="western"><surname>Glazer</surname>
<given-names>P. M.</given-names>
</name>
, <name name-style="western"><surname>Sarkar</surname>
<given-names>A. N.</given-names>
</name>
, <name name-style="western"><surname>Chisholm</surname>
<given-names>G. E.</given-names>
</name>
, & <name name-style="western"><surname>Summers</surname>
<given-names>W. C.</given-names>
</name>
(1987) <italic toggle="yes">Mol.</italic>
<italic toggle="yes">Cell.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">7</italic>
, 218−224.</mixed-citation>
</ref>
<ref id="atyp_ref8"><mixed-citation><name name-style="western"><surname>Gopalakrishnan</surname>
<given-names>V.</given-names>
</name>
, <name name-style="western"><surname>Peliska</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Benkovic</surname>
<given-names>S.</given-names>
</name>
(1992) <italic toggle="yes">Proc.</italic>
<italic toggle="yes">Natl.</italic>
<italic toggle="yes">Acad. Sci.</italic>
<italic toggle="yes">U.S.A.,</italic>
<italic toggle="yes">89</italic>
, 10763−10767<pub-id pub-id-type="doi">10.1073/pnas.89.22.10763</pub-id>
</mixed-citation>
</ref>
<ref id="atyp_ref9"><mixed-citation><name name-style="western"><surname>Goodenow</surname>
<given-names>M.</given-names>
</name>
, <name name-style="western"><surname>Huet</surname>
<given-names>T.</given-names>
</name>
, <name name-style="western"><surname>Saurin</surname>
<given-names>W.</given-names>
</name>
, <name name-style="western"><surname>Kwok</surname>
<given-names>S.</given-names>
</name>
, <name name-style="western"><surname>Sninsky</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Wain-Hobson</surname>
<given-names>S.</given-names>
</name>
(1989) <italic toggle="yes">J.</italic>
<italic toggle="yes">Acquired</italic>
<italic toggle="yes">Immune</italic>
<italic toggle="yes">Defic.</italic>
<italic toggle="yes">Syndr.</italic>
<italic toggle="yes">2</italic>
, 344−352</mixed-citation>
</ref>
<ref id="atyp_ref10"><mixed-citation><name name-style="western"><surname>Hsieh</surname>
<given-names>J.-C.</given-names>
</name>
, <name name-style="western"><surname>Zinnen</surname>
<given-names>S.</given-names>
</name>
, & <name name-style="western"><surname>Modrich</surname>
<given-names>P.</given-names>
</name>
(1993) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">268</italic>
, 24607−24613.</mixed-citation>
</ref>
<ref id="atyp_ref11"><mixed-citation><name name-style="western"><surname>Jacobo-Molina</surname>
<given-names>A.</given-names>
</name>
, <name name-style="western"><surname>Ding</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Nanni</surname>
<given-names>R. G.</given-names>
</name>
, <name name-style="western"><surname>Clark</surname>
<given-names>A. D.</given-names>
<suffix>Jr.</suffix>
</name>
, <name name-style="western"><surname>Lu</surname>
<given-names>X.</given-names>
</name>
, <name name-style="western"><surname>Tantillo</surname>
<given-names>C.</given-names>
</name>
, <name name-style="western"><surname>Williams</surname>
<given-names>R. L.</given-names>
</name>
, <name name-style="western"><surname>Kamer</surname>
<given-names>G.</given-names>
</name>
, <name name-style="western"><surname>Ferris</surname>
<given-names>A. L.</given-names>
</name>
, <name name-style="western"><surname>Clark</surname>
<given-names>P.</given-names>
</name>
, <name name-style="western"><surname>Hizi</surname>
<given-names>A.</given-names>
</name>
, <name name-style="western"><surname>Hughes</surname>
<given-names>S. H.</given-names>
</name>
, & <name name-style="western"><surname>Arnold</surname>
<given-names>E.</given-names>
</name>
(1993) <italic toggle="yes">Proc.</italic>
<italic toggle="yes">Natl.</italic>
<italic toggle="yes">Acad.</italic>
<italic toggle="yes">Sci.</italic>
<italic toggle="yes">U.S.A.</italic>
<italic toggle="yes">90</italic>
, 6320−6324.<pub-id pub-id-type="doi">10.1073/pnas.90.13.6320</pub-id>
</mixed-citation>
</ref>
<ref id="atyp_ref12"><mixed-citation><name name-style="western"><surname>Ji</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Loeb</surname>
<given-names>L. A.</given-names>
</name>
(1992) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">31</italic>
, 954−958.</mixed-citation>
</ref>
<ref id="atyp_ref13"><mixed-citation><name name-style="western"><surname>Ji</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Loeb</surname>
<given-names>L. A.</given-names>
</name>
(1994) <italic toggle="yes">Virology</italic>
<italic toggle="yes">199</italic>
, 323−330.</mixed-citation>
</ref>
<ref id="atyp_ref14"><element-citation publication-type="journal"><name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
<source>Methods Enzymol.</source>
<year>1986</year>
<volume>134</volume>
<fpage>677</fpage>
<lpage>705</lpage>
</element-citation>
</ref>
<ref id="atyp_ref15"><element-citation publication-type="journal"><name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
<source>The Enzymes</source>
<year>1992</year>
<volume>20</volume>
<fpage>1</fpage>
<lpage>61</lpage>
</element-citation>
</ref>
<ref id="atyp_ref16"><element-citation publication-type="journal"><name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
<source>Annu. Rev. Biochem.</source>
<year>1993</year>
<volume>62</volume>
<fpage>685</fpage>
<lpage>713</lpage>
<pub-id pub-id-type="doi">10.1146/annurev.bi.62.070193.003345</pub-id>
</element-citation>
</ref>
<ref id="atyp_ref17"><mixed-citation><name name-style="western"><surname>Kati</surname>
<given-names>W. M.</given-names>
</name>
, <name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
, <name name-style="western"><surname>Jerva</surname>
<given-names>L. F.</given-names>
</name>
, & <name name-style="western"><surname>Anderson</surname>
<given-names>K. S.</given-names>
</name>
(1992) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">267</italic>
, 25988−25997.</mixed-citation>
</ref>
<ref id="atyp_ref18"><mixed-citation><name name-style="western"><surname>Kohlstaedt</surname>
<given-names>L. A.</given-names>
</name>
, <name name-style="western"><surname>Wang</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Friedman</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Rice</surname>
<given-names>P. A.</given-names>
</name>
, & <name name-style="western"><surname>Steitz</surname>
<given-names>T. A.</given-names>
</name>
(1992) <italic toggle="yes">Science</italic>
<italic toggle="yes">256</italic>
, 1783−1790.</mixed-citation>
</ref>
<ref id="atyp_ref19"><mixed-citation><name name-style="western"><surname>Kuchta</surname>
<given-names>R. D.</given-names>
</name>
, <name name-style="western"><surname>Mizrahi</surname>
<given-names>V.</given-names>
</name>
, <name name-style="western"><surname>Benkovic</surname>
<given-names>P. A.</given-names>
</name>
, <name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
, & <name name-style="western"><surname>Benkovic</surname>
<given-names>S. J.</given-names>
</name>
(1987) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">26</italic>
, 8410−8417.</mixed-citation>
</ref>
<ref id="atyp_ref20"><mixed-citation><name name-style="western"><surname>Kuchta</surname>
<given-names>R. D.</given-names>
</name>
, <name name-style="western"><surname>Benkovic</surname>
<given-names>P.</given-names>
</name>
, & <name name-style="western"><surname>Benkovic</surname>
<given-names>S. J.</given-names>
</name>
(1988) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">27</italic>
, 6716−6725.</mixed-citation>
</ref>
<ref id="atyp_ref21"><mixed-citation><name name-style="western"><surname>Mansky</surname>
<given-names>L. M.</given-names>
</name>
, & <name name-style="western"><surname>Temin</surname>
<given-names>H. M.</given-names>
</name>
(1995) <italic toggle="yes">J. Virol.</italic>
<italic toggle="yes">69</italic>
, 5087−5094.</mixed-citation>
</ref>
<ref id="atyp_ref22"><mixed-citation><name name-style="western"><surname>Mendelman</surname>
<given-names>L. V.</given-names>
</name>
, <name name-style="western"><surname>Boosalis</surname>
<given-names>M. S.</given-names>
</name>
, <name name-style="western"><surname>Petruska</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Goodman</surname>
<given-names>M. F.</given-names>
</name>
(1989) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">264</italic>
, 14415−14423.</mixed-citation>
</ref>
<ref id="atyp_ref23"><mixed-citation><name name-style="western"><surname>Mendelman</surname>
<given-names>L. V.</given-names>
</name>
, <name name-style="western"><surname>Petruska</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Goodman</surname>
<given-names>M. F.</given-names>
</name>
(1990) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">265</italic>
, 2338−2346.</mixed-citation>
</ref>
<ref id="atyp_ref24"><mixed-citation><name name-style="western"><surname>Müller</surname>
<given-names>B.</given-names>
</name>
, <name name-style="western"><surname>Restle</surname>
<given-names>T.</given-names>
</name>
, <name name-style="western"><surname>Weiss</surname>
<given-names>S.</given-names>
</name>
, <name name-style="western"><surname>Gautel</surname>
<given-names>M.</given-names>
</name>
, <name name-style="western"><surname>Sczakiel</surname>
<given-names>G.</given-names>
</name>
, & <name name-style="western"><surname>Goody</surname>
<given-names>R. S.</given-names>
</name>
(1989) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">264</italic>
, 13975−13978.</mixed-citation>
</ref>
<ref id="atyp_ref25"><mixed-citation><name name-style="western"><surname>Patel</surname>
<given-names>S. S.</given-names>
</name>
, <name name-style="western"><surname>Wong</surname>
<given-names>I.</given-names>
</name>
, & <name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
(1991) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">30</italic>
, 511−525.</mixed-citation>
</ref>
<ref id="atyp_ref26"><mixed-citation><name name-style="western"><surname>Perrino</surname>
<given-names>F.</given-names>
</name>
, <name name-style="western"><surname>Preston</surname>
<given-names>B. D.</given-names>
</name>
, <name name-style="western"><surname>Sandell</surname>
<given-names>L. L.</given-names>
</name>
, & <name name-style="western"><surname>Loeb</surname>
<given-names>L. A.</given-names>
</name>
(1989) <italic toggle="yes">Proc.</italic>
<italic toggle="yes">Natl.</italic>
<italic toggle="yes">Acad.</italic>
<italic toggle="yes">Sci.</italic>
<italic toggle="yes">U.S.A</italic>
. <italic toggle="yes"> 86</italic>
, 8343−8347</mixed-citation>
</ref>
<ref id="atyp_ref27"><mixed-citation><name name-style="western"><surname>Preston</surname>
<given-names>B. D.</given-names>
</name>
, <name name-style="western"><surname>Poiesz</surname>
<given-names>B. J.</given-names>
</name>
, & <name name-style="western"><surname>Loeb</surname>
<given-names>L. A.</given-names>
</name>
(1988) <italic toggle="yes">Science</italic>
<italic toggle="yes">242</italic>
, 1168−1171.</mixed-citation>
</ref>
<ref id="atyp_ref28"><element-citation publication-type="journal"><name name-style="western"><surname>Preston</surname>
<given-names>B. D.</given-names>
</name>
<name name-style="western"><surname>Keulen</surname>
<given-names>W.</given-names>
</name>
<name name-style="western"><surname>Nijhuis</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Schuurman</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>Berkhout</surname>
<given-names>B.</given-names>
</name>
<name name-style="western"><surname>Boucher</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Balzarini</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Pelemans</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>De Clercq</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Karlsson</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Kleim</surname>
<given-names>J.-P.</given-names>
</name>
<name name-style="western"><surname>Prasad</surname>
<given-names>V. R.</given-names>
</name>
<name name-style="western"><surname>Drosopoulos</surname>
<given-names>W. C.</given-names>
</name>
<name name-style="western"><surname>Wainberg</surname>
<given-names>M. A.</given-names>
</name>
<source>Science</source>
<year>1997</year>
<volume>275</volume>
<fpage>228</fpage>
<lpage>231</lpage>
</element-citation>
</ref>
<ref id="atyp_ref29"><element-citation publication-type="journal"><name name-style="western"><surname>Reardon</surname>
<given-names>J. E.</given-names>
</name>
<source>Biochemistry</source>
<year>1992</year>
<volume>31</volume>
<fpage>4473</fpage>
<lpage>4479</lpage>
</element-citation>
</ref>
<ref id="atyp_ref30"><element-citation publication-type="journal"><name name-style="western"><surname>Reardon</surname>
<given-names>J. E.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1993</year>
<volume>268</volume>
<fpage>8743</fpage>
<lpage>8751</lpage>
</element-citation>
</ref>
<ref id="atyp_ref31"><mixed-citation><name name-style="western"><surname>Ricchetti</surname>
<given-names>M.</given-names>
</name>
, & <name name-style="western"><surname>Buc</surname>
<given-names>H.</given-names>
</name>
(1990) <italic toggle="yes">The</italic>
<italic toggle="yes">EMBO</italic>
<italic toggle="yes">J</italic>
. <italic toggle="yes"> 9</italic>
, 1583−1593.</mixed-citation>
</ref>
<ref id="atyp_ref32"><mixed-citation><name name-style="western"><surname>Rittinger</surname>
<given-names>K.</given-names>
</name>
, <name name-style="western"><surname>Divita</surname>
<given-names>G.</given-names>
</name>
, & <name name-style="western"><surname>Goody</surname>
<given-names>R. S.</given-names>
</name>
(1995) <italic toggle="yes">Proc.</italic>
<italic toggle="yes">Natl.</italic>
<italic toggle="yes">Acad. Sci.</italic>
<italic toggle="yes">U.S.A.</italic>
<italic toggle="yes">92</italic>
, 8046−8049.<pub-id pub-id-type="doi">10.1073/pnas.92.17.8046</pub-id>
</mixed-citation>
</ref>
<ref id="atyp_ref33"><mixed-citation><name name-style="western"><surname>Roberts</surname>
<given-names>J. D.</given-names>
</name>
, <name name-style="western"><surname>Bebenek</surname>
<given-names>K.</given-names>
</name>
, & <name name-style="western"><surname>Kunkel</surname>
<given-names>T. A.</given-names>
</name>
(1988) <italic toggle="yes">Science</italic>
<italic toggle="yes">242</italic>
, 1171−1173</mixed-citation>
</ref>
<ref id="atyp_ref34"><mixed-citation><name name-style="western"><surname>Spence</surname>
<given-names>R. A.</given-names>
</name>
, <name name-style="western"><surname>Kati</surname>
<given-names>W. M.</given-names>
</name>
, <name name-style="western"><surname>Anderson</surname>
<given-names>K. S.</given-names>
</name>
, & <name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
(1995) <italic toggle="yes">Science</italic>
<italic toggle="yes">267</italic>
, 988−993.</mixed-citation>
</ref>
<ref id="atyp_ref35"><mixed-citation><name name-style="western"><surname>Stahlhut</surname>
<given-names>M.</given-names>
</name>
, <name name-style="western"><surname>Li</surname>
<given-names>Y.</given-names>
</name>
, <name name-style="western"><surname>Condra</surname>
<given-names>J. H.</given-names>
</name>
, <name name-style="western"><surname>Fu</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Gotlib</surname>
<given-names>L.</given-names>
</name>
, <name name-style="western"><surname>Graham</surname>
<given-names>D. J.</given-names>
</name>
, & <name name-style="western"><surname>Olsen</surname>
<given-names>D.</given-names>
</name>
(1994) <italic toggle="yes">Protein</italic>
<italic toggle="yes">Expression</italic>
<italic toggle="yes">Purif.</italic>
<italic toggle="yes">6</italic>
, 614−621.</mixed-citation>
</ref>
<ref id="atyp_ref36"><mixed-citation><name name-style="western"><surname>Stephenson</surname>
<given-names>C.</given-names>
</name>
, & <name name-style="western"><surname>Karran</surname>
<given-names>P.</given-names>
</name>
(1989) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">264</italic>
, 21177−21182.</mixed-citation>
</ref>
<ref id="atyp_ref37"><mixed-citation><name name-style="western"><surname>Sugimoto</surname>
<given-names>N.</given-names>
</name>
, <name name-style="western"><surname>Nakano</surname>
<given-names>S.</given-names>
</name>
, <name name-style="western"><surname>Katoh</surname>
<given-names>M.</given-names>
</name>
, <name name-style="western"><surname>Matsumura</surname>
<given-names>A.</given-names>
</name>
, <name name-style="western"><surname>Nakamuta</surname>
<given-names>H.</given-names>
</name>
, <name name-style="western"><surname>Ohmichi</surname>
<given-names>T.</given-names>
</name>
, <name name-style="western"><surname>Yoneyama</surname>
<given-names>M.</given-names>
</name>
, & <name name-style="western"><surname>Saski</surname>
<given-names>M.</given-names>
</name>
(1995) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">34</italic>
, 11211−11216.</mixed-citation>
</ref>
<ref id="atyp_ref38"><mixed-citation><name name-style="western"><surname>Takeuchi</surname>
<given-names>Y.</given-names>
</name>
, <name name-style="western"><surname>Nagumo</surname>
<given-names>T.</given-names>
</name>
, & <name name-style="western"><surname>Hoshing</surname>
<given-names>H.</given-names>
</name>
(1988) <italic toggle="yes">J.</italic>
<italic toggle="yes">Virol.</italic>
<italic toggle="yes">62</italic>
, 3900−3902.</mixed-citation>
</ref>
<ref id="atyp_ref39"><mixed-citation><name name-style="western"><surname>Wang</surname>
<given-names>A. C.</given-names>
</name>
, <name name-style="western"><surname>Kim</surname>
<given-names>S.</given-names>
</name>
, <name name-style="western"><surname>Flynn</surname>
<given-names>P.</given-names>
</name>
, <name name-style="western"><surname>Chou</surname>
<given-names>S.-H.</given-names>
</name>
, <name name-style="western"><surname>Orban</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Reid</surname>
<given-names>B. R.</given-names>
</name>
(1992) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">31</italic>
, 3940−3946.</mixed-citation>
</ref>
<ref id="atyp_ref40"><mixed-citation><name name-style="western"><surname>Weber</surname>
<given-names>J.</given-names>
</name>
, & <name name-style="western"><surname>Grosse</surname>
<given-names>F.</given-names>
</name>
(1989) <italic toggle="yes">Nucleic.</italic>
<italic toggle="yes">Acids</italic>
<italic toggle="yes">Res.</italic>
<italic toggle="yes">17</italic>
, 1379−1393.<pub-id pub-id-type="doi">10.1093/nar/17.4.1379</pub-id>
</mixed-citation>
</ref>
<ref id="atyp_ref41"><mixed-citation><name name-style="western"><surname>Wong</surname>
<given-names>I.</given-names>
</name>
, <name name-style="western"><surname>Patel</surname>
<given-names>S. S.</given-names>
</name>
, & <name name-style="western"><surname>Johnson</surname>
<given-names>K. A.</given-names>
</name>
(1991) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">30</italic>
, 526−537.</mixed-citation>
</ref>
<ref id="atyp_ref42"><mixed-citation><name name-style="western"><surname>Yu</surname>
<given-names>H.</given-names>
</name>
, & <name name-style="western"><surname>Goodman</surname>
<given-names>M. F.</given-names>
</name>
(1992) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">267</italic>
, 10888−10896.</mixed-citation>
</ref>
<ref id="atyp_ref43"><mixed-citation><name name-style="western"><surname>Zhu</surname>
<given-names>L.</given-names>
</name>
, <name name-style="western"><surname>Salazar</surname>
<given-names>M.</given-names>
</name>
, & <name name-style="western"><surname>Reid</surname>
<given-names>R. R.</given-names>
</name>
(1995) <italic toggle="yes">Biochemistry</italic>
<italic toggle="yes">34</italic>
, 2372−2380.</mixed-citation>
</ref>
<ref id="atyp_ref44"><mixed-citation><name name-style="western"><surname>Zinnen</surname>
<given-names>S.</given-names>
</name>
, <name name-style="western"><surname>Hsieh</surname>
<given-names>J. C.</given-names>
</name>
, & <name name-style="western"><surname>Modrich</surname>
<given-names>P.</given-names>
</name>
(1994) <italic toggle="yes">J.</italic>
<italic toggle="yes">Biol.</italic>
<italic toggle="yes">Chem.</italic>
<italic toggle="yes">269</italic>
, 24195−24202.</mixed-citation>
</ref>
<ref id="bi9713851b00001"><mixed-citation><comment>Abbreviations used: dNTP, deoxynucleoside 5‘-triphosphate; dATP, deoxyadenosine 5‘-triphosphate; dCTP, deoxycytidine 5‘-triphosphate; dGTP, deoxyguanosine 5‘-triphosphate; dTTP, deoxythymidine 5‘-triphosphate; EDTA, ethylenediaminetetraacetic acid; HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase.</comment>
</mixed-citation>
</ref>
</ref-list>
</back>
</article>
</istex:document>
</istex:metadataXml>
<mods version="3.6"><titleInfo><title>RNA Dependent DNA Replication Fidelity of HIV-1 Reverse Transcriptase: Evidence of Discrimination between DNA and RNA Substrates†</title>
</titleInfo>
<titleInfo contentType="CDATA"><title>RNA Dependent DNA Replication Fidelity of HIV-1 Reverse Transcriptase: Evidence of Discrimination between DNA and RNA Substrates†</title>
</titleInfo>
<name type="personal"><namePart type="family">KERR</namePart>
<namePart type="given">Stephen G.</namePart>
<affiliation>Department of Pharmacology, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520-8066</affiliation>
<affiliation> Present address: Massachusetts College ofPharmacy & AlliedHealth Sciences, 179 Longwood Ave., Boston, MA 02115.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal" displayLabel="corresp"><namePart type="family">ANDERSON</namePart>
<namePart type="given">Karen S.</namePart>
<affiliation>Department of Pharmacology, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520-8066</affiliation>
<affiliation> Author to whom correspondence should be addressed.Telephone:(203)-785-4526. Fax: (203)-785-7670. email:karen.anderson@yale.edu.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
<genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre>
<originInfo><publisher>American Chemical Society</publisher>
<dateCreated encoding="w3cdtf">1997-11-18</dateCreated>
<dateIssued encoding="w3cdtf">1997-11-18</dateIssued>
<copyrightDate encoding="w3cdtf">1997</copyrightDate>
</originInfo>
<note type="footnote" ID="bi9713851AF2"> This work was supported by NIH Grant GM 49551 to K.S.A.</note>
<note type="footnote" ID="bi9713851AF7"> Abstract published in Advance ACS Abstracts, November 1, 1997.</note>
<language><languageTerm type="code" authority="iso639-2b">eng</languageTerm>
<languageTerm type="code" authority="rfc3066">en</languageTerm>
</language>
<abstract>The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template−primer is employed as compared with the corresponding DNA/DNA template−primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 × 106 for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template−primer substrates, HIV-1 RT exhibits approximately a 10−60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.</abstract>
<note type="footnote" ID="bi9713851AF2"> This work was supported by NIH Grant GM 49551 to K.S.A.</note>
<note type="footnote" ID="bi9713851AF7"> Abstract published in Advance ACS Abstracts, November 1, 1997.</note>
<relatedItem type="host"><titleInfo><title>Biochemistry</title>
</titleInfo>
<titleInfo type="abbreviated"><title>Biochemistry</title>
</titleInfo>
<genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre>
<identifier type="ISSN">0006-2960</identifier>
<identifier type="eISSN">1520-4995</identifier>
<identifier type="acspubs">bi</identifier>
<identifier type="coden">BICHAW</identifier>
<identifier type="uri">pubs.acs.org/biochemistry</identifier>
<part><date>1997</date>
<detail type="volume"><caption>vol.</caption>
<number>36</number>
</detail>
<detail type="issue"><caption>no.</caption>
<number>46</number>
</detail>
<extent unit="pages"><start>14056</start>
<end>14063</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref1" displayLabel="bib1"><name type="personal"><namePart type="family">ABBOTTS</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">JAJU</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">WILSON</namePart>
<namePart type="given">S.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">AbbottsJ., JajuM., & WilsonS. (1991) J. Biol. Chem. 266, 3937−3943.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref2" displayLabel="bib2"><name type="personal"><namePart type="family">BEBENEK</namePart>
<namePart type="given">K.</namePart>
</name>
<name type="personal"><namePart type="family">ABBOTTS</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">ROBERTS</namePart>
<namePart type="given">J. D.</namePart>
</name>
<name type="personal"><namePart type="family">WILSON</namePart>
<namePart type="given">S. H.</namePart>
</name>
<name type="personal"><namePart type="family">KUNKEL</namePart>
<namePart type="given">T. A.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">BebenekK., AbbottsJ., RobertsJ. D., WilsonS. H., & KunkelT. A. (1989) J. Biol. Chem. 264, 16948−16956.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref3" displayLabel="bib3"><name type="personal"><namePart type="family">BEBENEK</namePart>
<namePart type="given">K.</namePart>
</name>
<name type="personal"><namePart type="family">ABBOTTS</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">WILSON</namePart>
<namePart type="given">S. H.</namePart>
</name>
<name type="personal"><namePart type="family">KUNKEL</namePart>
<namePart type="given">T. A.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">BebenekK., AbbottsJ., WilsonS. H., & KunkelT. A. (1993) J. Biol. Chem. 268, 10324−10334</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref4" displayLabel="bib4"><name type="personal"><namePart type="family">BOYER</namePart>
<namePart type="given">J. C.</namePart>
</name>
<name type="personal"><namePart type="family">BEBENEK</namePart>
<namePart type="given">K.</namePart>
</name>
<name type="personal"><namePart type="family">KUNKEL</namePart>
<namePart type="given">T. A.</namePart>
</name>
<titleInfo><title>Proc.</title>
</titleInfo>
<note type="content-in-line">BoyerJ. C., BebenekK., & KunkelT. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6919−6923</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref5" displayLabel="bib5"><name type="personal"><namePart type="family">COFFIN</namePart>
<namePart type="given">J. M.</namePart>
</name>
<titleInfo><title>Cell</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Coffin J. M. Cell 1986 46 1 4</note>
<part><date>1986</date>
<detail type="volume"><caption>vol.</caption>
<number>46</number>
</detail>
<extent unit="pages"><start>1</start>
<end>4</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref6" displayLabel="bib6"><name type="personal"><namePart type="family">FEDOROFF</namePart>
<namePart type="given">O. Y.</namePart>
</name>
<name type="personal"><namePart type="family">SALAZAR</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">REID</namePart>
<namePart type="given">B. R.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">FedoroffO. Y., SalazarM., & ReidB. R. (1993) J. Mol. Biol. 233, 509−523.10.1006/jmbi.1993.1528</note>
<identifier type="doi">10.1006/jmbi.1993.1528</identifier>
</relatedItem>
<relatedItem type="references" ID="atyp_ref7" displayLabel="bib7"><name type="personal"><namePart type="family">GLAZER</namePart>
<namePart type="given">P. M.</namePart>
</name>
<name type="personal"><namePart type="family">SARKAR</namePart>
<namePart type="given">A. N.</namePart>
</name>
<name type="personal"><namePart type="family">CHISHOLM</namePart>
<namePart type="given">G. E.</namePart>
</name>
<name type="personal"><namePart type="family">SUMMERS</namePart>
<namePart type="given">W. C.</namePart>
</name>
<titleInfo><title>Mol.</title>
</titleInfo>
<note type="content-in-line">GlazerP. M., SarkarA. N., ChisholmG. E., & SummersW. C. (1987) Mol. Cell. Biol. 7, 218−224.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref8" displayLabel="bib8"><name type="personal"><namePart type="family">GOPALAKRISHNAN</namePart>
<namePart type="given">V.</namePart>
</name>
<name type="personal"><namePart type="family">PELISKA</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">BENKOVIC</namePart>
<namePart type="given">S.</namePart>
</name>
<titleInfo><title>Proc.</title>
</titleInfo>
<note type="content-in-line">GopalakrishnanV., PeliskaJ., & BenkovicS. (1992) Proc. Natl. Acad. Sci. U.S.A., 89, 10763−1076710.1073/pnas.89.22.10763</note>
<identifier type="doi">10.1073/pnas.89.22.10763</identifier>
</relatedItem>
<relatedItem type="references" ID="atyp_ref9" displayLabel="bib9"><name type="personal"><namePart type="family">GOODENOW</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">HUET</namePart>
<namePart type="given">T.</namePart>
</name>
<name type="personal"><namePart type="family">SAURIN</namePart>
<namePart type="given">W.</namePart>
</name>
<name type="personal"><namePart type="family">KWOK</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">SNINSKY</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">WAIN-HOBSON</namePart>
<namePart type="given">S.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">GoodenowM., HuetT., SaurinW., KwokS., SninskyJ., & Wain-HobsonS. (1989) J. Acquired Immune Defic. Syndr. 2, 344−352</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref10" displayLabel="bib10"><name type="personal"><namePart type="family">HSIEH</namePart>
<namePart type="given">J.-C.</namePart>
</name>
<name type="personal"><namePart type="family">ZINNEN</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">MODRICH</namePart>
<namePart type="given">P.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">HsiehJ.-C., ZinnenS., & ModrichP. (1993) J. Biol. Chem. 268, 24607−24613.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref11" displayLabel="bib11"><name type="personal"><namePart type="family">JACOBO-MOLINA</namePart>
<namePart type="given">A.</namePart>
</name>
<name type="personal"><namePart type="family">DING</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">NANNI</namePart>
<namePart type="given">R. G.</namePart>
</name>
<name type="personal"><namePart type="family">CLARK</namePart>
<namePart type="given">A. D.</namePart>
</name>
<name type="personal"><namePart type="family">LU</namePart>
<namePart type="given">X.</namePart>
</name>
<name type="personal"><namePart type="family">TANTILLO</namePart>
<namePart type="given">C.</namePart>
</name>
<name type="personal"><namePart type="family">WILLIAMS</namePart>
<namePart type="given">R. L.</namePart>
</name>
<name type="personal"><namePart type="family">KAMER</namePart>
<namePart type="given">G.</namePart>
</name>
<name type="personal"><namePart type="family">FERRIS</namePart>
<namePart type="given">A. L.</namePart>
</name>
<name type="personal"><namePart type="family">CLARK</namePart>
<namePart type="given">P.</namePart>
</name>
<name type="personal"><namePart type="family">HIZI</namePart>
<namePart type="given">A.</namePart>
</name>
<name type="personal"><namePart type="family">HUGHES</namePart>
<namePart type="given">S. H.</namePart>
</name>
<name type="personal"><namePart type="family">ARNOLD</namePart>
<namePart type="given">E.</namePart>
</name>
<titleInfo><title>Proc.</title>
</titleInfo>
<note type="content-in-line">Jacobo-MolinaA., DingJ., NanniR. G., ClarkA. D.Jr., LuX., TantilloC., WilliamsR. L., KamerG., FerrisA. L., ClarkP., HiziA., HughesS. H., & ArnoldE. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6320−6324.10.1073/pnas.90.13.6320</note>
<identifier type="doi">10.1073/pnas.90.13.6320</identifier>
</relatedItem>
<relatedItem type="references" ID="atyp_ref12" displayLabel="bib12"><name type="personal"><namePart type="family">JI</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">LOEB</namePart>
<namePart type="given">L. A.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">JiJ., & LoebL. A. (1992) Biochemistry 31, 954−958.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref13" displayLabel="bib13"><name type="personal"><namePart type="family">JI</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">LOEB</namePart>
<namePart type="given">L. A.</namePart>
</name>
<titleInfo><title>Virology</title>
</titleInfo>
<note type="content-in-line">JiJ., & LoebL. A. (1994) Virology 199, 323−330.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref14" displayLabel="bib14"><name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>Methods Enzymol.</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Johnson K. A. Methods Enzymol. 1986 134 677 705</note>
<part><date>1986</date>
<detail type="volume"><caption>vol.</caption>
<number>134</number>
</detail>
<extent unit="pages"><start>677</start>
<end>705</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref15" displayLabel="bib15"><name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>The Enzymes</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Johnson K. A. The Enzymes 1992 20 1 61</note>
<part><date>1992</date>
<detail type="volume"><caption>vol.</caption>
<number>20</number>
</detail>
<extent unit="pages"><start>1</start>
<end>61</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref16" displayLabel="bib16"><name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>Annu. Rev. Biochem.</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Johnson K. A. Annu. Rev. Biochem. 1993 62 685 713 10.1146/annurev.bi.62.070193.003345</note>
<identifier type="doi">10.1146/annurev.bi.62.070193.003345</identifier>
<part><date>1993</date>
<detail type="volume"><caption>vol.</caption>
<number>62</number>
</detail>
<extent unit="pages"><start>685</start>
<end>713</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref17" displayLabel="bib17"><name type="personal"><namePart type="family">KATI</namePart>
<namePart type="given">W. M.</namePart>
</name>
<name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<name type="personal"><namePart type="family">JERVA</namePart>
<namePart type="given">L. F.</namePart>
</name>
<name type="personal"><namePart type="family">ANDERSON</namePart>
<namePart type="given">K. S.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">KatiW. M., JohnsonK. A., JervaL. F., & AndersonK. S. (1992) J. Biol. Chem. 267, 25988−25997.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref18" displayLabel="bib18"><name type="personal"><namePart type="family">KOHLSTAEDT</namePart>
<namePart type="given">L. A.</namePart>
</name>
<name type="personal"><namePart type="family">WANG</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">FRIEDMAN</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">RICE</namePart>
<namePart type="given">P. A.</namePart>
</name>
<name type="personal"><namePart type="family">STEITZ</namePart>
<namePart type="given">T. A.</namePart>
</name>
<titleInfo><title>Science</title>
</titleInfo>
<note type="content-in-line">KohlstaedtL. A., WangJ., FriedmanJ., RiceP. A., & SteitzT. A. (1992) Science 256, 1783−1790.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref19" displayLabel="bib19"><name type="personal"><namePart type="family">KUCHTA</namePart>
<namePart type="given">R. D.</namePart>
</name>
<name type="personal"><namePart type="family">MIZRAHI</namePart>
<namePart type="given">V.</namePart>
</name>
<name type="personal"><namePart type="family">BENKOVIC</namePart>
<namePart type="given">P. A.</namePart>
</name>
<name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<name type="personal"><namePart type="family">BENKOVIC</namePart>
<namePart type="given">S. J.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">KuchtaR. D., MizrahiV., BenkovicP. A., JohnsonK. A., & BenkovicS. J. (1987) Biochemistry 26, 8410−8417.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref20" displayLabel="bib20"><name type="personal"><namePart type="family">KUCHTA</namePart>
<namePart type="given">R. D.</namePart>
</name>
<name type="personal"><namePart type="family">BENKOVIC</namePart>
<namePart type="given">P.</namePart>
</name>
<name type="personal"><namePart type="family">BENKOVIC</namePart>
<namePart type="given">S. J.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">KuchtaR. D., BenkovicP., & BenkovicS. J. (1988) Biochemistry 27, 6716−6725.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref21" displayLabel="bib21"><name type="personal"><namePart type="family">MANSKY</namePart>
<namePart type="given">L. M.</namePart>
</name>
<name type="personal"><namePart type="family">TEMIN</namePart>
<namePart type="given">H. M.</namePart>
</name>
<titleInfo><title>J. Virol.</title>
</titleInfo>
<note type="content-in-line">ManskyL. M., & TeminH. M. (1995) J. Virol. 69, 5087−5094.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref22" displayLabel="bib22"><name type="personal"><namePart type="family">MENDELMAN</namePart>
<namePart type="given">L. V.</namePart>
</name>
<name type="personal"><namePart type="family">BOOSALIS</namePart>
<namePart type="given">M. S.</namePart>
</name>
<name type="personal"><namePart type="family">PETRUSKA</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">GOODMAN</namePart>
<namePart type="given">M. F.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">MendelmanL. V., BoosalisM. S., PetruskaJ., & GoodmanM. F. (1989) J. Biol. Chem. 264, 14415−14423.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref23" displayLabel="bib23"><name type="personal"><namePart type="family">MENDELMAN</namePart>
<namePart type="given">L. V.</namePart>
</name>
<name type="personal"><namePart type="family">PETRUSKA</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">GOODMAN</namePart>
<namePart type="given">M. F.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">MendelmanL. V., PetruskaJ., & GoodmanM. F. (1990) J. Biol. Chem. 265, 2338−2346.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref24" displayLabel="bib24"><name type="personal"><namePart type="family">MüLLER</namePart>
<namePart type="given">B.</namePart>
</name>
<name type="personal"><namePart type="family">RESTLE</namePart>
<namePart type="given">T.</namePart>
</name>
<name type="personal"><namePart type="family">WEISS</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">GAUTEL</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">SCZAKIEL</namePart>
<namePart type="given">G.</namePart>
</name>
<name type="personal"><namePart type="family">GOODY</namePart>
<namePart type="given">R. S.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">MüllerB., RestleT., WeissS., GautelM., SczakielG., & GoodyR. S. (1989) J. Biol. Chem. 264, 13975−13978.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref25" displayLabel="bib25"><name type="personal"><namePart type="family">PATEL</namePart>
<namePart type="given">S. S.</namePart>
</name>
<name type="personal"><namePart type="family">WONG</namePart>
<namePart type="given">I.</namePart>
</name>
<name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">PatelS. S., WongI., & JohnsonK. A. (1991) Biochemistry 30, 511−525.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref26" displayLabel="bib26"><name type="personal"><namePart type="family">PERRINO</namePart>
<namePart type="given">F.</namePart>
</name>
<name type="personal"><namePart type="family">PRESTON</namePart>
<namePart type="given">B. D.</namePart>
</name>
<name type="personal"><namePart type="family">SANDELL</namePart>
<namePart type="given">L. L.</namePart>
</name>
<name type="personal"><namePart type="family">LOEB</namePart>
<namePart type="given">L. A.</namePart>
</name>
<titleInfo><title>Proc.</title>
</titleInfo>
<note type="content-in-line">PerrinoF., PrestonB. D., SandellL. L., & LoebL. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8343−8347</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref27" displayLabel="bib27"><name type="personal"><namePart type="family">PRESTON</namePart>
<namePart type="given">B. D.</namePart>
</name>
<name type="personal"><namePart type="family">POIESZ</namePart>
<namePart type="given">B. J.</namePart>
</name>
<name type="personal"><namePart type="family">LOEB</namePart>
<namePart type="given">L. A.</namePart>
</name>
<titleInfo><title>Science</title>
</titleInfo>
<note type="content-in-line">PrestonB. D., PoieszB. J., & LoebL. A. (1988) Science 242, 1168−1171.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref28" displayLabel="bib28"><name type="personal"><namePart type="family">PRESTON</namePart>
<namePart type="given">B. D.</namePart>
</name>
<name type="personal"><namePart type="family">KEULEN</namePart>
<namePart type="given">W.</namePart>
</name>
<name type="personal"><namePart type="family">NIJHUIS</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">SCHUURMAN</namePart>
<namePart type="given">R.</namePart>
</name>
<name type="personal"><namePart type="family">BERKHOUT</namePart>
<namePart type="given">B.</namePart>
</name>
<name type="personal"><namePart type="family">BOUCHER</namePart>
<namePart type="given">C.</namePart>
</name>
<name type="personal"><namePart type="family">BALZARINI</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">PELEMANS</namePart>
<namePart type="given">H.</namePart>
</name>
<name type="personal"><namePart type="family">DE CLERCQ</namePart>
<namePart type="given">E.</namePart>
</name>
<name type="personal"><namePart type="family">KARLSSON</namePart>
<namePart type="given">A.</namePart>
</name>
<name type="personal"><namePart type="family">KLEIM</namePart>
<namePart type="given">J.-P.</namePart>
</name>
<name type="personal"><namePart type="family">PRASAD</namePart>
<namePart type="given">V. R.</namePart>
</name>
<name type="personal"><namePart type="family">DROSOPOULOS</namePart>
<namePart type="given">W. C.</namePart>
</name>
<name type="personal"><namePart type="family">WAINBERG</namePart>
<namePart type="given">M. A.</namePart>
</name>
<titleInfo><title>Science</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Preston B. D. Keulen W. Nijhuis M. Schuurman R. Berkhout B. Boucher C. Balzarini J. Pelemans H. De Clercq E. Karlsson A. Kleim J.-P. Prasad V. R. Drosopoulos W. C. Wainberg M. A. Science 1997 275 228 231</note>
<part><date>1997</date>
<detail type="volume"><caption>vol.</caption>
<number>275</number>
</detail>
<extent unit="pages"><start>228</start>
<end>231</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref29" displayLabel="bib29"><name type="personal"><namePart type="family">REARDON</namePart>
<namePart type="given">J. E.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Reardon J. E. Biochemistry 1992 31 4473 4479</note>
<part><date>1992</date>
<detail type="volume"><caption>vol.</caption>
<number>31</number>
</detail>
<extent unit="pages"><start>4473</start>
<end>4479</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref30" displayLabel="bib30"><name type="personal"><namePart type="family">REARDON</namePart>
<namePart type="given">J. E.</namePart>
</name>
<titleInfo><title>J. Biol. Chem.</title>
</titleInfo>
<genre type="journal">journal</genre>
<note type="content-in-line">Reardon J. E. J. Biol. Chem. 1993 268 8743 8751</note>
<part><date>1993</date>
<detail type="volume"><caption>vol.</caption>
<number>268</number>
</detail>
<extent unit="pages"><start>8743</start>
<end>8751</end>
</extent>
</part>
</relatedItem>
<relatedItem type="references" ID="atyp_ref31" displayLabel="bib31"><name type="personal"><namePart type="family">RICCHETTI</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">BUC</namePart>
<namePart type="given">H.</namePart>
</name>
<titleInfo><title>The</title>
</titleInfo>
<note type="content-in-line">RicchettiM., & BucH. (1990) The EMBO J. 9, 1583−1593.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref32" displayLabel="bib32"><name type="personal"><namePart type="family">RITTINGER</namePart>
<namePart type="given">K.</namePart>
</name>
<name type="personal"><namePart type="family">DIVITA</namePart>
<namePart type="given">G.</namePart>
</name>
<name type="personal"><namePart type="family">GOODY</namePart>
<namePart type="given">R. S.</namePart>
</name>
<titleInfo><title>Proc.</title>
</titleInfo>
<note type="content-in-line">RittingerK., DivitaG., & GoodyR. S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 8046−8049.10.1073/pnas.92.17.8046</note>
<identifier type="doi">10.1073/pnas.92.17.8046</identifier>
</relatedItem>
<relatedItem type="references" ID="atyp_ref33" displayLabel="bib33"><name type="personal"><namePart type="family">ROBERTS</namePart>
<namePart type="given">J. D.</namePart>
</name>
<name type="personal"><namePart type="family">BEBENEK</namePart>
<namePart type="given">K.</namePart>
</name>
<name type="personal"><namePart type="family">KUNKEL</namePart>
<namePart type="given">T. A.</namePart>
</name>
<titleInfo><title>Science</title>
</titleInfo>
<note type="content-in-line">RobertsJ. D., BebenekK., & KunkelT. A. (1988) Science 242, 1171−1173</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref34" displayLabel="bib34"><name type="personal"><namePart type="family">SPENCE</namePart>
<namePart type="given">R. A.</namePart>
</name>
<name type="personal"><namePart type="family">KATI</namePart>
<namePart type="given">W. M.</namePart>
</name>
<name type="personal"><namePart type="family">ANDERSON</namePart>
<namePart type="given">K. S.</namePart>
</name>
<name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>Science</title>
</titleInfo>
<note type="content-in-line">SpenceR. A., KatiW. M., AndersonK. S., & JohnsonK. A. (1995) Science 267, 988−993.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref35" displayLabel="bib35"><name type="personal"><namePart type="family">STAHLHUT</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">LI</namePart>
<namePart type="given">Y.</namePart>
</name>
<name type="personal"><namePart type="family">CONDRA</namePart>
<namePart type="given">J. H.</namePart>
</name>
<name type="personal"><namePart type="family">FU</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">GOTLIB</namePart>
<namePart type="given">L.</namePart>
</name>
<name type="personal"><namePart type="family">GRAHAM</namePart>
<namePart type="given">D. J.</namePart>
</name>
<name type="personal"><namePart type="family">OLSEN</namePart>
<namePart type="given">D.</namePart>
</name>
<titleInfo><title>Protein</title>
</titleInfo>
<note type="content-in-line">StahlhutM., LiY., CondraJ. H., FuJ., GotlibL., GrahamD. J., & OlsenD. (1994) Protein Expression Purif. 6, 614−621.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref36" displayLabel="bib36"><name type="personal"><namePart type="family">STEPHENSON</namePart>
<namePart type="given">C.</namePart>
</name>
<name type="personal"><namePart type="family">KARRAN</namePart>
<namePart type="given">P.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">StephensonC., & KarranP. (1989) J. Biol. Chem. 264, 21177−21182.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref37" displayLabel="bib37"><name type="personal"><namePart type="family">SUGIMOTO</namePart>
<namePart type="given">N.</namePart>
</name>
<name type="personal"><namePart type="family">NAKANO</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">KATOH</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">MATSUMURA</namePart>
<namePart type="given">A.</namePart>
</name>
<name type="personal"><namePart type="family">NAKAMUTA</namePart>
<namePart type="given">H.</namePart>
</name>
<name type="personal"><namePart type="family">OHMICHI</namePart>
<namePart type="given">T.</namePart>
</name>
<name type="personal"><namePart type="family">YONEYAMA</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">SASKI</namePart>
<namePart type="given">M.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">SugimotoN., NakanoS., KatohM., MatsumuraA., NakamutaH., OhmichiT., YoneyamaM., & SaskiM. (1995) Biochemistry 34, 11211−11216.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref38" displayLabel="bib38"><name type="personal"><namePart type="family">TAKEUCHI</namePart>
<namePart type="given">Y.</namePart>
</name>
<name type="personal"><namePart type="family">NAGUMO</namePart>
<namePart type="given">T.</namePart>
</name>
<name type="personal"><namePart type="family">HOSHING</namePart>
<namePart type="given">H.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">TakeuchiY., NagumoT., & HoshingH. (1988) J. Virol. 62, 3900−3902.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref39" displayLabel="bib39"><name type="personal"><namePart type="family">WANG</namePart>
<namePart type="given">A. C.</namePart>
</name>
<name type="personal"><namePart type="family">KIM</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">FLYNN</namePart>
<namePart type="given">P.</namePart>
</name>
<name type="personal"><namePart type="family">CHOU</namePart>
<namePart type="given">S.-H.</namePart>
</name>
<name type="personal"><namePart type="family">ORBAN</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">REID</namePart>
<namePart type="given">B. R.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">WangA. C., KimS., FlynnP., ChouS.-H., OrbanJ., & ReidB. R. (1992) Biochemistry 31, 3940−3946.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref40" displayLabel="bib40"><name type="personal"><namePart type="family">WEBER</namePart>
<namePart type="given">J.</namePart>
</name>
<name type="personal"><namePart type="family">GROSSE</namePart>
<namePart type="given">F.</namePart>
</name>
<titleInfo><title>Nucleic.</title>
</titleInfo>
<note type="content-in-line">WeberJ., & GrosseF. (1989) Nucleic. Acids Res. 17, 1379−1393.10.1093/nar/17.4.1379</note>
<identifier type="doi">10.1093/nar/17.4.1379</identifier>
</relatedItem>
<relatedItem type="references" ID="atyp_ref41" displayLabel="bib41"><name type="personal"><namePart type="family">WONG</namePart>
<namePart type="given">I.</namePart>
</name>
<name type="personal"><namePart type="family">PATEL</namePart>
<namePart type="given">S. S.</namePart>
</name>
<name type="personal"><namePart type="family">JOHNSON</namePart>
<namePart type="given">K. A.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">WongI., PatelS. S., & JohnsonK. A. (1991) Biochemistry 30, 526−537.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref42" displayLabel="bib42"><name type="personal"><namePart type="family">YU</namePart>
<namePart type="given">H.</namePart>
</name>
<name type="personal"><namePart type="family">GOODMAN</namePart>
<namePart type="given">M. F.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">YuH., & GoodmanM. F. (1992) J. Biol. Chem. 267, 10888−10896.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref43" displayLabel="bib43"><name type="personal"><namePart type="family">ZHU</namePart>
<namePart type="given">L.</namePart>
</name>
<name type="personal"><namePart type="family">SALAZAR</namePart>
<namePart type="given">M.</namePart>
</name>
<name type="personal"><namePart type="family">REID</namePart>
<namePart type="given">R. R.</namePart>
</name>
<titleInfo><title>Biochemistry</title>
</titleInfo>
<note type="content-in-line">ZhuL., SalazarM., & ReidR. R. (1995) Biochemistry 34, 2372−2380.</note>
</relatedItem>
<relatedItem type="references" ID="atyp_ref44" displayLabel="bib44"><name type="personal"><namePart type="family">ZINNEN</namePart>
<namePart type="given">S.</namePart>
</name>
<name type="personal"><namePart type="family">HSIEH</namePart>
<namePart type="given">J. C.</namePart>
</name>
<name type="personal"><namePart type="family">MODRICH</namePart>
<namePart type="given">P.</namePart>
</name>
<titleInfo><title>J.</title>
</titleInfo>
<note type="content-in-line">ZinnenS., HsiehJ. C., & ModrichP. (1994) J. Biol. Chem. 269, 24195−24202.</note>
</relatedItem>
<relatedItem type="references" ID="bi9713851b00001" displayLabel="bibbi9713851b00001"><titleInfo><title>Abbreviations used: dNTP, deoxynucleoside 5‘-triphosphate; dATP, deoxyadenosine 5‘-triphosphate; dCTP, deoxycytidine 5‘-triphosphate; dGTP, deoxyguanosine 5‘-triphosphate; dTTP, deoxythymidine 5‘-triphosphate; EDTA, ethylenediaminetetraacetic acid; HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase.</title>
</titleInfo>
<note type="content-in-line">Abbreviations used: dNTP, deoxynucleoside 5‘-triphosphate; dATP, deoxyadenosine 5‘-triphosphate; dCTP, deoxycytidine 5‘-triphosphate; dGTP, deoxyguanosine 5‘-triphosphate; dTTP, deoxythymidine 5‘-triphosphate; EDTA, ethylenediaminetetraacetic acid; HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase.</note>
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