(Received for publication, March 27, 1995; and in revised form, June 21, 1995)
From the
In order to determine the catalytic role of Arg of
HIV-1 reverse transcriptase (RT), we carried out site-directed
mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were
purified and characterized. With Arg to Ala substitution the k
of the polymerase reaction was reduced by
nearly 100-fold with poly(rA) template, but only about 5-15-fold
with poly(rC) and poly(dC) templates. The Arg to Lys substitution
exhibited a qualitatively similar pattern, although the overall
reduction in k
was less severe. Most
interestingly, we noted a large difference in the rate constant of the
first and second nucleotide incorporation by R72A, suggesting that
Arg
participates in the reaction after the formation of
the first phosphodiester bond. We propose this step to be the
pyrophosphate binding and removal step following the
nucleotidyltransferase reaction. Support for this proposal is obtained
from the observation that the R72A mutant (i) exhibited a pronounced
translocation defect in the processivity analysis, (ii) lacked the
ability to catalyze pyrophosphorolysis, and (iii) showed complete
resistance to phosphonoformate, an analog of PP
. Arg
is the first residue of HIV-1 RT proposed to be involved in the
pyrophosphate binding/removal function of RT.
HIV-1 reverse transcriptase (RT) ()is a
multifunctional heterodimeric enzyme that catalyzes the incorporation
of deoxyribonucleotides using both RNA and DNA templates. It is
processed initially from the pol gene product as a 66-kDa
polypeptide(2, 3, 4) . Subsequent proteolytic
cleavage of the p66/p66 homodimer yields the p66/p51
heterodimer(2, 3, 4) . HIV-1 RT shares some
common features with other DNA polymerases, including a large cleft
that accommodates the TP, formed by structurally similar
three-dimensional motifs which have been called palm, fingers, and
thumb(5, 6) . In the cleft of the enzyme resides the
``catalytic triad'' of three acidic residues. These residues
are Asp
, Asp
, and Asp
of the
p66 subunit of HIV-1 RT(7) . They are proposed to bind to the
divalent metal ion(s) required for the polymerase
activity(5, 7) , thereby, indirectly affecting the
binding of the dNTP
Mg complex. In addition, other neighboring
residues are expected to participate in other vital processes, such as
dNTP binding, pyrophosphate product removal, and translocation along
the template primer. To our knowledge, with the exception of Q151(16),
no other residue in HIV-1 RT has been experimentally shown to
participate either in the dNTP binding function or in the PP
removal function. The observation that resistance to a number of
nucleoside-analog inhibitors in RT is associated with mutation(s) of
some residue(s) in the region spanning 65-74 has suggested a
possible influence of this region in the dNTP binding function.
Furthermore, an antibody against an oligopeptide containing the amino
acid sequence of this region inhibited the polymerase activity
competitively, with respect to dNTP(8) . However, the
activities of most of these mutants were either unaffected or only
slightly reduced(9) . In the case of L74V the K
for ddATP was found to be increased but
no change in K
for dATP was
apparent(10) , implying no direct role for Leu
in
dNTP binding. Recent studies have suggested that this region could be
involved in the binding of template-strand binding (11) rather
than of dNTP. Therefore, a considerable degree of uncertainty exists
regarding the role of residues in this region.
We have chosen
Arg as a target for in vitro site-directed
mutagenesis to investigate the role of this region in general and that
of Arg
in particular. The choice was based on the fact
that our comparative modeling studies of the Klenow fragment (KF) and
HIV-1 RT have suggested a spatially equivalent location of Arg
of RT with that of Lys
of KF(12) . Earlier
studies on Lys
of KF had implicated it in both the dNTP
binding function and in the translocation of enzyme across the
template(13) . The present work provides experimental evidence
regarding the role of Arg
, using the R72A and R72K mutants
of HIV-1 RT. Furthermore, in order to determine the subunit
specificity, if any, we examined the properties of reconstituted hybrid
heterodimers, containing mutations only in one subunit
(p66
/p51
and
p66
/p51
). Our results show that the
mutational effects of Arg
are expressed through the p66
(and not the p51) subunit. In addition, Arg
does not
appear to be required for dNTP or DNA binding. The severe impairment in
the processive mode of DNA synthesis by R72A together with the
inability to catalyze pyrophosphorolysis and the complete resistance to
phosphonoformate (a PP
analog), strongly implicate
Arg
in the PP
binding/removal function of
HIV-1 RT.
Figure 4:
Qualitative processivity studies on
poly(rA)(dT)
and 18/47. The two labeled TPs were
poly(rA)
5`-
P-(dT)
(A) and
47/5`-
P-18-mer (B). Enzymes carrying mutation in
either of the subunits were incubated with the labeled TP as described
under ``Experimental Procedures.'' Processive reactions were
initiated by the addition of dNTP and DNA trap together and incubated
at 25 °C for 1 (lane 1) and 10 min (lane 2).
Total DNA synthesis (processive and distributive) was initiated by the
addition of dNTP alone (no trap) and incubated as above for 1 (lane
3) and 10 min (lane 4). The position of 18-mer primer is
shown at the extreme right.
2) The
quantitative processivity studies were essentially as before (26) . Reaction mixtures (final volume 0.5 ml) contained 40
nM WT or 60 nM mutant RTs, 200 nM poly(rA)(dT)
(annealed at a 1:1 ratio with
respect to the 5`-ends), 0.03 mM
[
H]dNTP, 80 mM NaCl, 1 mM DTT,
50 mM HEPES, pH 8.0, and 0.1 mg/ml bovine serum albumin.
MgCl
was added to a final concentration of 2 mM to
initiate control reactions, MgCl
and 100 µM
non-substrate homopolymeric trap to initiate the processivity
reactions. The mixture was incubated at 25 °C, and aliquots of 25
µl were removed at indicated times and processed as above. We
observed no significant effect in the processivity of R72A at dNTP
concentrations up to 100 µM.
Figure 1:
Binding of template-primer to wild type
and R72A HIV-1 RT enzymes. 60 nM of WT (squares) or
R72A (circles) were incubated with varying concentrations of
5`-P-18/47-mer (2-100 nM) in a standard
reaction mixture (50 µl) and subjected to UV exposure. Samples were
processed as described under ``Experimental Procedures.'' The
data are plotted in the Scatchard (A) and semilogarithmic
graph format (B). K
values were
calculated from the formula: slope (in graph A) =
-1/K
. A nonlinear least-squares
program was used to fit the data in B to a binding isotherm.
[(Bound DNA) = maxBound DNA * Free DNA/K
+ Free DNA]. Points in the graphs represent the
mean values for at least duplicate samples. Standard error was
<15%.
Figure 2:
Pyrophosphorolysis reaction by wild type
and R72A. Reactions were performed as described under
``Experimental Procedures'' for the pyrophosphorolysis of P-labeled 47/18-mer TP (1
10
disintegrations/min/pmol of 18-mer) in the presence (lanes
2, 4, and 6) or absence (lanes 1, 3, and 5) of 1 mM PP
. WT is
shown in lanes 1 and 2,
p66
/p51
in lanes 3 and 4, and p66
/p51
in lanes 5 and 6. Lane 7 contains only
P-labeled 18/47-mer.
Figure 3:
Processivity studies on
poly(rA)(dT)
for wild type and R72A RTs. Reactions
were performed as described under ``Experimental Procedures''
for incorporation of TMP into poly(rA)
(dT)
, by WT (A) and R72A (B). The curves represent the best fit
of the data N/E = (k
/k
)(1 -
exp(-k
t)). N/E is the
number of nucleotides added per processive cycle by an enzyme molecule (y axis)(27) . The amount of background incorporation
with DNA trap preincubation was <8% of the respective activities in
unchallenged experiments (filled triangles). The corrected
values for the processive conditions in the presence of trap are
presented in the graphs (open
squares).
Similar,
though less dramatic, results were observed with 47/18-mer as TP. In
the presence of trap it is observed that the majority of extended
primers by p66/p51
in 1 or 10 min are of
the n+1 size (n = size of original
primer) (Fig. 4B, lanes 1 and 2 of p66
/p51
). However,
without DNA trap, the same enzyme is able to add a few more nucleotides (Fig. 4B, lanes 3 and 4 of p66
/p51
). Once again, a
substantial number of WT RT molecules (Fig. 4B, lanes 1-3 of p66
/p51
) are able to copy
the full-length of template in the presence or absence of trap.
The
reduced processivity of R72A probably resulted from the extended
stalling of p66/p51
by remaining bound to
the TP, followed by dissociation from the TP. Evidence for the former
is the observed increase of the product size with time under processive
conditions (increasingly higher intensity of lower bands in Fig. 4A, lanes 1versus2,
for p66
/p51
). Under these
conditions (trap present), extension of product can only be done by the
same enzyme molecule, as the trap binds effectively all free and
dissociating enzyme molecules (the effectiveness of trap to arrest free
enzyme is shown in Fig. 4(extreme right lane), where no
polymerization by WT was observed in 10 min, when the trap was added at
the binding step). The effects of the Arg
mutation are
exerted through the p66 subunit, as judged by the processivity profiles
of p66
/p51
and
p66
/p51
. The mutation in p51 affects
neither the activity nor the processivity, whereas mutation in p66
alone, decreases both processivity and polymerization activity.
Figure 5:
Rates of first and second nucleotide
incorporation by wild type and R72A RT. The rate of incorporation of
first nucleotide (A) was measured by monitoring the
incorporation of [-
P]TMP into 18/47 (see Fig. 4for nucleotide sequence) in a reaction mixture containing
50 mM HEPES, pH. 7.5, 2 mM MgCl
, 400
nM enzyme, and 20 µM
[
-
P]TTP (1.4 µCi/nmol). Reactions were
carried at 25 °C and aliquots were removed at indicated times and
the amount of [
P]TMP incorporated was determined
as described under ``Experimental Procedures.'' The rate of
second nucleotide (dATP) addition (B) was measured in a
similar manner, except that 100 µM of unlabeled first
nucleotide (TTP) was also added along with 20 µM of
[
-
P]dATP (1.4 µCi/nmol). Open
squares and filled circles represent values for WT and
R72A, respectively. The first-order rate of single nucleotide
incorporation in E/template-primer was measured from the slope
of the first-order plot of
ln{[RT-TP]
/[RT-TP]
} versus time (slope= -k/2.3).
[RT-TP]
is estimated from the amount of
dNTP incorporated in E-TP at prolonged incubation (30 min),
assuming 1:1 stoichiometry. [RT-TP]
is
estimated from the difference between [RT-TP]
and the amount incorporated at time t.
The characterization of the available large number of HIV-1
RT mutants (9, 28, 29, 38) has not
been extensive with regard to the polymerization mechanism. Hence, the
role of the mutated residues in the catalytic mechanism of RT has
remained elusive. Furthermore, with the exception of a few
studies(7, 30) , the vast majority of mutagenesis
studies have not been subunit specific, leaving an uncertainty
regarding the specific residue responsible for the observed effect. We
have shown in our laboratory (12) that 10 residues of KF of E. coli DNA polymerase I have equivalent counterparts in the
three-dimensional structure of HIV-1 RT, suggesting functional
equivalence of these residues. In this study, Lys of the
KF was reported to be spatially equivalent to Arg
of RT.
However, with the availability of the new crystal structure coordinates
for KF(31) , we now find that Arg
is the
equivalent residue to Arg
. (
)Nevertheless, the
catalytic roles of both Arg
or Lys
, as
judged by site-directed mutagenesis appear similar(32) .
Furthermore, Lys
has been extensively characterized
through chemical modification and site-directed mutagenesis
studies(33, 13) . The mutational studies suggested
its involvement in (a) dNTP binding and (b)
translocation along the template strand(13) . In addition, the
recent crystallographic studies of KF complexed with PP
and
dNTP (31) have implicated Lys
as a probable
binding site for the
-
phosphates (PP
) of dNTP.
With regard to Arg
of KF, its importance in the catalytic
function is implied by a significant loss of activity of
R754A(32) . Furthermore, it was also suggested to play a major
role in PP
binding (32) . Since in RT, Arg
is now proposed to be a counterpart of Arg
, we
examined the role of Arg
in the catalysis of DNA synthesis
by RT. We constructed and characterized RTs with conservative and
non-conservative mutations at Arg
. Incidentally, a
conservative R72K mutant had been constructed previously and moderate
reduction in the activity of cell extracts containing the mutant enzyme
was reported, without further investigation of the role of this residue (9) .
We found Arg to be required for efficient
polymerization. Interestingly, the catalytic properties of Arg
could be partially compensated by lysine, as judged by the small
reduction in the k
of R72K (Table 1).
These results suggest that the participation of Arg
in the
catalytic process is of electrostatic nature. Nevertheless, the
significant loss of polymerase activity associated with the R72A
mutation could not be accounted for by the minor changes in the K
for dNTP. We therefore examined if the
R72A has reduced DNA binding ability. The x-ray structure of RT
complexed with double-stranded DNA had suggested a role for the
Arg
containing region in the binding of DNA(6) .
Our examination clearly showed that Arg
does not
participate in DNA binding, as the K
and K
for DNA TP of R72A, calculated from
kinetic and DNA cross-linking experiments (Table 1) were
comparable to those of the WT enzyme. Thus, the defect in the
polymerase reaction catalyzed by the mutant enzyme and presumably the
functional role of Arg
appeared to lie at a step beyond
the E
TP complex formation. We therefore considered a
possible shift from processive to distributive mode of DNA synthesis by
R72A. Our experiments ( Fig. 3and Fig. 4), clearly show
that the p66
/p51
enzyme lost its ability to
polymerize in the processive mode (Fig. 4). Furthermore, as
expected Arg
of only the p66 subunit is necessary for
processive polymerization. Additional insight into the catalytic defect
of R72A was provided by kinetic experiments. First-order kinetics (Fig. 5) showed that the first nucleotide is added by the mutant
and WT enzymes at comparable rates (Fig. 5). However, the rate
of subsequent nucleotide addition was significantly reduced only in the
case of R72A. Possible affected steps between the addition of first and
second nucleotides are either the removal of pyrophosphate or the
translocation along the template strand. Our experimental evidence
argues for involvement of Arg
in the former step, as R72A
exhibits a total resistance to PFA, a PP
analog (see
below). Furthermore, our pyrophosphorolysis results strongly support
the involvement of Arg
in the pyrophosphate binding
function, as in the absence of Arg
the mutant is severely
impaired in its ability to catalyze pyrophosphorolysis of DNA (Fig. 2). In addition, computer-assisted modeling of a
prepolymerization complex consisting of HIV-1 RT, TP, and Mg-dNTP (16) shows that the Arg
side chain is oriented
toward the PP
moiety of dNTP. Finally, the crystal
structures of KF with PP
or dNTPs show the side chains of
Arg
as well as Lys
(the equivalent of
Arg
residue in the RT enzyme) providing the
PP
-binding site(31) . A recent report on the
properties of R754A has also suggested a role for R754 in PP
binding(32) . Therefore, the primary role for Arg
in catalysis appears to be related to binding and/or removal of
PP
. A plausible consequence of such a defect could very
well be seen in the inability of R72A to translocate across the
template; however, the possibility of participation of Arg
in the PP
-binding-independent translocation event
cannot be excluded.
In our drug susceptibility studies, R72A and WT
RTs were found to be equally sensitive to nucleotide analog as well as
nevirapine, implying non-involvement of Arg in the binding
of these inhibitors. In regard to PP
inhibition, a small
but consistent increase in the K
for
PP
was noted with R72A (Table 2). However, the most
prominent effect of the R72A mutation was seen in the form of total
resistance of mutant enzyme to PFA (Table 2). Despite the
similarity in the structures of PP
and PFA, we find R72A to
discriminate among these two inhibitors. The differences in the
inhibitory effects may be attributed to the different size and charge
of the two molecules, which presumably causes the large difference in
their K
values, even for the WT enzyme (Table 2, and (18) ).
In addition to Arg,
other RT residues (Glu
, Asp
,
Ala
, and Tyr
) (34, 35) may
also be involved in the mechanism of resistance to PFA. From these
mutants, Glu
Gly and Tyr
Asn have been shown
to be almost totally resistant to PFA, similar to Arg
Ala
reported in this communication. As these three residues
(Glu
, Arg
, and Tyr
) are
significantly apart in the three-dimensional
structure(5, 6) , it is unlikely that they directly
interact with PFA at a common binding site. The following reasoning
shows that only Arg
could participate directly in the
PFA/PP
binding: as the Tyr
interacts through
its phenyl ring in a hydrophobic manner (Tyr
Phe has WT
properties)(36) , it seems unlikely for this residue to
directly interact with the polar PFA molecule. In addition, our
modeling studies show the Tyr
side chain poised to
interact with the ribose ring of the dNTP which is significantly away
from the pyrophosphate moiety of dNTP. Similarly, the negative charge
of Glu
makes any direct interaction with PFA unlikely. The
fact that Arg
Lys retains sensitivity to PFA, suggests that
the RT-PFA interaction is based on electropositive charge at the 72
position. While examining the metal preferences of WT and mutant
enzymes, we observed a 2-3-fold increase in the Mn/Mg activity
ratio (results not shown). This change in metal specificity is
consistent with Arg
interacting with the metal-binding
-
phosphate moiety of the dNTP.
In conclusion, we have
shown that Arg is a catalytically important residue for
RT, required for PP
binding, release, and the translocation
function. To our knowledge, this is the first residue of HIV-1 RT,
outside the thumb subdomain(37) , experimentally shown to
contribute to the processive mode of polymerization. It therefore
appears that the ability for processive polymerization is not
exclusively due to residues of the thumb subdomain, but to other amino
acids, as well.