Background
Human immunodeficiency virus type-1 reverse
transcriptase (HIV-1 RT) is a product of the gag-pol
polyprotein precursor, which is subsequently cleaved by the
pol -encoded protease to yield the
active form of the enzyme [ 1 2 ] . This multifunctional
enzyme is responsible for copying the single stranded viral
RNA genome into double stranded proviral DNA [ 3 4 ] .
HIV-1 RT is a heterodimer consisting of a 66 and 51 kDa
polypeptide chain designated as p66 and p51, respectively.
The p51 subunit is generated via endoproteolytic cleavage
of the p66 subunit between Phe 440 and Tyr 441 [ 5 6 ] .
The larger subunit (p66) contains both polymerase and RNase
H activities, while the smaller subunit (p51) lacks these
functions, in context of the heterodimer [ 7 8 ] . However,
both the p66 and p51 monomers are functionally inactive
when dissociated from each other [ 9 ] . Several years have
passed since it was first suggested that agents that could
specifically disrupt the dimerization of HIV-1 RT might
prove a worthwhile antiretroviral strategy [ 10 ] , though
such agents have yet to be developed.
Despite the fact that p51 shares an identical amino acid
sequence with the N-terminal portion of p66, the two
subunits assume different global folding patterns in the
formation of the asymmetric heterodimer [ 9 ] . Structural
determination through X-ray crystallography has revealed
that the p66 subunit of HIV-1RT has its polymerase domain
in an "open" conformation, with its subdomains forming a
large cleft which accommodates DNA. In contrast, the p51
subunit assumes a compact folded conformation that causes
the active site residues in this subunit to be buried and
therefore, nonfunctional [ 11 12 13 ] . It has been
proposed that the open conformation of p66 is supported by
interactions with a closed and compact p51 molecule [ 12 14
15 ] . The two subunits interact mainly via their
connection subdomains. Additional contacts, between the
thumb subdomain of p51 and RNase H subdomain of p66 are
also substantial [ 11 12 ] .
Although there have been conflicting reports regarding
the DNA polymerase activity of recombinant preparations of
the p51 homodimer [ 16 17 ] , it has become clear that p51
mainly plays a supportive role in context of the p66/p51
heterodimer. Assembly of chimeric heterodimers formed by
mixing subunits of HIV-1 RT and FIV-1 RT, has demonstrated
that the p51 subunit of HIV-1 RT helps to preserve the
functional integrity of the HIV-1 RT heterodimer [ 18 ] .
Despite the fact that several functions have been proposed
for the p51 subunit, the mechanism whereby p51 performs
these functions has remained largely undefined. Some of the
proposed functions for p51 include: (i) stabilizing the
t-RNA primer binding for the initiation of reverse
transcription [ 11 ] , (ii) enhancement of strand
displacement DNA synthesis [ 19 20 ] , and (iii) as a
processivity factor in DNA synthesis [ 21 ] .
Cys→Ser mutation at position 280 in the p51 subunit has
been shown to alter the RNase H activity of the
heterodimeric enzyme, indicating that this residue in the
thumb subdomain of p51 plays an important role in support
of the RNase H activity of p66 [ 22 ] . The emergence of a
strain of HIV-1 resistant to the non-nucleoside RT
inhibitor TSAO (Tertbutyldimethyl silyspiro amino
oxathioledioside) displaying Glu→Lys mutation at position
138 in the p51 subunit of HIV-1 RT has also been reported [
23 ] , thus implicating p51 to play a more direct role in
drug binding and/or the enzymatic activities of HIV-1RT.
This report was initially surprising, since Glu138 of p51
was thought to be quite distant from the purported
dNTP-binding pocket of HIV-1RT, as well as the NNRTI
binding pocket. However, in light of our recent findings
implicating this loop region of p51 as a critical
structural element supporting the catalytic functions of
p66, it seems feasible that mutation at position 138 in p51
effectively altered the binding of TSAO through its
influence on the p66 catalytic subunit [ 14 ] .
Examination of the crystal structure of HIV-1 RT reveals
the presence of a small groove like region on the floor of
the polymerase cleft of p66 [ 11 ] . The β7-β8 loop of p51,
comprising of six amino acids denoted as SINNET appears to
fit into this groove-like region and likely stabilizes the
polymerase domain of p66. In an earlier communication, we
have shown that the p51 subunit of HIV-1 RT is required to
load the p66 subunit on to the template primer for DNA
synthesis [ 14 ] . Our recent studies indicate that the
β7-β8 loop of the p51 subunit is essential for the
catalytic function of the p66 subunit. Deletion of this
loop or substitution of four amino acid residues with
alanine within the β7-β8 loop of p51 severely impaired the
DNA polymerase activity of the enzyme as a consequence of
the inability of the enzyme to form stable dimers [ 15 ] .
These findings clearly establish the absolute requirement
of the β7-β8 loop of p51 for RT dimerization.
Nonetheless, the question regarding the optimal size and
composition requirement of this loop for efficient
dimerization remains unanswered. In the present article, we
have addressed the impact of increasing the size of the
β7-β8 loop on the dimerization process. As a preamble to
these studies, we have increased the size of this loop by
repeating its six amino acid sequence in tandem. The
rationale for duplicating the loop sequence was to increase
the size of this loop without significantly disrupting the
interactions seen with the wild type β7-β8 loop. The
resulting mutant derivatives of HIV-1 RT containing
insertion of six amino acids in the β7-β8 loop in either or
both the subunits were analyzed for their ability to form
stable dimers and other biochemical characteristics. In
this article, we present evidence that HIV-1 RT mutants,
carrying insertion of six amino acids in the β7-β8 loop
specifically in the second subunit, do not form stable
dimers. This inability to dimerize substantially decreases
the enzymes affinity for DNA consequently impairing its
polymerase and RNase H activities.
Results
Glycerol gradient ultra-centrifugation
analysis
The ability of the HIV-1 RT mutants, carrying
insertion of six residues in the β7-β8 loop in either one
or both the subunits was analyzed by glycerol gradient
sedimentation analysis. Fractions of 200 μL were
collected from the bottom of the tube and aliquots of
every third fraction were subjected to SDS PAGE. The
results shown in Figure 1A, indicate that the
sedimentation peak for the wild type p66/p66 homodimer
was between fractions 16-19 of the gradient (Panel A),
whereas the wild type p51 species sedimented between
fractions 22-28, as a monomeric protein (Panel C). The
sedimentation pattern for the p66 WT/p51 INSmutant
indicated two distinct peaks. While the p66 WTsubunit
predominantly sedimented in between fractions 16-19, at
the predicted position of the homodimer, the p51
INSsubunit sedimented between fractions 22-28, indicating
a monomeric conformation (Panel D). This sedimentation
profile indicates that the p66 WT/p51 INSmutant carrying
the 6 amino acid insertion in the β7-β8 loop of its p51
subunit is unable to form stable heterodimer. Under these
conditions, the p66 WTsubunit of the p66 WT/p51 INSmutant
tends to homodimerize. The mutants, p66 INS/p66 INS(Panel
B) and p66 INS/p51 INS(Panel F), exhibited a distinct
sedimentation peak between fractions 22-28 of the
gradient, indicating that these enzymes are unable to
form stable dimers. In contrast, the p66 INS/p51 WTmutant
(Panel E), in which only the p66 subunit carried an
insertion in the β7-β8 loop sedimented between fractions
16-19 of the gradient, indicating that these two subunits
can form stable heterodimers. These analyses imply that
the proper size of the β7-β8 loop in the second subunit
of HIV-1 RT is important for the formation of a stable
dimeric enzyme.
In order to correlate the sedimentation profile of
these insertion mutants with their functional activity,
we analyzed the polymerase activity in the various
gradient fractions. These results are presented in Fig.
1B. The polymerase activity profile of the gradient
fractions of the wild-type p66/p66 and the p66 INS/p51
WTmutant revealed major polymerase activity peaks
corresponding to fractions 16-19 (Fig. 1B). This activity
peak correlates with the protein band intensity seen in
Fig. 1A(panels A and E) and is also in agreement with the
sedimentation pattern of these two enzymes.
Interestingly, the activity profile of the p66 WT/p51
INSmutant also yielded a peak corresponding to gradient
fractions 16-19 (Fig. 1B), thus substantiating our
contention that the p66 WTsubunit of the p66 WT/p51
INSmutant tends to self-dimerize and form the
catalytically active p66 homodimer. The wild type p51 and
the two mutants, p66 INS/p66 INSand p66 INS/p51 INS, the
sedimentation profile of which indicated a monomeric
conformation (Fig. 1A) were conspicuously devoid of any
polymerase activity (Fig. 1B). These results imply that
the β7-β8 loop of the second subunit of HIV-1 RT is
critical in forming functionally active dimeric
enzyme.
DNA polymerase activities of wild type HIV-1 RT and
its insertion mutants
DNA polymerase activity of the wild type HIV-1 RT and
its mutant derivatives were quantitatively determined on
both heteropolymeric RNA (U5-PBS RNA) and DNA (49-mer)
templates, primed with 32P labeled 17-mer PBS primer. As
documented in Table 1, insertion of the six amino acid
segment in both the subunits (p66 INS/p66 INSor p66
INS/p51 INS), resulted in significant loss of polymerase
activity corresponding to greater than 90% reduction.
Interestingly, when the p66 INSmutant was dimerized with
the wild type p51 (p66 INS/p51 WT), the polymerase
activity was restored to wild type levels. The same
result was obtained when p66 INSwas dimerized with p51
having Asp→Ala mutation at amino acid position 186 (one
of the catalytically crucial carboxylate triad) but
having an intact β7-β8 loop. The rationale for using p51
D186Amutant was to ascertain that the wild type
polymerase activity observed with p66 INS/p51 WTwas not
due to residual polymerase activity of p51. These results
suggest that insertion of six residues in the β7-β8 loop
of p51 but not in p66 is detrimental to the function of
the heterodimeric enzyme.
As shown in figure 2, evaluation of the polymerase
activity of the wild type HIV-1RT and its insertion
mutants by primer extension assay generally mirrored the
results observed in the TCA precipitation assay,
summarized in Table 1. Insertion of six amino acid
residues in the β7-β8 loops of both the subunits (p66
INS/p66 INSor p66 INS/p51 INS) resulted in significant
impairment of polymerase activity of the mutant enzymes.
On an RNA template, both these enzymes exhibited total
lack of polymerase activity (Fig. 2B), though the p66
INS/p51 INSmutant exhibited residual primer extension
capability on a DNA template (Fig. 2A), while the p66
INSp/66 INSmutant was inactive on this template.
Consistent with the results of the TCA precipitation
assay, both the mutants, p66 INS/p51 WTand p66 INS/p51
D186A, exhibited near wild type (p66 WT/p51 WT)
polymerase activity on both RNA and DNA templates. Thus,
the impairment of the polymerase activity seen with these
mutants carrying insertion in either both the subunits or
specifically in the second subunit may be related to the
inability of these mutants to form stable dimers
resulting in a closed polymerase cleft.
Effect of insertion in either or both the subunits
of HIV-1 RT on the DNA binding function of the
enzyme
Earlier we have shown that substitution of four amino
acids on the β7-β8 loop with alanine reduced the DNA
binding ability of the enzyme. Similar results were
obtained when four amino acids were deleted from the
loop. We therefore, concluded that the effect was exerted
via p51 since alanine substitution or deletion
specifically in the p66 subunit had no effect on DNA
binding and polymerase activity of the enzyme. We
speculated that alanine substitution or deletion in the
loop of the p51 subunit may shorten the interacting
sphere of the loop which may not be able to induce
opening of the polymerase cleft in p66, which is
essential for DNA binding. Given the fact that shortening
of this loop in p51 prevented DNA binding, we were
interested in examining the effect of six amino acids
insertion on DNA binding. Therefore, we determined the
equilibrium dissociation constants (K
d ) of E-TP binary complexes for the
wild type enzyme and its mutant derivatives by gel
mobility shift assay. For this purpose, we used a 33-mer
heteropolymeric DNA template primed with 5' 32P-labeled
21-mer DNA. Results shown in figure 3and table 2indicate
a 24-fold reduction in DNA binding affinity when both the
subunits carried insertion (p66 INS/p51 INS) in their
β7-β8 loops. Similar results were obtained with the p66
INS/p66 INSmutant. Interestingly, the DNA binding
affinity was restored to the wild type levels, when the
mutant p66 subunit was dimerized with the wild type p51
(p66 INS/p51 WT). We expected that the p66 WT/p51
INSmutant would also exhibit reduction in its affinity
for DNA, since insertion of six residues in the p51
subunit was speculated to be detrimental for the
dimerization process. However, the DNA binding affinity
of the p66 WT/p51 INSmutant was similar to the wild type
enzyme. Our glycerol gradient sedimentation analysis had
revealed that the p66 WT/p51 INSmutant does not form
stable dimers, rather the p66 WTsubunit tends to self
dimerize. Thus, the wild type DNA binding affinity seen
in case of the p66 WT/p51 INSmutant may be attributed to
the presence of these p66 WT/p66 WThomodimeric species.
Based on our observations from the sedimentation
analysis, we propose that the loss of DNA binding
function in case of the HIV-1RT insertion mutants results
from a failure to form stable dimers. The wild type p51
alone exhibited very low DNA binding affinity with a K
d[DNA] 1300-fold higher than the wild
type heterodimeric enzyme. These results suggest that the
optimal size of the β7-β8 loop in the second subunit of
the HIV-1 RT dimer is crucial for opening the polymerase
cleft of the p66 subunit.
Ternary complex formation by the wild type and
mutant enzymes
In the crystal structures of the ternary complex of
HIV-1 RT (E-DNA-dNTP) the finger subdomain moves by 20Å
towards the palm subdomain [ 24 ] . In this finger
closing conformation, the DNA is locked in a stable
ternary complex poised for catalysis. An
in vitro assay using dideoxy
terminated primer annealed with the template which allows
the next correct dNTP to bind in the ternary complex
without actual DNA synthesis has recently been reported [
25 ] . Using this assay system, we have evaluated the
ability of the insertion mutants to form the ternary
complexes and the effect of DNA trap on such complexes.
Since binding of dNTP to the enzyme is an ordered
mechanism which occurs only after DNA binding, the extent
of labeled TP remaining bound to the enzyme in the
presence of dNTP and DNA trap represents the extent of
ternary complex formed. The E-TP binary complex was
formed at enzyme concentrations which binds 100% of the
labeled template primer. The preformed E-TP complex was
then incubated in the presence of next correct dNTP
followed by addition of 300-fold molar excess of
unlabeled TP as the DNA trap. We found that E-TP binary
complex was completely competed out by the DNA trap (data
not shown) while a significant amount of the E-TP binary
complex converted to E-TP-dNTP ternary complex was
resistant to competition with DNA trap (Fig. 4)
suggesting the stability of the ternary complex. Table
2lists the apparent dNTP binding affinity for the WT
enzyme and its insertion mutants determined from data
shown in Fig. 4. It was observed that although the DNA
binding affinity was severely affected in case of the p66
INS/p51 INSmutant, its apparent dNTP binding affinity in
the ternary complex did not change with respect to the
wild type enzyme. However, the p66 INS/p66 INSmutant was
unable to form a ternary complex. These data suggest that
these two mutants may have a different conformation and
mode of interaction in the ternary complex. It is
apparent that the p66 INS/p66 INSbinds to TP in a
nonproductive manner which may have a direct impact on
dNTP binding in the ternary complex.
Steady state kinetic analysis of HIV-1 RT and its
insertion mutants
In order to determine whether alteration in DNA
binding without any change in the apparent dNTP binding
affinity of the insertion mutants is consistent with
their kinetic parameters, we analyzed the steady-state
kinetic parameters of these mutants. The results of this
investigation are summarized in Table 3. On poly
(rA).(dT)
18 , only the p66 INS/p66 INSmutant
showed a significant increase in K
m [dNTP] . This observation is in
agreement with the apparent dNTP binding affinity data in
the ternary complex, where p66 INS/p66 INSmutant was
found to be defective in forming a productive ternary
complex. This observation is also consistent with our
suggestion that p66 INS/p66 INSbinds nonproductively to
TP that may influence the formation of ternary complexes.
Interestingly, the p66 INS/p51 INSmutant carrying
insertion in both the subunits did not display the same
reduction in dTTP binding affinity. However, the p66
INS/p66 INSand p66 INS/p51 INSmutants displayed nearly
6,000-fold and 400-fold reduction in catalytic efficiency
(k
cat /Km) compared to their wild type
counterparts, respectively, on this template primer. A
10-fold reduction in catalytic efficiency in case of the
p66 INS/p51 WTwas noted only on poly (rA). (dT)
18 and may be template-primer
specific. None of the enzymes displayed a significant
reduction in K
m [dNTP] when the heteropolymeric
DNA\DNA template primer was used, although the p66
INS/p66 INSand p66 INS/p51 INSmutants exhibited drastic
reduction in catalytic efficiency. The p66 WT/p51
INSdisplayed no change in either dNTP binding or
catalytic efficiency. These results are in keeping with
our analysis of the polymerase and DNA binding assay for
this mutant. Once again, we believe that the wild type
p66 subunit in this enzyme preparation tends to
homodimerize since the p51 INSfails to participate in
stable dimer formation. This phenomenon masks the
deleterious effect of the insertion mutation.
RNase H activity of the insertion mutants
Since the polymerase activity of the homo- and
hetero-dimeric enzymes carrying insertion in the β7-β8
loop in both the subunits (p66 INS/p66 INSand p66 INS/p51
INS) was drastically impaired, it was of interest to
examine how this insertion affects their RNase H
activity. To evaluate this, we employed a 30-mer RNA-DNA
hybrid, and examined the cleavage of the 5'- 32P-RNA
strand of the duplex by the wild type enzyme and its
mutant derivatives. The result of this analysis is
presented in Fig. 5. Similar to their polymerase
activities, the RNase H activities of the p66 INS/p66
INSand p66 INS/p51 INSmutants were severely impaired.
This is not surprising, since our analysis of the DNA
binding function of these two mutants had indicated a
substantial loss of DNA binding affinity, which in turn
is expected to affect both the polymerase and RNase H
functions. Dimerization of the p66 INSsubunit with the
wild type p51 (p66 INS/p51 WT) carrying an intact β7-β8
loop resulted in substantial recovery of the RNase H
activity. The RNase H activity seen in case of the p66
WT/p51 INSmutant was not surprising since the p66
WTsubunit of the p66 WT/p51 INSmutant tends to
self-dimerize and form the catalytically active p66
homodimer.
Discussion
In an earlier investigation on the role of the p51
subunit of HIV-1 RT, we demonstrated that decrease in size
of its β7-β8 loop impairs the catalytic function of the
heterodimer [ 15 ] . In the present studies, we demonstrate
that maintaining the wild type size of this loop in the p51
subunit is critical for dimerization of the enzyme and its
catalytic activity. Duplication of the β7-β8 loop sequence
selectively in the p66 subunit did not affect the dimer
formation, DNA binding or polymerase activity of the p66
INS/p51 WTmutant. However, insertion of the same amino acid
residues in the β7-β8 loop of p51 prevented stable
dimerization of the p51 INSsubunit with either p66 INSor
p66 WTand adversely impacted the DNA binding, polymerase
and RNase H activities. Earlier, we have shown that p51
facilitates the loading of the p66 subunit on to the
template primer [ 14 ] . Therefore, the impaired polymerase
activity and template-primer binding affinity of HIV-1 RT
mutants carrying insertion in p51 may be due to their
inability to load the catalytic p66 (p66 INS) on the
template primer. These altered biophysical/enzymatic
properties of these insertion mutants may be attributed to
the reduced dimer stability.
Crystal structures of HIV-1 RT show that p66 and p51
assume different folding patterns and tertiary structures [
11 12 ] . It has been proposed that p66 in a monomeric form
exists in a closed conformation similar to p51 [ 26 ] .
Following dimerization with another molecule of p51 (or
p66), it assumes an open conformation [ 11 14 15 ] . The
polymerase domain of p51 is buried within its core. This
difference in tertiary structures between the two subunits
makes the dimer asymmetric. The amino acid residues at the
contact interface differ with respect to their position and
location in the 3-D structure. The counterpart of amino
acid residues of p66 located at the contact interface are
buried in the p51 folded conformation, whereas those of p51
are scattered in the p66 'open' conformation (Fig. 6).
Of the several domain interactions between p66 and p51,
the β7-β8 loop of p51 is strategically positioned to
interact with the residues on the floor of the palm
subdomain of p66. It has been suggested that the stability
of the dimer is related to the buried surface area between
the two subunits [ 11 12 ] . In the nevirapine-bound HIV-1
RT crystal structure, the total contact surface area
between the subunits is approximately ~4600 Å 2. The two
major contact regions between the subunits which provide it
stability are their connection subdomains and the thumb of
p51 and RNase H domain of p66. These contacts account for
approximately two third of the total buried surface area.
Interestingly, the marginal decrease in the total surface
area due to deletion of four residues in the β7-β8 loop
does not account for the dimer instability, thus suggesting
that polar interactions of residues in the β7-β8 loop of
p51 with the palm subdomain of p66 may play a role in
conferring stability to the heterodimer. The observation
that a single point mutation at L289 of p66, a residue not
in direct contact with p51, also destabilizes the dimer [
27 28 ] indicates that other factors may also contribute
towards dimer stability.
In order to analyze the impact of inserting the six
amino acid peptide in the β7-β8 loop of p51, we used the
molecular modeling approach. A search in the database of
known protein structures employing the 'loop-search'
algorithm of SYBYL yielded 100 loops, of which only five
were sterically permissible. The loop exhibiting the best
homology was incorporated in the modeled structure (Fig.
7). This loop of 9 amino acids (RFNAHGDVN) from the protein
S. lectin formed a short anti-parallel two strand β-sheet.
This inserted loop lies in the vicinity of the palm
subdomain of p66 and exhibits additional hydrophobic and
polar interactions with residues in the palm subdomain of
p66, not seen in the wild type structure. These additional
interactions are expected to enhance the stability of the
dimer. However, sedimentation analysis indicates that the
insertion mutants form unstable dimer. This implies that
the insertion may have altered the relative position of the
subdomains in the two subunits thereby perturbing the dimer
stability. In summary, the β7-β8 loop of p51 is an
important structural element involved in imparting
stability to the heterodimer and in opening the polymerase
cleft of p66 for catalysis.
Materials and methods
Plasmid and clones
The expression vector pET-28a and
E. coli expression strain BL21
(DE3) were obtained from Novagen. The HIV-1RT expression
clones (pKK223-3 RT66 and pET-28a-RT51) constructed in
this laboratory [ 29 30 31 ] were used for PCR
amplification and construction of the insertion mutants
in the p66 and p51 subunits of HIV-1 RT. An HIV-RNA
expression clone pHIV-PBS was a generous gift from Dr. M.
A. Wainberg [ 32 ] .
Insertion of 6 amino acid residues in the β7-β8
loop
The pKK-RT66 clone containing two unique restriction
sites,
Hpa1 and
Stu1 , at codons 136 and 140 in the
RT coding region [ 15 ] was used for insertion of 6
amino-acid residues in the β7-β8 loop of the p66 and p51
subunit. The pKK-RT66 clone was digested with
HpaI restriction enzyme to generate
a blunt end at codon 136. For insertion, two
complementary pre-kinased 18-mer synthetic DNA oligos
having the following sequences: 5'-ATA AAC AAT GAG ACA
ATA-3 (sense strand) and 3'-TAT TTG TTA CTC TGT TAT-5'
(antisense strand) were hybridized. The 18-mer duplex DNA
encoding the insertion peptide (Ile-Asn-Asn-Glu-Thr-Ile)
was ligated with
Hpa1 digested pKK-RT66 in between
codon 135 and 136. The positive clones were screened in
E. coli HB101 by the absence of an
Hpa1 site and the correct
orientation of the insertion was confirmed by DNA
sequencing. This construct expresses the p66+6aa subunit
without His tag sequences. A His-tag at the N-terminal of
the p66+6aa subunit was introduced by sub cloning the
Bal-I and
Hind III fragment of pKKRT66+6aa
into pET-28a-RT66 expression cassette. A unique
Sac I site was also introduced in
pKK-RT66 template at codon 440. The construction of
P51+6aa was carried out by removal of the 360 bp fragment
from pKK-RT66+6aa by restriction digestion with
SacI followed by re-ligation of the
vector ends. The insertion mutant in pET28a and pKK223-3
vectors were introduced into
E. coli BL-21 (DE3) pLys S and
E. coli JM109, respectively, for
expression. Induction of the enzyme protein was carried
out as described before for the wild type HIV-1RT [ 29 ]
. The enzyme with the hexahistidine-tag was purified from
bacterial lysates by immobilized metal affinity
chromatography [ 33 ] , while non-hexahistidine-tagged
enzyme was purified using the phosphocellulose and
Q-Sepharose columns as described previously [ 24 ] .
Preparation of the heterodimeric enzyme with
subunit specific insertion
The p51 subunit with a hexahistidine-tag and a
non-tagged p66 were used to generate the heterodimers
containing insertion in either or both of the subunits.
For each set of heterodimers, 260 μg of p51 was mixed
with 660 μg of p66 in the buffer containing 50 mM Tris
HCl, pH 7.8, 60 mM KCl and 5 mM MgCl
2 . The rationale for using a 1:3
ratio of p51 to p66 was to saturate the His-tagged p51
with the non-tagged p66, ensuring heterodimer formation
and eliminating excess p66 during IMAC purification. The
mixture was incubated for 16 hours at 4°C and applied to
(0.5 mL) Ni 2+iminodiacetic-Sepharose (IDA-Sepharose)
column, which was pre-equilibrated with the binding
buffer (20 mM Tris HCl pH 7.8, 500 mM NaCl and 5 mM
Imidazole). The column was washed with 15 mL of the same
buffer to remove the excess of p66 that was not dimerized
with p51 bound to the IDA-sepharose column. The
heterodimeric RT was then eluted from the column with
elution buffer (20 mM Tris HCl pH 7.8, 500 mM NaCl and
250 mM imidazole). Fractions of 0.5 mL were collected and
an aliquot of each fraction was analyzed by SDS-PAGE
using Coomassie Blue stain. The fractions containing
approximately equal band intensity of p66 and p51 were
dialyzed against a storage buffer (50 mM Tris HCl pH 7.0,
200 mM NaCl and 50% Glycerol) and this enzyme preparation
was used in all experiments.
Glycerol gradient ultra centrifugation
Fifty micrograms of the enzyme protein in 100 μL of
buffer (50 mM Tris HCl, pH 7.8, 1 mM DTT and 400 mM NaCl)
was carefully loaded onto 5 mL of 10-30% glycerol
gradients prepared in the same buffer. The gradients were
centrifuged at 48,000 rpm in an SW48 rotor for 22 h at
4°C. Fractions (200 μL) were collected from the bottom of
the tube and aliquots of these fractions were
electrophoresed using SDS PAGE and Coomassie Blue stain
to identify the protein peak.
The polymerase activity in the gradient fractions were
analyzed by extension of the labeled (dT)
18 annealed to poly (rA) template.
Every third fraction between 7 and 33 of the glycerol
gradient was diluted 10-fold and analyzed for its
polymerase activity. Reactions were carried out at 37°C
for 2 min at 20 μM dTTP concentration and quenched with
Sanger's gel loading dye [ 35 ] . The reaction products
were resolved by denaturing polyacrylamide-urea gel
electrophoresis and analyzed on a PhosphorImager
(Molecular Dynamics, Inc.).
DNA polymerase assay
Polymerase activity of the HIV-1RT WT and insertion
mutant enzymes was determined using two different
template-primers: U-5PBS HIV-1 RNA and synthetic 49-mer
U5-PBS DNA templates primed with the 17-mer PBS primer [
36 ] . Assays were carried out in a 50 μL volume
containing 50 mM Tris HCl, pH 7.8, 100 μg/mL bovine serum
albumin, 5 mM MgCl
2 , 1 mM dithiothreitol, 60 mM KCl,
100 nM template-primer, 50 μM of each of the four dNTPs
with one of them being 32P-labeled (0.1 μCi/nmol dNTP)
and 21 nM enzyme. Reactions were incubated at 37°C for 3
min and terminated by the addition of ice-cold 5%
trichloroacetic acid containing 5 mM inorganic
pyrophosphate. Following termination, the reaction
mixtures were filtered on Whatman GF/B filters. The
filters were then dried, immersed in scintillation fluid
and counted in a liquid scintillation counter.
Gel analysis of RNA and DNA dependent polymerase
activities
The U5-PBS HIV-1 RNA and heteropolymeric synthetic
U5-PBS HIV-1 DNA templates primed with the 17-mer PBS DNA
primer were used to assess the polymerase activities of
the wild type and mutant heterodimeric enzymes. The
primers were 5'-labeled using γ- 32P-ATP and T4
polynucleotide kinase according to the standard protocol
[ 37 ] . Polymerase reactions were carried out by
incubating 2.5 nM template primer with 50 nM of the wild
type HIV-1RT or its mutant derivative in a total reaction
volume of 6 μL containing 25 mM Tris-HCl, pH 7.5, 10 mM
dithiothreitol, 100 μg/mL bovine serum albumin, 5 mM MgCl
2 and 50 μM of each dNTP. Reactions
were initiated by the addition of enzyme and terminated
by the addition of an equal volume (6 μL) of Sanger's gel
loading dye [ 35 ] . The reaction products were resolved
by denaturing poly acrylamide-urea gel electrophoresis
and analyzed on a PhosphorImager (Molecular Dynamics,
Inc.).
Template-Primer (TP) binding affinity of the wild
type enzyme and its mutant derivatives
The dissociation constants (K
d ) of the E-TP binary complexes of
the wild type HIV-1 RT and its mutant derivatives were
determined as described by Tong et al. [ 25 ] . The
heteropolymeric 33-mer DNA (0.4 nM) annealed to 5'-
32P-labeled 21-mer primer (0.3 nM) was incubated with
varying concentrations of the wild type enzyme and its
mutant derivatives in a total volume of 10 μL containing
50 mM Tris-HCl, pH 7.8, 5 mM MgCl
2 and 0.01 % BSA. Following incubation
of the mixture for 10 min at 4°C, equal volume of 2×
gel-loading dye containing 0.25% bromophenol blue and 20%
glycerol was added. The E-TP binary complexes formed were
resolved at 4°C on 6% native polyacrylamide gel using
Tris-Borate buffer (85 mM Tris, 85 mM Boric acid, pH
8.0). The amounts of the TP in the binary complex (E-TP)
and in free form with respect to the varying
concentrations of the enzyme protein were determined by
PhosphorImager (Molecular Dynamics, Pharmacia) analysis
of the gel. The fraction of the bound DNA was plotted
against enzyme concentration and the K
d [DNA] value was determined as the RT
concentration at which 50% of DNA is bound.
Ternary complex formation assay
The ternary complex (E-DNA-dNTP) formation was
assessed by incubating the binary complexes of enzyme and
dideoxy terminated template primer in the presence of
next correct dNTP [ 25 ] . The binary complexes were
formed by incubating 10-50 nM of the wild type enzyme or
its mutant derivatives with 0.3 nM of 5'- 32P-labeled
dideoxy terminated 33-mer/21-mer template-primer as
described above. The chosen concentration of enzyme was
such that resulted in almost complete shift during E-TP
complex formation. The E-TP-dNTP ternary complex
formation was assessed by the addition of dNTP
complementary to the next template base (in this case
dGTP, 200 μM). Following incubation with dNTP at 4°C for
10 min, 300 fold molar excess of a DNA trap was added to
the incubation mixture to assess the stability of the
binary and ternary complexes formed by the enzyme. The
complexes were resolved on a 6% native polyacrylamide
followed by phosphorImaging. The extent of E-TP-dNTP
ternary complexes formed was quantified using ImageQuant
software.
RNase H activity assay
We used a 5'- 32P labeled 30-mer synthetic U5-PBS RNA
template annealed with a complementary 30-mer DNA to
determine the RNase H activity of the enzymes [ 31 ] .
The reaction mixture contained labeled RNA-DNA hybrid (10
K Cerenkov cpm), 50 mM Tris-HCl pH 8.0, 60 mM KCl, 10 mM
dithiothreitol, 0.1 mg/ml bovine serum albumin, 5 mM MgCl
2 , and 20 ng of enzyme in a final
volume of 5 μl. Reactions were carried out at 37°C for 30
sec and 1 min and terminated by the addition of equal
volume of Sanger's gel loading dye [ 35 ] . The cleavage
products were analyzed on an 8% denaturing
polyacrylamide-urea gel and scanned on a phosphorImager
(Molecular Dynamics Inc.).
