(Received for publication, October 30, 1996)
From the Sealy Center for Molecular Science, The
University of Texas Medical Branch, Galveston, Texas 77555-1071, the § Institute of Molecular Biology, University of Oregon,
Eugene, Oregon 97403-1229, and the ¶ Laboratory of Molecular
Genetics, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709
Details of the interactions between the human
immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA
were probed both by introducing site-specific and stereospecific
modifications into DNA and by altering the structure of potential
critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer
DNAs containing R and S enantiomers of styrene
oxide at N2-guanine were ligated with two additional
oligonucleotides to create 63-mers that served as templates for HIV-1
reverse transcriptase replication. Oligonucleotides that primed
synthesis 5 bases 3 to the adducts could be extended up to 1 base 3
and opposite the lesion. However, when the positions of the 3
-OH of
the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases
downstream of the styrene oxide guanine adducts, replication was
initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases
beyond the lesion. The sites of this adduct-induced termination
corresponded to the position of the DNA where
-helix H makes contact
with the DNA minor groove, 3-5 bases upstream of the growing 3
end. In addition, mutants of the polymerase in
-helix H (W266A and G262A)
alter the termination probabilities caused by these DNA adducts,
suggesting that
-helix H is a sensitive monitor of modifications in
the minor groove of newly synthesized template-primer DNA several bases
distal to the 3
-OH.
During the last 10 years, the use of site-specifically and
stereospecifically modified oligonucleotides has played a major role
not only in defining which DNA adducts are mutagenic but also in
instructing us how different DNA polymerases interact at those sites.
We have expanded these studies to use DNA lesions as a probe of
polymerase structure and function. The decision to use
HIV-11 RT as the polymerase of choice is
warranted not only because the cocrystal structure is known for an
intact enzyme/DNA complex (1) but also because of the tremendous
biological significance of RT. Previously using this strategy, we
demonstrated that the major groove DNA adducts, N6-adenine
SO lesions, were readily bypassed by wild type RT, only to experience
termination/pause sites 3-5 bases beyond the site of the lesion (2).
These sites of enhanced termination probabilities corresponded to the
adduct being positioned in the template-primer duplex near the thumb
subdomain of the polymerase, where the DNA is in close contact with an
-helix and is making the transition from A to B form DNA. Our
results could be rationalized because in this bend, the minor groove is
widened with a compensatory narrowing of the major groove. The presence
of an adduct in the major groove at this position was hypothesized to
be the likely source of the enhanced termination through alteration of
the minor groove
-helix H contacts, 3-5 bases upstream of the
3
-OH.
To further test whether DNA adducts can give insight into the structure
and functioning of HIV-1 RT, we have carried out a study in which
monocyclic, minor groove adducts known to cause no significant
perturbation to the local DNA structure (3) were used to probe
DNA-protein contacts. This study was designed to accomplish the
following: 1) determine the effects that these minor groove DNA lesions
might have on HIV-1 RT replication, 2) monitor the movement of DNA at a
distance from the catalytic site as it traverses through the
polymerase, and 3) identify key amino acid residues important for
adduct bypass when the adducts are positioned in the template-primer
duplex. To accomplish these objectives, we chose to build site-specific
and stereospecific SO N2-guanine minor groove adducts into
63-mer oligonucleotides (Fig. 1). The R
enantiomer of SO is oriented in the 3 direction relative to the
template strand such that it is pointing toward the 3
-OH of the
elongating primer (3). The S enantiomer has the opposite orientation such that it is pointing in the same direction as DNA
synthesis. Consequently, although both R and S
adducts report specific interactions within the minor groove, the two
enantiomers have nearly opposite spatial orientations. This
stereospecificity in the SO lesions offers an opportunity to probe
different polymerases-DNA contacts within the context of an
otherwise identical template-primer.
Also, throughout these studies, we used three forms of HIV-1 RT: the
wild type heterodimer (i.e. p66/p51) and two mutant
heterodimers, W266A and G262A, whose amino acid alterations are located
in -helix H of the thumb subdomain. Analysis of the cocrystal
structure suggests that this
-helix H makes contact with the minor
groove of the newly synthesized DNA as it undergoes a 45 ° bend and
transition from A to B form DNA (Fig. 2).
Alanine-scanning mutagenesis has revealed that these two residues make
important DNA contacts and that alanine substitution drastically
elevates koff and reduces processivity and
frameshift fidelity (4, 5). In contrast, alanine-scanning mutagenesis
of
-helix I, which sits over the template phosphate backbone (Fig.
2), has suggested that these residues do not make critical DNA contacts
(6).
Wild type HIV-1 RT and mutants were prepared as described previously (4).
Preparation of the SO-adducted TemplatesThe 11-mer
oligonucleotides adducted with R and S
enantiomers of styrene oxide at the second position of codon 12 (N2-guanine) of N-ras were synthesized,
purified, and supplied by Thomas and Connie Harris (Vanderbilt
University) as described previously (7). Two 63-mer templates were
constructed by enzymatic ligation (Fig. 1). The SO-adducted 11-mer was
ligated to a 20-mer at the 3 termini and to a 32-mer oligomer at the
5
termini, in the presence of a 46-mer scaffold. A nonadducted,
control 63-mer was also synthesized. Purification of the 63-mers was
performed on a 12% denaturing polyacrylamide gel. The sequence of the
63-mer template was as follows, in which the underlined G is the site of adduction:
5
-GAATGTGCAAGATACTGTGGGCAGTGGTGAATGGTCTGGGCAATGTCGTGACTGGGAAAAC-3
.
The oligonucleotide primers,
ranging in length from 17- to 21-mers, were 5 end-labeled with
[
-32P]ATP (6000 Ci/mmol, DuPont NEN) using T4 DNA
kinase (New England Biolabs, Beverly, MA). A 12-fold excess of template
over the labeled primer was annealed by heating the template-primer mix
in the polymerization reaction buffer (33 mM Tris-OAc pH
7.5, 66 mM KOAc, 10 mM MgOAc) for 2 min at
65 °C, followed by slow cooling to <35 °C. Native polyacrylamide
gels (10%) were run to confirm that greater than 90% of the primers
were annealed to their respective templates. Deoxynucleotides were
added to the template-primer mix to a final concentration of 500 µM in a reaction buffer, (33 mM Tris-OAc pH
7.5 at 37 °C, 66 mM KOAc, 10 mM MgOAc, 0.2 mg/ml bovine serum albumin, 1 mM dithiothreitol). Wild type
HIV-1 RT and the mutant polymerases were added to the reaction mix (10 µl of total volume) at concentrations unique for each enzyme. All
reactions were carried out at 37 °C. Extensions were terminated by
the addition of 5 µl of stop buffer (95% (v/v) formamide, 20 mM EDTA, 0.05% (w/v) bromphenol blue, 0.05% (w/v) xylene
cyanol)/2.5 µl of reaction mixture. DNAs were separated through 15%
polyacrylamide-8 M urea gels and were run at 2000 V for
3 h. The relative intensities of the termination sites were
determined by PhosphorImager technologies.
Using unadducted R and S
styrene oxide-adducted 63-mers as templates, HIV-1 RT replication was
initiated on the primers shown in Fig. 3. The primers
are referred to by the position of the 3-OH relative to the adduct
site in the complementary strand, such that the
5 primer initiates
synthesis five nucleotides 3
to the adduct site, the 0 primer is
located opposite the lesion, and all the positively numbered primers
contain the adducted base within the template-primer duplex.
Under replication conditions defining single hit encounters
(i.e. conditions leaving most of the primer unextended, 20%
utilization) between the polymerase and the primer, wild type HIV-1 RT
was readily able to extend the 5 primer (Fig. 4) on
unadducted templates to full-length products (Fig. 4, lane
1), whereas under these experimental conditions, there was no
evidence that either the R- or S-SO lesions could
be bypassed (Fig. 4, lanes 2 and 3,
respectively). Addition of greater amounts of the polymerases revealed
that all primers could be extended consistent with the ratio of
template and primer being 10:1. The major pause site occurred 1 base
prior to the lesion, with only slight incorporation opposite the
lesion. However, using vast excesses of enzyme under multiple hit
conditions allowed for modest levels of bypass of these lesions (8).
The reduced processivity of the W266A and G262A mutants replicating unadducted templates is apparent by numerous pause sites at positions less than full-length DNAs (Fig. 4, lanes 4 and
7, respectively). These pause sites were generally in runs
of template purines, and both of these mutants are sensitive to
premature termination at the same sites. Similar to the wild type
enzyme, under these experimental conditions, neither the W266A or G262A
were able to replicate past the R or S adduct
site but terminated predominantly 1 base 3
to the lesion (Fig. 4,
lanes 5 and 6 and lanes 8 and 9, respectively). No major differences were noted in the
extent or site of termination between the oppositely oriented
R- or S-SO enantiomers.
Minor Groove Adducts Result in Strong Termination 4-7 Bases beyond the Adduct Site When Primers Are Positioned 0 to +2
Because both
the R and S enantiomers of SO blocked replication
3 and opposite the lesion, this result prevented an analysis of the
effects that these minor groove adducts might have as they translocate
through the HIV-1 RT. To get around this problem, we designed
oligonucleotide primers 0 to +2 (Figs. 3 and 4) to "mimic"
synthesis opposite and beyond the template lesion. Lanes 1-9 of Fig. 5 show the results of replication
reactions using reaction conditions consistent with multiple hit
conditions with wild type enzyme on unadducted (U) and
adducted templates (R-SO (R) and S-SO
(S)), which were primed with +2, +1, and 0, respectively. For comparison purposes, single-hit conditions for the unadducted templates are shown in Fig. 4 and indicate processive synthesis by wild
type enzyme. Because all of these primers are 17-mers, the length of
the full-length products on the unadducted templates increases by 1 base each (Fig. 5, lanes 1, 4, and 7).
In strong contrast to the data presented in Fig. 4, the replication
beyond the R enantiomer shows dramatic termination at
positions 4, 5, 6, and 7 beyond the lesion (Fig. 5, lanes 2,
5, and 8 for the +2, +1, and 0 primers,
respectively). Under the reaction conditions used in these experiments,
it is of interest to note that the R-SO-containing template
could be readily extended using the 0 primer (Fig. 5, lane
9), whereas no synthesis on the S-SO-containing template was observed (Fig. 5, lane 8). It is also of
interest to note that the insertion of the seventh base beyond the
adduct always results in a very diffuse band, suggesting that
different nucleotides can be incorporated at that site (Fig. 5,
lanes 2, 5, and 8).
Qualitatively similar results were obtained for extension of primers
using S-SO-containing templates. However, the following significant differences were observed. Using the +2 and +1 primers, termination occurred primarily at 5 and 6 bases 3 to the lesion with
minor termination at the 4th base position. Additionally, no extension
was observed 7 bases beyond the lesion as was seen with the
R enantiomer, and the diffuse band was located 6 bases beyond the lesion rather than 7 bases beyond the lesion.
The identical series of experiments was carried out using alanine mutants of HIV-1 RT, W266A (Fig. 5, lanes 10-18) and G262A (Fig. 5, lanes 19-27). Using the W266A mutant to replicate beyond the R-SO enantiomer, significant differences were observed relative to the wild type enzyme. Strong termination was observed only at 4, 5, and 6 bases beyond the lesion and the extent of primer utilization decreased significantly from the +2 to 0 primer, such that no synthesis could be observed using the 0 primer. Even more dramatic effects were observed with the templates containing the S enantiomer. Replication was observed only with the +2 primer with termination occurring 4, 5, and 6 bases beyond the lesion. With both the R and S enantiomers, a diffuse band was observed 6 bases beyond the lesion, indicating that replication was being differentially modulated by the alanine substitution at position 266.
With the G262A mutant polymerase, termination sites were again
qualitatively similar to W266A in that only the +2 primer was effectively utilized (Fig. 5, lanes 20, 21, and
23). No replication was ever observed beyond 6 bases past
the lesion, and primer utilization decreased dramatically because
the 3-OH was positioned near the lesion (Fig. 5, lanes 24,
26, and 27).
The data
presented in Fig. 6 show the results that were obtained
when primers were positioned at sites 3, 4, 5, and 6 bases beyond the
unadducted site (U) or the R enantiomer
(R) with the wild type enzyme. The 5 ends of these primers
were held constant relative to the +2 primer, and thus their lengths
increased from 18 to 21 bases. The amount of enzyme and time of
reaction were sufficient for the wild type polymerase to completely
utilize all primers on unadducted templates and resulted in full-length products. However, using the identical reaction conditions, the +3 to
+6 primers on the adducted templates were extended very poorly (Fig. 6,
lanes 2, 4, 6, and 8,
respectively). In most cases, a single base addition represents >95%
of the synthesis that occurred. This result suggests that the
polymerase is able to load onto the template-primer duplex but that
translocation relative to the DNA did not occur. The exception to this
trend was obtained using the +3 primer, in which synthesis was very
poor overall, but when it was initiated, the termination sites were at
4, 5, 6, and 7 bases beyond the lesion (Fig. 6, lane 2), a
result consistent with data obtained with the 0 to +2 primers.
Mutations in
In the previous section, it was demonstrated
that wild type HIV-1 RT only very poorly utilized primers in which a
minor groove adduct was positioned within the template-primer stem 3-5
bases from the 3-OH. To ascertain whether mutations in the
-helix that contact this bend might affect replication, we utilized the +3 and
+4 primers on unadducted and R-SO enantiomer-containing templates, comparing wild type (1-h primer extension) (Fig.
7, lanes 1 and 2, +3 primer;
lanes 3 and 4, +4 primer), W266A (5-min primer
extension) (lanes 5 and 6, +3 primer; lanes
7 and 8, +4 primer), and W266A (1 h primer extension)
(lanes 9 and 10, +3 primer; lanes 11 and 12 +4 primer). Under the multiple hit conditions used
here, all unadducted templates were extended to completion with both
sets of primers. In contrast to what was observed using the wild type
enzyme, the +3 primer supported significant DNA synthesis at 5 min and
1 h by W266A (Fig. 7, lanes 6 and 8,
respectively). Termination occurred at 4, 5, and 6 bases downstream of
the lesion. Although extension of the +4 primer by W266A in 5 min was
equal to the wild type after 1 h (Fig. 7, lanes 4 and
8, respectively), the W266A mutant extended this primer
efficiently after 1 h (lane 12).
The overall processivity of the HIV-1 RT can be modulated by a
number of factors including both the structure of the polymerase (4, 6)
and the sequences that are being actively replicated (9) or those that
are located in the template-primer stem (10, 11). More specifically, it
has been established that many of the pause sites often occur in
homopolymeric runs and synthesis through these sites can lead to
frameshift and base substitutions through a template-primer slippage
intermediate (9-11). Although the fidelity of these reactions was
originally expected to be associated with the templating base at the
catalytic site, these studies suggested that single nucleotide
differences in the template-primer double-stranded region, which were
as far away as 6 nucleotides, could influence fidelity (10). The x-ray
crystallographic structure of a RT-DNA complex suggests that -helix
H contacts the minor groove of the template-primer DNA ~4-6 base
pairs from the catalytic site. This "action at a distance" has been
previously observed as HIV-1 RT replicated past DNA major groove
site-specific and stereospecific styrene oxide lesions on
N6 adenines (2, 8). In these experiments, it was shown that termination probabilities were enhanced up to 6-fold at positions 3-5
base pairs beyond the site of the lesion. These data suggested that the
presence of the lesions in the major groove modulated important
polymerase contacts with the DNA.
To gain understanding of the effects that specific minor groove base
alterations might have on the polymerase processivity, we chose to
monitor the effects that monocyclic adducts might have on termination
and the previously described action at a distance. The data presented
in Fig. 5 dramatically display the strong termination of wild type
replication when the R and S adducts are
positioned 4-7 and 4-6 base pairs downstream of the catalytic site,
respectively. It is also obvious that these lesions affect the ability
of the various primers to be used effectively for replication such that initiation of synthesis from the +2 primer was greater than the amount
of replication observed with the +1 primer and decreased further with
the 0 primer. This decreased primer utilization was further enhanced
when either of the mutant forms of the polymerase (i.e.
G262A and W266A) were used. There was no synthesis using the
S-SO enantiomer template with either the +1 or 0 primer or the R-SO enantiomer-containing template with the 0 primer.
These data suggest that alanine substitution perturbs important DNA contacts within the region where the bend in the DNA is observed to
occur. The pertubations are probably residue-specific, because in one
case the alanine substitution is increasing the size of the side chain
(i.e. G262A) and in the other case dramatically reducing it
(i.e. W266A). The exact identity of these two side chains is
probably crucial because these residues are highly conserved within the
retroviral reverse transcriptases (12). Thus, one of the roles of
-helix H may be to monitor the integrity of the newly replicated DNA
by facilitating a conformational change in the DNA such that unmodified
DNAs are sufficiently malleable to allow progression of processive
synthesis, whereas modified DNAs are conformationally constrained to
cause pausing and replication termination. For this model and
consistent with our data, the presence of monocyclic minor groove
adducts near or at the catalytic site are insufficient distortions to
completely block the initiation of replication, but adducts near the
active site, coupled with
-helix mutations, are sufficient to
effectively prevent either initiation or elongation.
The question arises whether specific contacts between specific residues
in -helix H and the various SO lesions can be inferred from these
data. Careful inspection of the relative termination probabilities from
the utilization of the +2 primer give insight into this issue. The
percentage of termination for positions 4-7 were calculated for both
the R- and S-SO templates for each of the
polymerases used (Table I). The residue that gives the
greatest differential change is Trp266, which when changed
to alanine caused a 2-5 fold enhanced probability to terminate
synthesis 4 base pairs beyond the catalytic site. In contrast the G262A
mutant only displayed moderate differences in termination probabilities
with the R-SO lesion, and those effects were manifested
primarily 5 base pairs downstream of the catalytic site. These results
are similar to the effect these mutations have on frameshift fidelity
where the G262A mutant enhances slippage errors in longer homopolymeric
runs than W266A (6).
|
The NMR solution structures have been previously solved for both of the
11-mer SO-containing oligonucleotides in these reactions (3). The
relative orientations of the R and S SO
enantiomers on the template DNA were shown to be 3 and 5
,
respectively (3). This nonintercalative groove binding was solved on
duplex DNA and thus in the majority of this study is likely to
accurately represent the structure of the DNA prior to polymerase
binding, using the +1 to +6 primers. The orientation of the adducts had their most prominent effect on the wild type enzyme in which the 3
-oriented R-SO enantiomer allowed a further progression of
the polymerase up to 7 bases beyond the lesion, whereas the 5
-oriented S-SO enantiomer blocked synthesis consistently 1 base
shorter (Fig. 4, lanes 1-9). This orientation difference is
somewhat surprising because the distances between the lesion and the
terminally incorporated nucleoside were significantly different for the
two lesions. For example, the distance between the R-SO and
the terminal nucleoside spans ~8 bases, whereas this distance is
predicted to be only 5 bases on the S-SO lesion. Resolution
of this unexpected finding will require the solving of the co-crystal
structure of HIV-1 RT with each of these adduct-containing DNAs or
solving the NMR solution structure of the complexes.
Finally, we have consistently noted that the S-SO-containing templates support lower levels of overall synthesis relative to the R-SO or unadducted templates. Preliminary studies suggest that the kon rate is roughly equivalent but that the koff is distinctly different when the S-SO adduct is in the template-primer stem.2 This suggests that these adducted oligonucleotides could potentially be used as competitive inhibitors of the action of HIV-1 RT.
Thus, overall we conclude that it is possible that replication of a lesion-containing DNA can be used in conjunction with wild type and mutant polymerases to not only gain insight into the parameters that define the molecular interactions between the two macromolecules but also to understand the structure-function relationships of the HIV-1 RT.
Special thanks go to Judy Daniels for the preparation of this manuscript and to Dr. Amanda McCullough for preparation of the figures.