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J. Biol. Chem., Vol. 278, Issue 29, 26526-26532, July 18, 2003
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Nonpolar Thymine Isosteres in the Ty3 Polypurine Tract DNA Template Modulate Processing and Provide a Model for Its Recognition by Ty3 Reverse Transcriptase*
From the Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702-1201
Received for publication, March 7, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (–)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T
F substitution and enhanced ribonuclease H (RNase
H) activity 
12–13 bp downstream. In the most pronounced case, dual
T F substitution at PPT positions –1/–2 redirects RNase H
cleavage almost exclusively to the novel site. The structural features of this
unusual base suggest that its insertion into the Ty3 PPT (–)-DNA
template weakens the duplex, inducing a destabilization that is recognized by
a structural element of Ty3 RT 12–13 bp from its RNase H catalytic
center. A likely candidate for this interaction is the thumb subdomain, whose
minor groove binding tract most likely contacts the duplex. The spatial
relationship derived from T F substitution also infers that Ty3 PPT
processing requires recognition of sequences in its immediate 5'
vicinity, thereby locating the RNase H catalytic center over the PPT-U3
junction, a notion strengthened by additional mutagenesis studies of this
paper. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although reverse transcriptase (RT)1-associated ribonuclease H (RNase H) activity degrades RNA of the RNA/DNA replication intermediate with little sequence specificity, it must precisely remove the tRNA and polypurine tract (PPT) primers of (–)-strand (1) and (+)-strand DNA synthesis (2), respectively, to generate sequences at the 5' and 3' termini of the double-stranded DNA recognized by the integration machinery (3–8). Since the PPT is most likely embedded in a considerably larger RNA/DNA hybrid, precise hydrolysis at the PPT-U3 junction observed in vitro (9) suggests unique structural features may participate by correctly positioning this junction in the RNase H catalytic center. Our recent chemical footprinting of HIV-1 PPT-containing RNA/DNA hybrids (10) and a comparison with the crystal structure of HIV-1 RT bound to a related duplex (11) support this notion. Nucleic acid in the RT-RNA/DNA co-crystal is distorted 8–14 bp upstream of the PPT-U3 junction, comprising weakly paired, unpaired, and mispaired bases (Fig. 1A). Subsequent chemical footprinting studies (10) revealed that template thymines of this region and thymine +1 (i.e. immediately 3' to the PPT) deviate from standard Watson-Crick base pairing in the absence of the retroviral enzyme. The finding that these naturally occurring HIV-1 PPT distortions are 10–14 bp apart was particularly intriguing, since this approximates the distance between the thumb subdomain and RNase H catalytic center of the heterodimer-associated p66 subunit (11–14). Therefore, it is possible that the A-tract-induced HIV-1 PPT distortion plays a role in sequestering RT via an interaction with structural elements at the base of the p66 thumb, thus positioning the RNase H catalytic center over the PPT-U3 junction for correct processing.
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To examine whether this hypothesis may account for the precision of PPT
processing in related long terminal repeat-containing elements, we focused
here on the Saccharomyces cerevisiae retrotransposon Ty3. Differences
in the primary structure of the HIV-1 and Ty3 RTs (the former is a p66/p51
heterodimer, whereas Ty3 RT is a 55-kDa monomer) as well as in the sequence of
their PPTs make the comparison between these two systems very interesting. Our
preliminary analysis of Ty3 RT has indicated that its DNA polymerase and RNase
H catalytic centers are separated by 21 bp
(15) rather than the 17 bp
observed in HIV-1 RT, suggesting a different spatial arrangement of thumb
subdomain and RNase H catalytic center. Moreover, the Ty3 PPT differs in both
size (12 bp) and sequence (Fig.
1B) from the HIV-1 counterpart. Whereas Ty3 RT processes
its PPT with the appropriate precision in vitro
(15,
16), we show here that it
fails to remove (+)-DNA and imprecisely processes (+)-RNA 3' to the
HIV-1 PPT, suggesting co-evolution of enzyme and substrate.
RNA/DNA hybrids, whose (–)-DNA template was both individually and
dually substituted with 2,4-difluoro-5-methylbenzene (F) for thymine, were
used here to investigate structural features of the Ty3 PPT, mediating its
recognition and processing. 2,4-Difluoro-5-methylbenzene is isosteric with
thymine but has severely reduced hydrogen bonding capacity
(17). Thus, F is a
particularly useful tool to study the role of hydrogen bonding and base
structure and has been extensively used to evaluate the fidelity of DNA
synthesis
(18–21).
However, to date there have been no studies on the recognition of
F-substituted RNA/DNA hybrids. In the present study, T F substitutions
were designed to introduce flexibility and, possibly, structural changes at
different positions of Ty3 PPT-containing RNA/DNA hybrids, without changing
the sequence of the primer. We show that subtle alterations to the structure
of the Ty3 PPT (+)-RNA/(–)-DNA hybrid reposition the RNase H domain,
inducing a novel but highly specific cleavage within the U3 region, 12 nt
downstream from the site of F insertion. This suggests that correct processing
of the (+)-strand primer may proceed through interaction of a structural
subdomain of Ty3 RT with sequences immediately 5' to the PPT and
–12 bp from the PPT-U3 junction.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—Ty3 RT was expressed and purified as described (16). Unsubstituted DNA oligonucleotides were obtained from Integrated DNA Technologies (Gaithersburg, MD). 2,4-Difluoro-5-methylbenzene was purchased as the phosphoramidite from Glen Research. The DNA oligonucleotides containing single and pairwise F substitutions were synthesized using the Expedite 8909 automated synthesizer (PerkinElmer Life Sciences). RNA oligonucleotides were purchased from Dharmacon (Boulder, CO). All other reagents were of the highest purity and purchased from Sigma.
PPT Selection—To evaluate HIV-1 PPT selection a 55-nt, (–)-strand DNA template (corresponding to nucleotides 9048–9103 of the HIV-1HXB2 genome) was hybridized to a 30-nt, 5'-end-labeled PPT-containing RNA extended by 10 ribonucleotides (HIV-1/PPT-R) or deoxyribonucleotides (HIV-1/PPT-D) beyond the authentic RNase H cleavage site (Fig. 2A). The primer/template mixture was annealed by heating to 90 °C and slow cooling in 10 mM Tris/HCl (pH 7.6), 25 mM NaCl. A reaction mixture containing 50 nM template-primer was prepared in 10 mM Tris/HCl (pH 7.8), 9 mM MgCl2, 80 mM NaCl, 5 mM dithiothreitol. Hydrolysis was initiated by the addition of enzyme to a final concentration of 150 nM in a volume of 80 µl. The reaction mixture was incubated at 37 °C. Ten-µl aliquots were removed at times indicated and mixed with an equal volume of 89 mM Tris borate, pH 8.3, 2 mM EDTA, and 95% (v/v) formamide containing 0.1% (w/v) bromphenol blue and xylene cyanol. Polymerization products were resolved by high voltage denaturing 15% polyacrylamide gel electrophoresis and visualized by phosphor imaging. Quantification was performed using Quantity One software (Bio-Rad).
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For Ty3 PPT selection, a 46-nt, (–)-strand, DNA template (corresponding to nucleotides 4780–4809 of the Ty3 genome) was hybridized to a 29-nt PPT-containing RNA; the 13 nt 3' to the authentic cleavage site were either ribonucleotide (Ty3 PPT-R) or deoxyribonucleotide (Ty3 PPT-D; Fig. 1B). The mixture was annealed as described above, and a reaction mixture containing 50 nM template-primer was prepared in 25 mM Tris/HCl (pH 7.8), 9 mM MgCl2, 80 mM NaCl, 5 mM dithiothreitol. Hydrolysis was initiated by the addition of RT to a final concentration of 150 nM in an 80-µl volume. The reaction mixture was incubated at 30 °C. Ten-µl aliquots were removed at the times indicated and processed as above.
PPT Selection with 2,4-Difluoro-5-methylbenzene-substituted Substrates—Ty3 PPT-R RNA primer (Fig. 3A) was hybridized to 46-nt, (–)-strand DNA templates harboring single or dual F substitutions, as indicated. Conditions for template-primer annealing, PPT processing, and sample evaluation were as described above. For all experiments evaluating PPT selection, the hydrolysis products were processed as described under "PPT Selection."
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Modification of RNA/DNA Heteroduplexes with
KMnO4—KMnO4 sensitivity of
Ty3 PPT templates harboring a single +2, or dual –1/–2,
–5/–7, or –9/–11 T F substitution was evaluated
by a modification of the protocol of Kvaratskhelia et al.
(10). RNA/DNA hybrids were
incubated at room temperature for 5 min in 20 mM Tris-HCl, pH 8.0,
100 mM NaCl, and 100 µM MgCl2. The total
reaction volume was 20 µl. Reactions were initiated by adding 2 µl of
freshly prepared 25 mM KMnO4 solution and terminated
after 30 s with 2 µl of 14 M 
-mercaptoethanol. After
ethanol precipitation, samples were treated with 100 µl of 1 M
piperidine for 30 min at 90 °C. Piperidine was removed by vacuum
desiccation. Nucleic acids were washed three times with 50 µl of water and
vacuum-dried after each resuspension. Samples were finally resuspended in 89
mM Tris borate, pH 8.3, 2 mM EDTA, and 95% formamide
containing 0.1% bromphenol blue and xylene cyanol and analyzed by
electrophoresis through 15% denaturing polyacrylamide gels. Modification
products were visualized by phosphor imaging.
Circular Dichroism Spectra and Thermal Melting
Profiles—Equimolar amounts (25 µM) of RNA primer
were hybridized to +2, –1/–2, –5/–7, and
–9/–11 F-substituted 46-nt, (–)-strand PPT-containing DNA
templates by heating to 90 °C and slow cooling in degassed 10
mM Na2HPO4/NaH2PO4, pH
7.0, 80 mM NaCl. Circular dichroism spectra were recorded at 30
°C with an AVIV 202 spectrophotometer using a 1-mm path length cuvette.
Correction for each spectrum was against the respective buffer-only spectrum.
Nucleic acid duplexes were scanned from 190 to 300 nm. For measurement of
melting temperatures (Tm), 10 µg/ml solutions
of the same substrates were analyzed in a Beckman DU 640 spectrophotometer.
E260 was measured at 0.2 °C intervals from 30 to 80
°C. The Tm of each hybrid was calculated by
the "first derivative" method described by the manufacturer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Altered Processing of the HIV-1 PPT by Ty3 RT—A clear difference between the PPTs of Ty3 and more extensively studied retroviruses is the presence of contiguous (rA:dT) and (rG: dC) tracts in the latter, which might provide a structural basis for recognition. This difference prompted us to investigate the manner in which Ty3 RT processes its cognate PPT and that of HIV-1, for which a considerable body of literature is available (5, 7–9, 22–24). Fig. 1C indicated that the Ty3 PPT is accurately released from (+) U3 RNA or DNA by Ty3 RT. We next evaluated HIV-1 PPTs extended at the 3' terminus by either (+)-RNA or (+)-DNA (HIV-1 PPT-R or HIV-1 PPT-D, respectively. Fig. 2A). The hydrolysis pattern obtained with HIV-1 RT on a duplex extended with (+)-RNA (Fig. 2B, i) is similar to the one that we (9) and others (7, 8, 22) have reported, namely preferential cleavage at the PPT-U3 RNA junction and minor cleavage on either side (we define positions –1 and +1 as the bases on each side of the processing site). However, Ty3 RT cleaved this HIV-1 substrate with significantly altered specificity. Hydrolysis occurred at three positions of the adjacent RNA/DNA hybrid, centered around +2, whereas the correct site was virtually uncleaved (Fig. 2B, ii). Because RNase H cleaves RNA at the PPT-(+)-DNA junction, we examined hydrolysis of an HIV-1 PPT extended by (+)-DNA at its 3' terminus. In this case, we can only observe cleavage at the PPT-U3 DNA junction, if specific cleavage occurs, or within the PPT itself. The data of Fig. 2B, iii, again show minimal hydrolysis at the PPT-U3 DNA junction. In Fig. 2C, dNTPs were included to examine hydrolysis of the HIV-1 PPT-(+) DNA duplex in the context of DNA synthesis. The results indicated that, despite efficient polymerization from the HIV-1 PPT-D substrate, the removal of U3 DNA was again impaired, even after a 1-h incubation with Ty3 RT, eliminating the possibility that structural features of the HIV-1 substrate prevented binding of Ty3 RT. Following DNA synthesis, the 3'-OH of the extended primer is located 20 bp from the PPT-U3 junction, which would be ideally situated for Ty3 RNase H-mediated hydrolysis (Ty3 RNase H cleaves RNA substrates around 11 and 21 nt from the extremity that directs binding (15)). Similarly, the 5'-end of the HIV-1 PPT-R does not direct hydrolysis, since the same cleavage pattern was observed using a substrate extended by 15 nt at the 5'-end of the PPT. Therefore, altered processing of HIV-1-PPT-R (Fig. 2B, ii) and lack of cleavage of HIV-1-PPT-D by Ty3 RT (Fig. 2B, iii) must reflect recognition of a structural feature assumed by the HIV-1 PPT. In a similar experiment, Ty3 PPT variants were completely and nonspecifically hydrolyzed by HIV-1 RT (data not shown).
Dual T F Substitutions of the Ty3 PPT DNA Template
Modulate Cleavage Specificity—The results of
Fig. 2 suggest that structural
features of the Ty3 PPT may contribute to the specificity of processing.
Therefore, we used the base analog 2,4-difluoro-5-methylbenzene (F;
Fig. 3A), which is
isosteric with thymine but fails to hydrogen-bond with adenine
(25). F was substituted for
several thymines of the DNA template complementary to the PPT
(3'-C-T-C-T-C-T-C-T-C-C-T-T-5'). Such a strategy subtly alters the
stability of the PPT-containing heteroduplex, at the site of substitution, and
allowed us to determine the impact on both the structure of the duplex and
cleavage specificity.
Initially, a series of doubly F-substituted Ty3 PPT RNA/DNA hybrids
(Fig. 3A) was examined
to determine whether localized destabilization of the nucleic acid duplex
affected either the kinetics or specificity of processing. Indeed, adjacent F
insertion at template positions –1/–2 had a profound effect,
redirecting the RNase H catalytic center primarily over position +10/+11 of
the non-PPT RNA/DNA hybrid (Fig.
3C, ii). This repositioning of Ty3 RT was also
observed with substrates containing dual –5/–7
(Fig. 3C,
iii) or –9/–11 substitutions
(Fig. 3C,
iv), which enhance cleavage at positions +6 and +3 of the RNA/DNA hybrid,
respectively. Although less dramatic than the effect observed by a
–1/–2 substitution, phosphor imaging and quantification
(Fig. 4D) indicated
that F-induced +6 and +3 cleavage is equivalent to or exceeds that at the
PPT-U3 junction (Fig.
4C, i). Therefore, cleavage at the PPT-U3
junction is affected differently by adjacent or interrupted T F
substitutions. Adjacent substitutions create a more pronounced local
destabilization that will sequester the majority of the enzyme at the new
recognition site. Alternatively, the distortion induced by the presence of two
adjacent T F substitutions might render the PPT-U3 junction
uncleavable. Notably, the combined data of
Fig. 4, B and
C, also show a constant spatial correlation of
12–13 bp between the site of F insertion and that of enhanced RNase H
activity.
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Characterization of F-substituted Ty3 PPT RNA/DNA
Hybrids—Three independent experiments were performed to evaluate if
F insertion affected the Ty3 PPT structure
(Fig. 4). Since the lack of
hydrogen bonding has been correlated with a substantial drop in the
Tm of shorter nucleic acid duplexes
(18,
20), we determined the melting
temperature of the F-substituted RNA/DNA hybrids. Wild type Ty3 RNA/DNA hybrid
had a Tm of 69 °C
(Fig. 4A). A single T
F substitution at position +2 (i.e. outside the PPT-containing
duplex) reduced this to 65.6 °C, whereas that of substrates harboring dual
substitutions varied from 63.0 to 60.5 °C. Thus, whereas T F
substitutions had the expected consequences on decreasing duplex stability,
the Tm of all RNA/DNA hybrids was considerably
higher than the temperature at which PPT processing was evaluated (30 °C).
Fig. 4B compares the
CD spectrum of each doubly substituted RNA/DNA hybrid to the wild type. The
spectra were in good agreement with published data on polypurine-containing
RNA/DNA hybrids, which assume an intermediate configuration between A-like and
B-like (8,
26,
27). Although minor
differences were noted in the peak and trough heights at 277 and 210 nm,
respectively, mutant substrates differed minimally from the wild type PPT.
Finally, in Fig. 4C,
we examined the sensitivity of template thymines to chemical modification by
KMnO4 following T F substitution. Previously, we
successfully applied this strategy to the HIV-1 PPT, illustrating that
template thymines +1 and –10 to –15 adopted a distorted structure
(10). In contrast, very little
KMnO4 sensitivity is observed in the wild type Ty3 PPT (lane
C), suggesting the absence of preexisting structural perturbations.
However, the possibility that these might be induced following enzyme binding
could not be excluded. Furthermore, the single +2 F
(Fig. 4C, lane
1) or dual –1/–2, –5/–7, and –9/–11 T
F substitutions (Fig.
4C, lanes 2–4, respectively) were
accommodated without altering the structure of neighboring A:T base pairs.
Since F is insensitive to KMnO4 oxidation, this prohibited any
direct evaluation on the structure of the dF:rA pair. The combined data of
Fig. 4 therefore provide a
strong argument that T F substitution within or adjacent to the Ty3 PPT
is not accompanied by global changes in structure but rather a subtle and
localized alteration in hydrogen bonding.
Single T F Substitutions of the Ty3 PPT
RNA/DNA Hybrid—To conduct a more detailed analysis of Ty3
PPT architecture, a second series of RNA/DNA hybrids was prepared containing
single T F substitutions from positions –1to –11 of the
(–) DNA template (Fig.
5A). In each case, processing at the PPT-U3 RNA junction
was preserved (Fig.
5B) but was accompanied by an alteration in specificity
that again directed cleavage 12 bp downstream. Introducing F as template
nucleotide –1 enhanced cleavage at position +10, and to a lesser extent
at position +11 (Fig. 5, B and
C, ii). This substitution also decreased
cleavage at the PPT-U3 RNA junction and increased cleavage at position +2 and
+3 (Fig. 5, B and
C, compare i with ii). A –2
substitution likewise resulted in significantly increased cleavage at position
+10 without affecting the physiological cleavage site. These results suggest
that Ty3 RT partitions between the correct recognition site and a second
artificially induced as a consequence of F insertion. A –1 F
substitution also promoted increased cleavage at several positions downstream
from the junction. (Fig.
5B, ii). The extent of +10 cleavage in the case
of both monosubstituted substrates exceeded that at the authentic junction,
and was comparable with what was observed with a dual –1/–2
substitution. Introducing T F substitutions at the 5'-end of the
(–)-DNA template (positions –9 and –11) had a similar
effect, inducing RNase H activity 12–13 bp from the site of
insertion (Fig.
5B, iv and v). Although –5 and
–7 T F substitutions yielded a similar result, the alteration in
cleavage specificity was not as pronounced
(Fig. 5C, iv
and v). These data provide additional support that T F
substitution of the PPT (–) DNA template induces a subtle alteration in
duplex architecture and promotes the interaction with a structural component
of Ty3 RT 12–13 bp from the RNase H active site. Moreover, the gradual
increase in RNase H activity on the non-PPT RNA/DNA hybrid as the position of
F substitution approaches the 3'-end of the (–)-DNA template
suggests that regions immediately upstream of the PPT may work in concert with
the F-induced destabilization.
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Altering Sequences 5' of the Ty3 PPT Affects
Processing— Taken together, the T F substitution data of
Figs. 3 and
5 imply that this
non-hydrogen-bonding thymine isostere introduces flexibility and possibly
localized structural changes in the RNA/DNA hybrid. This may serve as a
determinant for Ty3 RT binding that directs RNase H cleavage 12–13
bp from the site of substitution. However, it is important to consider these
observations with respect to the features of the wild type PPT-containing
duplex that sequester Ty3 RT and result in correct cleavage at the PPT-U3
junction.
Since the Ty3 PPT/(–) DNA hybrid is 12 bp
(Fig. 1B), our data
suggest that initial sequestration of the retrotransposon polymerase is
mediated by sequences adjacent to the 5'-end of the polypurine tract.
This possibility was tested by introducing two alterations immediately
5' of the Ty3 PPT, changing the sequence from
5'-rC-rC-rC-rU-3' to either 5'-rC-rU-rC-rU-3' (mutant
C U) or 5'-rU-rU-rU-rU-3' (mutant 3C 3U). The latter
sequence closely resembles the U-rich region 5' of the HIV-1 PPT, which
has been shown to play a role in its utilization
(31,
32). To control for potential
destabilization of the duplex resulting from C U substitutions, each
RNA primer was extended by an additional 7 nucleotides at its 5'
terminus (Fig. 6A). As
a consequence, the 5' flanking region of the PPT served as an additional
substrate for Ty3 RNase H, resulting in precise cleavage at both the 5'
and 3' PPT junctions (Fig.
6B, i). This was expected, because our previous
data on HIV-1 and Ty3 PPT selection indicated that they are accurately
processed at both the 5' and 3' termini from within a larger
RNA/DNA hybrid (9,
16). A single base pair
substitution, altering the sequence to 5'-rC-rU-rC-rU-3', resulted
in slightly increased 5' processing
(Fig. 6B,
ii). However, altering the sequence upstream of the Ty3 PPT to
5'-rU-rU-rU-rU-3' was accompanied by a significant increase in
cleavage at the 5' terminus of the PPT
(Fig. 6B,
iii). To more accurately assess the effect of these mutations and
eliminate the possibility that 3' cleavage events had been obscured, we
also radiolabeled the RNA substrates at their 3'-end. RNase H hydrolysis
products obtained after 30 s were quantified. The results
(Fig. 6C) indicated
that cleavage at the PPT-U3 junction of mutant 3C 3U represented only
30% of total cleavage at the PPT termini, compared with 80% for the
WT. This significant change in cleavage specificity suggests that the accuracy
and extent of Ty3 PPT processing are influenced by sequences adjacent to its
5'-end.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The molecular mechanism through which the PPT is recognized and specifically processed remains elusive, despite numerous studies describing the effects of altering either nucleic acid sequence (4, 7, 8, 22) or the structural motifs of RT (23, 24, 28). This is of particular importance, since the fidelity with which the PPT is processed has significant bearing on subsequent steps in replication. Previous studies with HIV-1 and equine infectious anemia virus (9) showed their PPTs were faithfully selected by RT when embedded within a considerably larger DNA/RNA hybrid (
120 bp) (i.e. where nucleic acid termini cannot
influence positioning of RT). These and other observations
(7,
8) implicate the PPT as an
active participant in its recognition and processing. Furthermore, despite
differences in nucleic acid sequence and RT architecture, a universal
mechanism for PPT selection by the cognate RT might be expected. In this
paper, the local flexibility of the Ty3 PPT was varied to determine its effect
on (+)-strand primer removal. Introducing the non-hydrogen-bonding thymine
isostere, F, consistently induces novel RNase H-mediated cleavage 12–13
bp from the site of substitution. For example, the dual –1/–2 T
F substitution redirects the RNase H domain almost exclusively to the
novel cleavage site (Fig.
3D, ii). Additional mutagenesis studies indicate
that altering sequences immediately 5' to the Ty3 PPT (and 13 bp
from the PPT-U3 junction) significantly alters the balance of 5' and
3' processing (Fig. 6).
Finally, Ty3 RT hydrolyzes the HIV-1 PPT-R substrate 13 bp downstream
from a region demonstrated by chemical footprinting and x-ray crystallography
to contain weakly paired, mispaired, and unpaired bases
(10,
11). These independent lines
of evidence suggest the coordinated action of two regions of Ty3 RT during PPT
selection: the RNase H domain, which must be positioned at the PPT-U3
junction, and a structural motif within or adjacent to the DNA polymerase
domain, which interacts with nucleic acid 13 bp upstream of the
processing site.
Although supporting structural evidence for this Ty3 RT motif is presently
unavailable, it is possible to extrapolate from crystallographic data for the
HIV-1 enzyme
(11–13)
to infer the region of the retrotransposon polymerase that interacts with
nucleic acid 13 bp from its RNase H catalytic center. In all nucleic
acid-containing structures of HIV-1 RT, extensive contacts are made between
the base of the p66 thumb subdomain and the substrate 3–7 bp behind from
the DNA polymerase catalytic center. The RNase H catalytic center of HIV-1 RT
must therefore be 10–14 bp downstream from the base of the thumb
subdomain. In particular, helix 
H of the thumb is partially embedded
within the minor groove of double-stranded DNA. Gly262,
Lys263, and Trp266 of helix H are part of the
minor groove binding track, a motif implicated in correct tracking of the
enzyme over nucleic acid (29).
Mutagenesis studies suggest that this motif could function as a
"sensor" of duplex configuration, detecting base pair alterations
introduced by lesions (30).
Since crystallographic (11)
and chemical footprinting data
(10) have both identified
distortions within the HIV-1 PPT, recognition of this structure by the minor
groove binding track is a plausible mechanism that helps position the RNase H
catalytic center directly over the PPT-U3 junction. Indeed, mutations in the
minor groove binding track have been correlated with altered specificity of
HIV-1 PPT processing (31). It
is very likely that Ty3 RT also has a thumb subdomain and a minor groove
binding track. This may be the structural element of Ty3 RT that interacts
with the F-induced structural perturbation. Indeed, secondary structure
analysis has identified a putative thumb subdomain for Ty3 RT and a motif
related to the HIV-1 minor groove binding
tract.2 In fact, the
data of Figs. 3 and
5 indicate that whereas T
F substitutions are not associated with major structural distortions,
the local perturbations they introduce are sufficient to sequester a Ty3
enzyme "scanning" the PPT-containing RNA/DNA hybrid and induce
cleavage 12–13 bp downstream. This hypothesis also explains the
hydrolysis profile obtained by Ty3 RT on the HIV-1 PPT. Positioning of the Ty3
RNase H catalytic active site over positions +2to +4
(Fig. 2B) locates the
thumb subdomain 12–13 bp upstream, around positions –10 to
–11. Our chemical footprinting analysis
(10) identified distortions
between positions –10 and –15
(Fig. 1A). Recognition
of such distortion by the putative thumb subdomain of Ty3 RT is consistent
with recognition of the T F-induced local structural
destabilization.
Data of Fig. 6 implicate a cis-acting element (i.e. the short dG:rC block immediately upstream of the (+) strand primer) in Ty3 PPT recognition. Regions 5' of the PPTs of murine leukemia (31) and simian immunodeficiency virus (32) have also been shown to control the efficiency and accuracy with which they are processed. Likewise, in vivo studies with another long terminal repeat-containing retrotransposon, Ty1, have shown that an A:T-rich region immediately upstream of the PPT participates in its selection (33). Thus, despite the absence of sequence homology between these long terminal repeat-containing elements, features of the RNA/DNA hybrid 5' to the Ty3 PPT appear to influence its selection. Although we show here that alteration of this upstream region is associated with changes in PPT processing, the underlying structural basis is not clear. However, since G:C tracts are associated with major groove compression (34), it is possible that subtle differences in groove width immediately preceding the Ty3 PPT serve to "lock" the RT thumb in position, thus ensuring that the RNase H domain is correctly positioned over the biologically relevant processing site. Initial NMR studies with a Ty3 PPT-containing RNA/DNA hybrid in the absence of Ty3 RT have suggested that it may adopt an unusual configuration,3 which would support our postulation. Although our work has exploited the hydrogen-bonding isostere F, several alternative modified nucleosides are now available to better understand the molecular basis of PPT and tRNA primer processing and selection in HIV-1 and Ty3, including 2-aminopurine, 2,6-diaminopurine, purine riboside, and the non-hydrogen-bonding cytosine analog 2-fluoro-4-methylbenzene (35). The latter analog, in combination with F, has been used to probe the structure of the HIV-1 PPT and elucidate the molecular basis of its selection.4 These studies show that introducing F or 2-fluoro-4-methylbenzene into the HIV-1 PPT induces novel cleavage 3–4 nt downstream the insertion site instead of the 12 nt observed for Ty3, suggesting that the molecular bases for Ty3 and HIV-1 PPT selection are different.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 301-846-5256; Fax:
301-846-6013; E-mail:
slegrice{at}ncifcrf.gov.
1 The abbreviations used are: RT, reverse transcriptase; RNase H,
ribonuclease H; PPT, polypurine tract; HIV, human immunodeficiency virus; F,
2,4-difluoro-5-methylbenzene; PPT-R, 5'-end-labeled PPT-containing RNA
extended by 10 ribonucleotides; PPT-D, 5'-end-labeled PPT-containing RNA
extended by 10 deoxyribonucleotides; WT, wild type; nt, nucleotide(s).
2 D. Lener and S. Le Grice, unpublished observations.
3 J. Marino, personal communication.
4 J. W. Rausch, J. Qu, H.-Y. Yi-Brunnozzi, E. T. Kool, and S. F. J. Le Grice,
submitted for publication.
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ACKNOWLEDGMENTS |
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We thank M. Powers (SAIC Frederick) for synthesis of F-substituted DNA oligonucleotides and S. Tarasov (Structural Biophysics Laboratory, NCI-Frederick) for technical assistance and helpful discussions on CD measurements. We also thank G. J. Klarmann for useful suggestions and helpful discussions of the data. We also acknowledge the NCI, National Institutes of Health, CCR Fellows Editorial Board for editing the manuscript.
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