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J. Biol. Chem., Vol. 281, Issue 11, 7421-7428, March 17, 2006
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123
4
From the
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, the ¶Department of Pathology, University of Washington, Seattle, Washington 98195, the ||Center for Advanced Biotechnology and Medicine (CABM) and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, and the **Laboratory of Structural Biology, National Institutes of Health, NIEHS, Research Triangle Park, North Carolina 27707
Received for publication, October 19, 2005 , and in revised form, January 18, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3-4 hairpin of the fingers subdomain provides key contacts for the templating nucleotide and the incoming dNTP (8). The importance of this region for dNTP selection and fidelity is further supported by reports of several mutations in the 3-4 hairpin that affect RT fidelity (9-12).
Whereas residues proximal to the dNTP-binding pocket can directly influence polymerase fidelity, the contribution of residues that contact template-primer duplex to fidelity is presumably indirect. Such residues primarily influence processes related to template-primer interaction such as the RT-template-primer affinity, polymerase translocation along the template during synthesis, and processivity (13-15). Mutations at such residues can also affect fidelity. For example, two well studied structural elements, 
H of the thumb subdomain and the template grip 5a in the palm subdomain of HIV-1 RT, which are known to affect polymerase processivity (15, 16) also affected fidelity (16, 17). Alanine substitutions of residues in the H helix that form a part of the minor groove-binding track in the thumb have been shown to alter frameshift fidelity (16, 18). The E89G variant in 5a of the template grip increases dNTP incorporation fidelity of HIV-1 RT (17).
Glu89 is of particular interest because of its unique location in the RT template grip (see below) and its established importance in HIV-1 replication and drug resistance. The E89G variant was first identified in an in vitro phenotypic screen of a random library of RT mutations (19, 20). HIV-1 virions containing the E89G mutation are replication competent and exhibit multidideoxynucleoside analog resistance (20). Although the E89G mutation is rarely observed alone in natural settings, it has been observed in association with M184V in (-)-2'-deoxy-3'-thiacytidine-resistant isolates from HIV-infected individuals (21). In the context of SIV replication in monkeys, the E89G mutation rendered the virus less fit forcing it to acquire secondary mutations (22). Mansky and colleagues (23) measured the mutation rate of HIV-1 containing the E89G mutation in a single-cycle replication assay and found that it led to a small reduction (20%) in mutation rate (23). Detailed analysis of mutation patterns in vitro suggests that the reduction in one type of RT error is compensated for by an increase in other types of errors (24).
The crystal structures of HIV-1 RT complexed with the double-stranded DNA show Glu89 to be a key anchor residue in the 5a template grip (7, 8). Glu89 is in close proximity to the sugar-phosphate backbone of the template strand near the penultimate base pair. Thus, substitution at Glu89 may affect polymerase fidelity by changing the conformation of the template-primer duplex, thereby altering the geometry of the dNTP-binding site (25). Additionally, it is also possible that Glu89 substitutions affect the positioning of the template-primer rather than its conformation.
A high frequency of single-base deletion errors, or -1 frameshift errors, is a hallmark of mutations by HIV-1 RT. This type of error occurs at rates 90-fold higher in homopolymeric runs of nucleotides than at non-runs (26). HIV-1 RT also generates +1 frameshifting at nucleotide runs, although at a 
5-fold lower frequency than -1 frameshifting (26). The -1 frameshifting is thought to be mediated by template slippage events, whereas the +1 frameshifting is caused by primer strand slippage during polymerization (27). The location of Glu89 is approximately where template slippage might occur, suggesting a role for the Glu89 in (-1) frameshifting. In addition, of the 5-6 residues in the 5a strand, only the Glu89 side chain is in close proximity to template strand.
Considering its biological importance in drug resistance (20, 21), we wished to study the effect of altering this interaction between template grip and template-primer on slippage-mediated events. Employing the previously reported Glu89 mutants (28), we investigated the influence of limited vertical scanning mutagenesis of Glu89 on the frequency of frameshifting by HIV-1 RT. We show here that all eight substitutions of Glu89 tested display decreased frequencies of -1 frameshifting. Unexpectedly, the decrease in -1 frameshifting was also accompanied by increased frequencies of +1 frameshifting. Crystal structures of HIV-1 RT complexed with duplex DNA show contacts between Lys154 and Glu89, thus predicting a possible role for Lys154 in -1 frameshifting. By using site-directed mutants of Lys154 to measure -1 frameshifting, we confirm this prediction. Furthermore, substitutions at Glu89 increased the fidelity of dNTP incorporation showing the influence of Glu89 on multiple types of replication errors.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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for scoring -1 frameshifts (5T-1 substrate) and a single-base deletion for scoring +1 frameshifts (7T+1 substrate) (32). Both M13mp2-gapped DNA substrates were generous gifts of T. A. Kunkel (National Institutes of Health, NIEHS, Research Triangle Park, NC). M13-gapped DNA substrates were filled in by wild-type and mutant RTs as described previously (26). Complete filling in was ensured by agarose gel electrophoresis and the successfully filled-in gapped duplex DNA was electroporated into Escherichia coli strain MC1061. Electroporations were performed on separate days for each of three trials for each DNA synthesis reaction. Cells were allowed to recover for 10 min, then mixed with E. coli CSH50 cells (
(pro-lac), thi, ara, strA/F'(proAB, lacIqZ_M15, traD36) in the presence of 0.25 mM isopropyl -thiogalactopyranoside (LabScientific) and soft agar containing 0.2 mM X-Gal (5-bromo-4-chloro-3-indolyl -D-galactopyranoside; LabScientific), and plated on M9 plates. Plates were incubated at 37 °C for 15 h and the plaques were screened for -galactosidase activity by looking for those displaying the dark blue reversion phenotype. Such revertant plaques were picked for sequencing and stored in 1 ml of 0.9% saline at 4 °C. Reversion frequencies were determined by dividing the number of revertants by the total number of plaques screened. All reversion frequencies were corrected for background reversion frequency of the substrates (1.30 x 10-5 for the 5T-1 substrate and 8.3 x 10-5 for the 7T+1 substrate (32)). The isolation of single-stranded DNA from revertant plaques and the determination of nucleotide sequences were as described previously (33). The forward mutation frequencies were determined using a wild-type lacZ-gapped duplex DNA as described previously (24). Single dNTP Exclusion Assay—The "single dNTP exclusion" or the "minus dNTP" assay were performed on DNA-DNA template (55-mer) and primer (28-mer) oligonucleotides (Table 1). A 5'-32P-labeled primer oligonucleotide was extended by purified wild-type or the eight Glu89 mutant RTs in the presence of all four combinations of three dNTPs.
The DNA template-primers were prepared by annealing the template (at a molar ratio of 2 template to 1 primer) to the end-labeled primer. Reactions were initiated by combining equal volumes (10 µl) of enzyme-template-primer solution and dNTP salt solution in which all four dNTPs were present, or in which one of the dNTPs was omitted. Each RT was used at two different concentrations (1x and 2.5x). The 1x concentrations for wild-type, E89A, and E89V were, respectively, 0.68, 0.09, and 0.18 nM. Enzyme concentrations for wild-type and mutant RTs were selected to be at equal levels of DNA-dependent DNA polymerase activities on the template-primer used here. Thus, the 1x concentration for each enzyme is different in terms of protein input or unit activity as measured on poly(rA)·oligo(dT) template-primer, but similar with respect to their enzyme activity on the DNA-DNA template-primer used. After mixing, reactions contained 100 nM template-primer, increasing concentrations of wild-type, and mutant RTs as described above, 250 µM dNTPs, 50 mM Tris-HCl, pH 8, 60 mM NaCl, 20 mM dithiothreitol, 0.05% IGEPAL (surfactant), and 10 mM MgCl2. The reactions were terminated after 6 min by the addition of 20 µl of stop solution (95% formamide, 20 mM EDTA). Eight microliters of boiled reaction mixture were subjected to electrophoresis in a 10% urea polyacrylamide gel for 2.5 h at 100 W. Autoradiograms were analyzed by densitometry, and band intensities were quantitated via ImageQuant. Total band intensity was calculated as the total intensity of the band immediately below the barrier site plus all the bands above it. Extension was calculated as the sum of all bands at and above the barrier site. Percentage extension as a fraction of the total band intensity was then calculated from total band intensity and extension.
Mispair Extension Reactions—Primer extension reactions were performed using four different 5'-32P-labeled DNA 31-mers (G:C, G:T, G:G, G:A; see Table 1) annealed to a 55-mer DNA template using wild-type and mutant RTs. As before, two different concentrations of each RT (selected to be equal in DNA-dependent DNA polymerase activity between different enzymes as tested on a DNA-DNA template-primer) were tested for each enzyme at 1x and 2.5x concentrations. The 1x concentrations used for the wild-type, E89A, E89V, E89D, and E89G were 0.68, 0.12, 0.18, 0.09, and 2.3 nM, respectively.
Reactions and buffer conditions were as described above for the dNTP exclusion assay, but in the presence of dGTP, dATP, and dTTP, each at 25 µM. Reactions were terminated after 10 min and analyzed as previously described for dNTP exclusion assay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5a strand in the palm subdomain of HIV RT is located near the site of template strand slippage. Thus, it is well positioned to either directly or indirectly influence -1 frameshifting. To investigate whether the presumptive interaction between the template grip Glu89 and the template strand influences frameshift mutagenesis by HIV-1 RT, we perturbed this interaction by substituting Glu89 with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys. We then determined the frequency at which polymerizing mutant RTs lead to -1 frameshifting, using an M13-based reversion assay that employs a lacZ variant as a reporter gene (26). In this assay, deletion of a base in a run of 5 thymidines causes reversion to the wild-type phenotype (dark blue plaques). Interestingly, all substitutions at Glu89 led to a decrease in the frequency of the -1 frameshift errors by HIV-1 RT (Table 2). Substitutions to Val, Lys, and Thr resulted in the greatest decreases (40-, 28- and 8-fold, respectively) in -1 frameshift frequency. Other substitutions (Asp, Gly, Ala, Ser, and Asn) led to smaller effects, ranging from 2- to 5-fold reduction from the wild-type frequency. It is possible that revertants can also result from +2 events because of primer slippage, which can also restore the reading frame. However, sequencing a sampling of 12 revertants from each of the 9 reactions revealed only -1 events. These results suggest that wild-type Glu89 leads to one of the highest rates of single-base deletion mutations, whereas all substitutions tested lead to reductions.
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such that the insertion of a base within a run of 7 Ts results in the reversion phenotype. Although both +1 and -2 (deletion of 2 bases via template slippage) mutations could restore a functional lacZ gene, previous analysis of a large number of clones has revealed no -2 mutations in this construct.6
In contrast to the results obtained for -1 frameshift frequency measurements, all Glu89 substitutions (except the Glu to Val substitution) led to an increase in the frequency of +1 frameshift errors (Table 2). The wild-type +1 frameshift frequency was about five times lower than the -1 frameshift frequency. The substitutions led to a 2.6-47-fold increase in +1 frameshift frequency over that of wild-type (Table 2). Thus, the majority of frameshift mutations by wild-type RT were of the -1 type, whereas the majority of frameshift mutations by all of the variant RTs were of the +1 type. Some of the substitutions, such as Ser, Thr, and Asn caused increases in total frameshifting (7-, 2-, and 6-fold, respectively) while maintaining the inverse ratio of +1 to -1 frameshifting compared with wild-type RT (Table 1).
Base Substitution Fidelity—It was surprising that the wild-type enzyme displayed the highest -1 frameshift mutation frequency, as one would expect the wild-type enzyme to have minimal slippage-mediated mutations, which have little evolutionary benefit for protein coding sequences. Therefore, we investigated the influence of wild-type Glu89 on base substitution fidelity. Base substitution errors (or misincorporation) result from two events during polymerization. First the insertion of an incorrectly paired dNTP (misinsertion) must occur. Such events result in a mispaired primer terminus and are generally not preferred by most DNA polymerases. For the misinserted base to become a misincorporated base, the mispaired primer terminus must be extended by correct insertion of a base opposite the next template base (mispair extension).
To investigate the effect of Glu89 on both misinsertion and mispair extension, we utilized a gel-based single dNTP exclusion assay or minus dNTP assay (1), which provides a gross estimate of the efficiency of base substitution for a polymerase without proofreading function. This assay involves copying a heteropolymeric DNA template using a 5'-end-labeled primer in the presence of only 3 dNTPs (absence of a single dNTP). Primer extension past a template site for which the complementary dNTP is missing depends on the ability of the polymerase to misinsert dNTPs as well as to extend the mispaired primer. Thus the extent of primer extension under these conditions allows a gross estimate of the ability of the polymerase to generate misincorporations (the combined result of both misinsertion and mispair extension events).
The assay was set up as described before (34). A 22-nucleotide 5'-end-labeled DNA primer was annealed to a 55-nucleotide oligonucleotide DNA. Absence of one dNTP in the reactions creates a barrier to primer extension at the template position where RT would need to incorporate the missing dNTP. For polymerases (e.g. RT), which lack exonucleolytic proofreading activity, one can obtain an estimate of efficiency of misincorporation, a combined sum of changes in the misinsertion and mispair extension efficiencies, by measuring the amounts of products extended past the barrier. For the results of this assay to be meaningful, it was critical that the enzyme inputs in terms of activity levels be nearly identical in all reactions. To this end, we ensured the presence of equivalent levels of RT in control reactions in which all four dNTP were included. As shown in Fig. 2A, the wild-type and mutant RTs synthesized similar amounts of full-length and intermediate sized products (Fig. 2A, panel labeled All dNTPs), indicating that similar inputs of all RTs, based on DNA-dependent DNA polymerase activity, were used in the reactions. When increasing amounts of enzyme were included in these reactions, the amount of full-length product increased with all RTs tested, indicating that the reactions were not rate-limiting. Wild-type HIV-1 RT efficiently extended the primer past the first barrier site when dATP, dCTP, or dGTP were missing (Fig. 2A, panels labeled Minus dATP, dCTP, dGTP, respectively). In each of the remaining cases of excluding a single dNTP, the wild-type enzyme was capable of passing through several barrier sites and performing DNA synthesis. In reactions without dATP or without dGTP, wild-type enzyme also synthesized a limited amount of full-length product (Fig. 2A, panels labeled Minus dATP and Minus dGTP). Reactions where dTTP was missing led to little or no extension by all enzymes.
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To estimate differences in base substitution fidelities of the Glu89 mutant panel, we quantified the DNA synthesis above the first barrier for all mutants. The sum of intensities of all products above the "no substrate" barrier for which the complementary dNTP is missing (presented as a fraction of the sum of the intensities of the barrier band plus those above it) shows that all substitutions at Glu89 decrease the efficiency of base misinsertion by RT (Fig. 2B). Quantitation of extension products representing polymerization beyond the barrier revealed that the trend in increased fidelity observed for misinsertion frequency mimics the trend for -1 frameshift fidelity (Table 2 and Fig. 2B). For example, E89V substitution resulted in the largest decrease in both -1 frameshifting and base substitution errors (Fig. 2), whereas E89A, E89S, and E89N displayed fidelities most similar to wild-type in both cases. In fact, graphical representations of changes in fidelity in comparison to wild-type for the eight Glu89 substitutions for -1 frameshifting and base substitution are superimposable (compare Table 2 and Fig. 2B). Although, at first, this seems to suggest that the decreased efficiency of misincorporation may be because of slippage-mediated synthesis, we believe this to be unlikely because of the fact that none of the barrier sites being studied (the first in each case) are in the nucleotide runs in the heteropolymeric DNA template used in this assay.
In addition to its error proneness for internal misinsertions and frameshifting events, HIV-1 RT has been shown to be capable of inserting non-templated dNTPs at blunt ends of both RNA-RNA and DNA-DNA template-primer (upon reaching the end of copying of a long template) at a high efficiency (35). Interestingly, we observed that some of the Glu89 mutants also display an absence of non-templated addition of nucleotides. For example, E89V showed a complete lack of non-templated addition (as shown by the light band at the top of the lane, above the dark band representing the full-length product), but E89A retained this activity. Similarly, E89D, E89G, E89K, and E89T all showed a complete lack of non-templated dNTP incorporation, whereas E89N and E89S retained levels of non-templated dNTP incorporation that were comparable with wild-type (data not shown).
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Neither wild-type nor mutant RTs were able to efficiently extend G:G or G:A mispairs, therefore, only the results of extension from the G:T-mispaired terminus and the correctly paired G:C terminus are described. All substitutions at Glu89 led to an increase in mispair extension fidelity (except E89A; see Fig. 3), although quantitation of extension products revealed that the trend in mispair extension fidelity did not completely mimic the trend in the -1 frameshift fidelity (Figs. 1 and 3). Substitutions with Asp, Val, Ser, or Thr reduced mispair extension frequency to similar degrees (about 50% of wild-type mispair extension), whereas the E89G and E89K substitutions displayed maximal reductions and Asn substitution had a minimal effect. In all, it appears that the increased fidelity of Glu89 mutants is because of reductions in both misinsertion and mispair extension events.
Overall Mutation Frequencies—It was of interest to ask whether the large changes observed in -1 and +1 frameshifting frequencies affect the overall mutation frequencies of RT mutants. To examine this, we selected two mutants, E89K and E89V, displaying the largest decreases in -1 frameshifting and mutant E89S, which showed the largest increase in +1 frameshifting. Using a forward mutation frequency assay, we measured the overall mutation frequency of these mutants and compared it to that of wild-type HIV-1 RT. As shown in Table 3, the mutation frequencies of E89K, E89V, and E89S were altered minimally, bringing about reductions of 1.2-, 1.3-, and 1.6-fold from that of wild-type RT, respectively (which correspond to reductions of 16, 25, and 38%, respectively).
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E is also part of the template grip. Interestingly, this residue contacts the same sugar moiety of the template as Glu89 approaching it from the other side of the same deoxyribose ring. A P157S mutation in HIV-1 RT has been shown to confer resistance to inhibition by nucleotide analogs such as 3TCTP ((--)-2'-deoxy-3'-thiacytidine triphosphate) and FTCTP (2',3'-dideoxy-5-fluoro-3'-thiacytidine triphosphate) (30). More importantly, the sequence specificity in sensitivity to inhibition by chain terminators displayed by the wild-type HIV-1 RT was altered by the P157S substitution, further confirming its ability to modulate template-primer conformation via interaction with the template strand. Therefore, we tested the effect of the P157S mutation on -1 frameshift fidelity. Our results show that the effect was minimal with a 1.8-fold reduction in frameshifting (Table 4). When compared with the effect of Glu89, revealed by Glu89 substitution mutants (2.5-40-fold decrease in -1 frameshifting), the role of Pro157 appears to be minor. However, one cannot exclude the possibility that more pronounced effects could result from substitutions with residues other than serine.
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E. This contact between Glu89 and Lys154 is likely to be transient and may contribute to structural constraints that may directly or indirectly affect the stability of frameshifted complexes of RT and template-primer. To investigate this hypothesis, we tested the influence Lys154 on -1 frameshifting. Lys154 is the only positively charged residue in the VLPQGWK motif in the 8-E loop at the junction of the fingers and palm subdomains. Among several substitutions of Lys154 that were previously tested for the effect on HIV-1 RT function (31), we selected two that retained wild-type-like polymerase activities, K154A and K154R substituted mutant RTs, and tested them for -1 frameshifting. Interestingly, as seen in Table 4, both mutations led to significant reductions in -1 frameshifting, with 10- and 7.5-fold reductions, respectively (Table 4). These results suggest that Lys154 is involved in the template-slippage-mediated frameshifting events. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5a in frameshift mutagenesis by HIV-1 RT. In the wild-type enzyme, Glu89 is positioned to allow a relative high frequency of -1 frameshifting (i.e. template slippage) with a 5 times lower frequency of +1 frameshifting (primer slippage). Substitutions at Glu89 appear to disturb this ratio resulting in greater +1 frameshifting events. Our analysis of wild-type and eight different substitutions at position 89 reveals a key role for Glu89 in many aspects of polymerase fidelity tested, including -1 and +1 frameshifting, dNTP misincorporations, and mispair extensions. It appears that all types of RT errors except +1 frameshifting are at a maximal level when the wild-type Glu residue is present. Because of the proximity of Glu89 with the template backbone, it is not surprising that substitution of this residue leads to a change in frameshifting because of either direct or indirect effects on template slippage. However, the decreased frequency of -1 frameshifting for all Glu89 substitutions was unexpected.
The characteristic high frequency of -1 frameshift errors in homopolymeric runs may be because of the ability of HIV-1 RT to stabilize intermediate nucleic acid structures that result in non-copying of template nucleotides that would yield slipped, mismatched, or unpaired bases. Such structural aberrations are likely to disrupt the positioning of the ribose and phosphate moieties of one or more template base(s). The relatively high -1 frameshifting frequency of wild-type RT suggests that Glu89 may directly or indirectly facilitate the proper conformation of the template grip leading to stabilization of aberrant template-primer duplexes in the template-primer cleft of RT. In a typical, heteropolymeric template sequence, transient misalignments between template and primer nucleotides can be resolved, at some probability, by sequence-specific Watson-Crick base pairing, resulting in correct copying of the sequence. However, within a run of identical nucleotides, the partial misalignment could resolve incorrectly with base pairing to the previous or following primer base.
An opposite effect was observed for +1 frameshift fidelity; all substitutions led to increased +1 frameshifting. It appears that the template- and primer-slippage events are inter-related and are apparently mutually exclusive. For example, wild-type Glu89 appears to facilitate template slippage (-1 frameshifting), but not primer slippage (+1 frameshifting). In contrast, all substitutions tested here appear to disfavor the template-slippage but promote primer-slippage events. The structural aberration(s) of nucleic acid during primer slippage will be different from that during template slippage and by extension, contacts with the enzyme should also be different. The effect of Glu89 mutations on the ability of RT to accommodate non-canonical nucleic acids is also likely to be different. Structural studies with the relevant mutants and/or relevant RT-DNA complexes may provide insights into the precise role of residue 89 in +1 frameshifting. It is also possible that suppression of template slippage leading to an increase in the frequency of primer slippage may be because of an innate property of the homopolymeric nucleic acid duplex, such as an unusual conformation, that forces slippage events at these sites. HIV-1 RT is reportedly sensitive to minor groove compression caused by homopolymeric runs of adenosine (36). The fact that several substitutions at Glu89 cause a reduction in -1 frameshifting suggests that whether the homopolymeric stretches of sequence leads to template or primer-slippage events is in part determined by the nature of the interaction of the residue 89 or the surrounding residues of template grip 5a with the nucleic acid.
Structural Insights into Frameshifting Determinants—To rationalize the influence of RT mutations on frameshifting, we modeled the various substitutions into the structure of HIV-1 RT complexed with DNA and dNTP. As mentioned before, the crystal structure of HIV-1 RT reveals an interaction between Glu89 and Lys154 residues (Fig. 4, A and B). Glu89 is located in the template grip, contacting the template sugar phosphate backbone near the n-2 base (where the templating base is denoted n). Lys154 is located within the 8-E loop forming the junction between the fingers and palm subdomains. Residues other than lysine or arginine at that position are known to significantly affect RT-DNA-dNTP complex formation (31). Interestingly, Lys154 is in a position to form a hydrogen bond with Glu89 (8) (Fig. 4B). We hypothesize that Lys154 and Glu89 act in concert to maintain the integrity of the template grip region and by extension the polymerase active site.
We hypothesize that +1 and -1 frameshifting rates can be explained by movements that are influenced by the nature of the protein-DNA contacts present along the two strands. Furthermore, contacts on the template and primer strand are correlated, in the sense that modifying the contacts along one strand may influence the precise pattern of contacts along the other strand because of the necessity for the duplex DNA to pass between the template and primer grips. Thus, substitutions at position 89 would affect interactions with the template strand.
It is common to visualize the precise change in DNA conformation during slippage events as a bulge in the duplex. However, for frameshifting to occur, RT must accommodate this bulge. Because an extra nucleotide can occur in either strand, specific structural "pockets" should be present in both template and primer grip elements, neither pockets are observed. In fact, when we attempted modeling a slippage intermediate, a bulge could not be accommodated near the Glu89 contact, but only 5-6 bases away from the primer 3' terminus near the end of the polymerase module part of the template cleft. Furthermore, if there was an extrahelical pocket, frameshifts mediated by HIV-1 RT should not be exclusively limited to nucleotide runs.
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Multiple Effects of Glu89 Substitutions—All tested substitutions at Glu89 led to an increase in base substitution fidelity, as demonstrated by the results of dNTP exclusion assay results (Fig. 2). The pattern of changes observed among various mutants for -1 frameshifts was similar to that of base substitutions. It has been proposed that subtle variations in the conformation and/or position of the template-primer duplex can influence the dNTP-binding pocket, as shown by altered nucleoside analog sensitivity of the E89G variant (25) and the impact of P157S mutant on sequence-specific susceptibility to chain terminators (30). Our results from the dNTP exclusion assay further corroborate that Glu89, in addition to affecting frameshifting, also influences the efficiency of dNTP misinsertion consistent with the previous report of a E89G mutant (17). These results are in agreement with the notion that template-contacting residues influence the geometry of the dNTP binding pocket. The effects of Glu89 substitutions on mispair extension were milder and did not correlate with the pattern of variation observed for -1 frameshifting. Thus, the contacts by Glu89 or the nearby residues that apparently affect DNA conformation and or positioning, do not appear to influence mispair extension frequencies to a considerable degree.
The fact that Glu89 substitutions led to decreases in both -1 frameshift frequency and base substitution frequency, and that the trends in fidelity for Glu89 mutants were strikingly similar suggests that these distinct mutational events are linked and probably result in a particular enzyme-template-primer conformation. It appears that the status of the duplex DNA that favors primer slippage also results in lower rates of misinsertion and mispair extension and that the wild-type RT (with a Glu at this position) seems to perform more template-slippage events as well as higher rates of misinsertion and mispair extension events. It is possible that the interaction of non-Glu amino acid residues with the template backbone affords a more stable (i.e. less flexible) DNA conformation within the dNTP-binding pocket. As the template strand forms a key surface of the dNTP binding pocket, this results in greater fidelity of dNTP incoporation.
It is not surprising that Glu89 is crucial for many aspects of polymerization, as it directly contacts the template in the template grip of HIV-1 RT (Fig. 4A). Its influence on nucleoside analog sensitivity and RT fidelity suggest that alteration of Glu89 imparts changes to the dNTP binding pocket that alter the ability of RT to discriminate between dNTPs and ddNTPs (dideoxynucleoside triphosphates). Here we have shown a new, specific role of Glu89 in polymerase fidelity of HIV-1 RT; namely, that Glu89 is a determinant of the high frequency of -1 frameshifting at nucleotide runs. Second, our results confirm the previous conclusion (17) that Glu89 determines the efficiency of correct dNTP incorporation and mispair extension fidelity and suggest that these effects are a consequence of the proximity of Glu89 to the template backbone.
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FOOTNOTES |
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1 Supported by National Institutes of Health Institutional Training Grant NIGMS T32-GM07491. Data are from a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. Present address: Residency Program, Pediatrics, Rhode Island Hospital/Brown University, Providence, RI.
2 Present address: 1680 Owens St., Gladstone Institute of Virology and Immunology, San Francisco, CA 94158.
3 Present address: Center for Biotechnology at Stony Brook, Stony Brook, NY 11794.
4 To whom correspondence should be addressed: 1300 Morris Park Ave., Rm. GB 401, Bronx, NY 10461. Tel.: 718-430-2517; Fax: 718-430-8976; E-mail: prasad{at}aecom.yu.edu.
5 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase.
6 T. A. Kunkel, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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