| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
J Biol Chem, Vol. 275, Issue 20, 15025-15033, May 19, 2000
§,,
,,**
From the Sealy Center for Molecular Science,
University of Texas Medical Branch, Galveston, Texas 77555-1071 and
the ¶ Laboratory of Structural Biology and the Laboratory
of Molecular Genetics, NIEHS, National Institutes of Health,
Research Triangle Park, North Carolina 27709
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Biochemical and molecular modeling studies of
human immunodeficiency virus type 1 reverse transcriptase (RT) have
revealed that a structural element, the minor groove binding track
(MGBT), is important for both replication frameshift fidelity and
processivity. The MGBT interactions occur in the DNA minor groove from
the second through sixth base pair from the primer 3'-terminus where
the DNA undergoes a structural transition from A-like to B-form DNA. Alanine-scanning mutagenesis had previously demonstrated that Gly262 and Trp266 of the MGBT contributes
important DNA interactions. To probe the molecular interactions
occurring in this critical region, eight mutants of RT were studied in
which alternate residues were substituted for Trp266. These
enzymes were characterized in primer extension assays in which the
template DNA was adducted at a single adenine by either R-
or S-enantiomers of styrene oxide. These lesions failed to
block DNA polymerization by wild-type RT, yet the Trp266
mutants and an alanine mutant of Gly262 terminated
synthesis on styrene oxide-adducted templates. Significantly, the sites
of termination occurred primarily 1 and 3 bases following adduct
bypass, when the lesion was positioned in the major groove of the
template-primer stem. These results indicate that residue 266 serves as
a "protein sensor" of altered minor groove interactions and
identifies which base pair interactions are altered by these lesions.
In addition, the major groove lesion must alter important structural
transitions in the template-primer stem, such as minor groove widening,
that allow RT access to the minor groove.
Rapid and efficient DNA polymerization requires stable
interactions between the polymerase and the template-primer to
facilitate highly processive DNA synthesis. When examined alone, the
leading/lagging strand polymerases from human, Escherichia
coli, and T4 bacteriophage are poorly processive, but become
highly processive after binding accessory proteins (1, 2). Each of
these replication machines also exhibits high fidelity. Although the
presence of an editing 3' Human immunodeficiency virus type 1 (HIV-1)1 reverse
transcriptase (RT) is a heterodimer of p66 and p51 subunits and
exhibits both polymerase and RNase H activities. However, RT does not
have an associated 3' Several crystal structures of RT have been reported, including the
apoprotein (8), co-crystals with non-nucleoside inhibitors (9-11),
co-crystal with DNA (12), and a ternary complex with bound DNA and dNTP
(13). These structures have identified substrate binding and active
sites for this multifunctional enzyme. Whereas the polymerase and RNase
H domains are formed by the amino- and carboxyl-terminal regions of the
p66 subunit, both the p66 and p51 subunits form an apparent DNA-binding
cleft. The primary sequence that links the polymerase and RNase H
domains has been termed the connection subdomain. The p51 subunit lacks
a RNase H domain. One subdomain (residues 244-322; referred to as
"thumb" using the hand analogy (see Ref. 9)) of the polymerase
domain, which is primarily Studies from the Wilson and Kunkel laboratories have provided strong
evidence that amino acid residues in Expression and Purification of Wild-type and Mutant Forms of
HIV-1 RT--
Mutant and wild-type forms of HIV-1 RT were prepared as
described previously (16, 17, 19).
Preparation of SO Adducted Templates--
Oligonucleotides
(11-mers) containing site- and stereospecifically modified
R- and S- enantiomers of SO at
N6-adenine were synthesized as described
previously (24) and were the gift of Drs. Thomas and Connie Harris
(Vanderbilt University, Nashville, TN). The site of adduction was the
third position within codon 11 of the human N-ras gene:
GGCAGGTGGTG, where the underlined A was the site of
adduction. These sequences, whether unadducted or adducted with
R- or S-SO N6-adenine,
were assembled into 63-mer oligonucleotides as illustrated in Fig. 1.
Both the 11- and 32-mers were fully phosphorylated with T4 DNA kinase
(New England Biolabs, Beverly, MA) and ATP. The 63-mer was assembled on
a 46-mer scaffold that was complementary to part of the 63-mer DNA.
Each of the three 63-mers were purified through 12% denaturing
polyacrylamide, 8 M urea gels.
Primer Extension Assays--
The oligonucleotide primers were 5'
end-labeled with [ In order to assess the consequence(s) of mutations in the MGBT of
RT on damaged DNA, the primer extension activity of each enzyme was
measured on a 63-mer template containing either no adduct, or the
R- or S-SO adduct coupled to the
N6 position of a specific adenine (Fig.
1). The methods for the chemical
synthesis of these unique lesions has been described elsewhere (23,
24). The solution structure of the R- and S-SO lesions within an oligodeoxynucleotide duplex revealed that the adducts, which lie in the major groove, are well accommodated by the
DNA duplex and do not distort the local helix geometry (21, 22).
However, the R- and S-SO stereoisomers differ in their spatial orientation; the R-SO
N6-adenine adduct is directed toward the 5'-end
of the template (21), whereas the S-SO adduct is directed
toward the 3'-terminus of the template (22). As a result, they have
nearly opposite orientations in duplex DNA, although the R-
and S-SO adducts are chemically identical. The two adducts,
therefore, may report protein-DNA interactions at different positions
within the template-primer, even though they are attached to the same
nucleotide in the template. Crystal structures of several DNA
polymerases (25-28), including RT (12, 13), indicate that the DNA is
A-like near the polymerase active site. Since DNA polymerases interact
with duplex DNA through the DNA backbone and minor groove, these
adducts allow us to assess whether there are structural perturbations
translated to the minor groove and/or whether the local DNA structure
is altered by these lesions as RT deforms the DNA substrate during
replication.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease activity contributes
significantly to this low error rate, evidence supports a general
correlation between polymerase processivity and fidelity (3).
5' exonuclease function (4) and is only moderately processive (5). RT is one of the most error-prone DNA
polymerases examined to date, averaging one error per 2 kilobase pairs
replicated (4, 6). RT appears to lack a true processivity factor
in vivo, although HIV nucleocapsid protein may operate in a
manner similar to single-stranded binding protein to increase RT
processivity through regions of DNA secondary structure (7). Since the
rapid mutation rate of HIV is widely considered a major stumbling block
in the development of therapies to combat acquired immunodeficiency
syndrome, an understanding of how RT structure is correlated with
function during DNA synthesis is a high priority.
-helical in character, is believed to be
important in RT double-stranded nucleic acid binding. Indeed,
comparisons of RT crystal structures with and without template-primer
suggest that this subdomain may move as much as 30 Å (8, 14).
Non-nucleoside inhibitor binding to RT has been shown to affect the
mobility of this subdomain (10, 11), offering one explanation for how this class of drugs may inhibit enzyme catalysis. However, the specific
protein contacts provided by RT to bind template-primer, along with a
detailed structural description of these interactions, remain elusive.
Molecular modeling has been successfully applied to the RT/DNA crystal
structure in an attempt to examine specific protein-DNA interactions
since the resolution of the crystal structure was insufficient to
define these interactions (15, 16). The refined structure of the
Fab/DNA/RT complex (12) and the x-ray crystal structure of a covalently
trapped DNA/dNTP/RT complex (13) confirm many of the predictions of
this model.
-helix H (H, residues 255-268) (17, 18), but not -helix I (I, residues 278-286) (19),
influence nucleic acid binding and frameshift fidelity of
polymerase-competent enzyme. Most importantly, several residues of H
(i.e. Gln258, Gly262, and
Trp266) are part of a motif referred to as the minor groove
binding track (MGBT) (15). The MGBT is situated in the minor groove of
the template-primer stem where DNA undergoes a 45° bend that accompanies the change from A-like to B-form DNA (20), and makes "nonspecific" sugar and base contacts two to six base pairs
upstream of the 3'-terminus of the primer (15, 16). An earlier study analyzing the effects of site-defined styrene oxide (SO) DNA adducts on
RT-mediated DNA synthesis suggested that this monocyclic aromatic hydrocarbon major groove adduct causes RT to terminate polymerization. Since these major groove lesions do not significantly alter the solution structure of DNA (21, 22), it was suggested that the adducts
may restrict DNA bending in the template-primer stem and/or interfere
with key RT-DNA contacts (possibly those supplied by MGBT residues)
(23). In this report, we examine whether the major groove SO lesions
induce structural alterations in the DNA minor groove that are
monitored by residues known to interact in the minor groove in the
vicinity of the DNA bend (15, 16). To monitor structural changes in the
minor groove, mutants have been created at residues Gly262
and Trp266 that are known to affect DNA binding (16-18).
Eight mutants at residue 266 have been analyzed and compared with
previous work (16) to ascertain whether the influence of the SO lesions
occurs in a systematic manner consistent with altered DNA interactions with this residue. By examining the replication profiles of wild-type RT and Gly262 and Trp266 mutant enzymes on
SO-adducted templates, we have manipulated potential nucleic acid
interactions at the protein surface as the position of the SO
lesion changes as the primer is progressively extended. Analyses of our
results further define the structural role of these residues, as well
as confirm their sites of interaction during nucleic acid synthesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (6000 Ci/mmol, NEN Life
Science Products) using T4 DNA kinase. The 17-mer primer 3' hydroxyl
was positioned 7 nucleotides upstream of the unadducted or adducted
adenine. Template-primers (10:1 molar ratios) were heated to 65 °C
for 25 min in the polymerization reaction buffer (33 mM
Tris-OAc, pH 7.2, 66 mM KOAc, 10 mM MgOAc) and
then allowed to cool slowly to less than 35 °C. By analyzing an
aliquot of the annealing mixture on a nondenaturing 10% polyacrylamide gel, it was determined than greater than 95% of the labeled primers were annealed to their respective templates. Deoxynucleoside
triphosphates were added to a final concentration of 500 µM, and the final reaction mixture also contained 200 µg/ml bovine serum albumin and 1 mM dithiothreitol.
Reactions were initiated at 37 °C in a 10-µl volume by enzyme
addition. Aliquots (2.5 µl) were removed and added to 5.0 µl of
stop buffer (95% (v/v) formamide, 20 mM EDTA, 0.05% (w/v)
bromphenol blue, 0.05% (w/v) xylene cyanol). DNA samples were
separated by electrophoresis through 15% polyacrylamide, 8 M urea gels at 2000 V for 3 h. Further reaction
details are indicated in the figure legends. The relative intensities
of the termination sites were quantified using either a Molecular
Dynamics PhosphorImager or STORM860 of autoradiographs. Termination
probabilities at each site were calculated as the ratio of products at
a site to the products at that site plus all greater length products. Percentages were calculated around a 10-nucleotide window around the
adducted site. All data averaging and analysis of standard deviations
represent the compilation of three or four independent experiments,
except for data on the G262A mutant polymerase, in which only two data
sets were collected.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Template construction and sequence. The
asterisk denotes the position of the
N6-adenine R- and S-SO
lesion.
To simplify analysis of the experiments, primer extension reactions were conducted under conditions allowing single encounters between RT and the template-primer (i.e. most of the primer is left unextended). Consequently, the data could be analyzed in terms of termination probabilities, or the percentage of polymerase molecules that dissociate at each template base (29).
R- and S-SO Lesions in a Template-Primer Construct Do Not
Significantly Terminate DNA Synthesis by Wild-type RT--
In a
previous report, we demonstrated that site-specific
N6-adenine R- or S-SO
lesions situated in a 33-mer template terminated RT extension of a
17-mer primer 3-5 bases after adduct bypass (23). However, extension
past these adducts by wild-type RT on a 63-mer template primed with a
17-mer primer was essentially unhindered by the SO modification, based
on limited termination observed in the vicinity of the lesion (Fig.
2). This apparent discrepancy is due to
the different template-primers used in these studies. The local
sequence context of the R- and S-SO lesions in
this work (5'-GGCAGGTGGTG-3', where the underlined adenine was modified) and the previous study
(5'-CGGACAAGAAG-3', where the SO adducts were
localized to one of the three underlined adenines) are distinct. Single
base changes in the nucleotides flanking the SO adduct can alter
termination probability of RT on these substrates by as much as 4-fold
(23). Thus, the difference in the local sequence contexts between the
adducted template-primer constructs could readily explain the
differences in adduct bypass.
|
Additionally, the 63-mer template shown in Fig. 1 would have a much longer single-stranded overhang than the template-primer substrate characterized previously (23) when RT encountered the adduct. It has been suggested that optimal RT binding to nucleic acid requires an overhang of 6 or 7 nucleotides (14, 30), yet the region 3-5 bases after adduct bypass where maximal RT termination was observed only provided a 3-6-nucleotide single-stranded overhang. Since that template-primer may not offer an "optimal" substrate for RT binding, the termination probabilities calculated at certain positions may have been amplified by the weak DNA interactions occurring near the end of the template.2 In contrast, the 63-mer template primed with a 17-mer primer studied here provides a 23-nucleotide overhang once the polymerase encounters the adduct. As a result, this template-primer construct is an optimal (i.e. fully stabilizing) substrate in the region analyzed. Since the termination probabilities in this study are analyzed relative to the unadducted template-primer, the reason for the apparent differences with the previous work does not affect our interpretations in this study.
G262A and W266A RT Are Less Processive and Exhibit More
Adduct-directed Termination than Wild-type RT--
Characterization of
a "horizontal"3 scan of
alanine mutants in H implicated Gly262 and
Trp266 as being important in RT binding to template-primer
(17). Although the activity of both mutants was comparable to wild-type
enzyme, the mutants displayed dissociation rate constants for
template-primer that were several orders of magnitude greater than
wild-type RT. These mutants also exhibited a lower fidelity than
wild-type enzyme for template-primer slippage initiated errors (18).
Since these results strongly suggested that Gly262 and
Trp266 influence RT interactions with template-primer, the
behavior of each alanine mutant at these two residues on SO-adducted
DNA was analyzed (Fig. 2). In contrast to the activity of wild-type RT
(lanes 1-3) on the SO-adducted substrates, both
the W266A (lane 6) and the G262A (lane
9) enzymes terminated DNA synthesis 1-3 bases after
bypassing the lesion (Fig. 2). The findings that the dominant
termination sites arose after synthesis past the adduct, as opposed to
opposite or one base 3' to the adduct, are consistent with earlier
results (23). As observed earlier with M13 DNA, both G262A RT and W266A
RT were poorly processive as compared with the wild-type enzyme (18).
The contrast between the wild-type RT and the mutant enzymes, G262A and W266A is more clearly illustrated in Fig. 2B, where termination probability is plotted at each nucleotide position within a 10-nucleotide window around the adducted site. The position of the SO adduct is designated 0, and DNA synthesis is from left to right. Although the termination probabilities manifested by wild-type RT (upper panel) were nearly indiscriminate of the SO lesion, the replication profiles of the G262A and W266A mutants were strongly influenced by one or both of the styrene adducts. For example, when W266A replicated DNA containing the S-SO adduct, significantly elevated termination was observed 1, 3, and 4 nucleotides after bypass of the lesion (sites designated +1, +3, and +4, respectively). This termination was severalfold greater for the template containing the S-SO adduct than for the unadducted template or the template containing the R-SO lesion. Similar results were observed with G262A in which the major termination sites were +1 and +2 with the S-SO template. Much like the W266A mutant, translesion synthesis past the S-SO adduct by G262A induced more termination than replication past the R-SO lesion.
"Vertical" Scan Mutants of Trp266 Differ Dramatically in Their Ability to Bypass R- and S-SO Adducts-- Given the key role of Trp266 in RT-nucleic acid interactions several base pairs upstream of the polymerase active site, we examined whether this side chain can recognize changes in the DNA minor groove during replication past the SO adduct. To do this, we examined the ability of seven vertical scan mutants of residue 266, in addition to W266A, to bypass the SO adducts. Alternate side-chain substitutions included two aromatic (W266Y, W266F), three aliphatic (W266I, W266L, W266V), and two charged side chains (W266R, W266E). These mutants have been extensively studied and demonstrated to alter RT-nucleic acid interactions in a manner that indicates that Trp266 contributes both van der Waals and hydrogen bonding interactions (16). We are therefore in a position to decipher whether translesional DNA synthesis also occurs in a manner consistent with altered interactions with residue 266.
Primer extension by these mutants is shown in Fig.
3A. The termination pattern by
the wild-type RT and two mutants with aromatic substitutions (W266F and
W266Y) was similar, in that both mutants bypassed the SO adducts with
termination probabilities comparable to the wild-type enzyme. Both
W266F and W266Y, however, demonstrated a slight elevated termination
probability at the +1 position after bypassing the S-SO
(i.e. when the adduct is one base into the template-primer
stem).
|
In contrast, synthesis by the aliphatic side-chain mutants, including W266A, revealed a marked increase in termination probability at numerous nucleotides beyond and including the SO-adducted base (Fig. 3B). The increases in adduct-dependent termination by these RT mutants were most prominent at the +1, +3, and sometimes +4 sites, when the adduct was positioned in the template-primer stem. In all cases, adduct-dependent increases in termination probability were more pronounced with the S-SO adduct than the R-SO adduct.
The W266R mutant bypassed the SO lesion with efficiencies comparable to
that of some of the aliphatic side-chain mutants. Again, the +1
position dominated the spectrum of termination sites, and
adduct-dependent increases were seen at 
1, +1, and +3.
Although the R-SO adduct did increase termination slightly
at some sites (1, +1, +3), the largest increases were caused by the
S-SO lesion. By comparison, the W266E mutant exhibited such
limited processivity under these conditions that most of the extended
primers never reached the adducted site. However, increased termination
probabilities were most evident at +3 for R-SO, and 0, +1,
and +3 for S-SO.
The termination probability at a target site is greater for a
polymerase with low processivity relative to a processive polymerase. The fraction of termination that is due to a DNA adduct cannot be
accurately calculated unless a correction is made for the intrinsic processivity of the polymerase (31). Without such a correction, a
polymerase with low processivity may exhibit a higher termination probability at an adducted site than a polymerase with a higher processivity simply because termination is highly probable at that
position, whether the adduct is present or not. To better appreciate
the consequences of the adduct on termination, we replotted the data in
Fig. 3B after dividing the adduct-dependent
termination probability at each template nucleotide position by the
termination probability at the corresponding position in the unadducted
template. This analysis adjusts for the sequence-specific,
adduct-independent termination on DNA, leaving the residual termination
probability that results from the effect of the lesion on replicative
bypass DNA synthesis. The results of these analyses are shown in Fig. 4 for the R-SO and
S-SO templates (panels A and
B, respectively). Although termination induced by
R-SO lesions was consistently lower than that induced by
S-SO lesions, the maximum
R-SO-dependent terminations for W266A, W266I,
and W226V were still quite large in some instances (e.g.
more than 5-fold at position +3, Fig. 4A). Indeed, the +3
position was the site of maximum termination probability on the
R-SO template for all six of the non-aromatic substitutions.
Similar results were obtained after analysis of the
S-SO-induced termination sites, except that the +1 position was the site of maximum termination probability on this template for
all of the substitutions at residue 266 except for W266E. Given that
the R- and S-SO adducts likely differ in their
orientation on DNA and in their ability to terminate RT polymerization,
the finding that both lesions induce termination after lesion bypass at
similar sites along the DNA template supports the conclusion that these
major groove lesions induce structural alterations that are monitored
in the DNA minor groove by residue 266 upstream of the polymerase
active site.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this report, we present evidence that residues Gly262 and Trp266 can act as "protein sensors" of structural perturbations in the DNA minor groove upstream of the polymerase active-site. By studying altered forms of the enzyme (via amino acid substitutions) and the template-primer (by placing site-specific DNA modifications in the template), one can modulate protein-DNA interactions from the perspectives of the protein and the DNA. In this way, the binding interactions between RT and DNA can be tightly coupled and uncoupled, presenting a more detailed view of how the two macromolecules interact.
The results show that the predominant sites of R- and S-SO adduct-directed termination by the G262A and W266A mutants of RT occurred not opposite or immediately 3' to the lesion, but instead, 1-3 bases after translesion DNA synthesis. This finding is in excellent agreement with the position of Trp266 relative to the DNA as revealed in the model of the RT-DNA complex, where Trp266 is nearest the +3 base pair (15, 16), and confirmed by x-ray crystal structures of RT-DNA complexes (12, 13). As a result, the data presented here indicate that residue 266 is precisely positioned to detect lesion-induced changes in the minor groove.
In general, the R-SO lesion induced the most relative termination of DNA synthesis by RT when situated at the +3 position, whereas the S-SO lesion caused the most relative termination at the +1 position (Fig. 4 and Table I). This difference in the position of relative termination between the two adducts is consistent with the orientations of the R and S enantiomers in duplex DNA as determined by 1H NMR. The styryl ring in R-SO points toward the 5'-end of the template (i.e. in the direction of polymerization) (21), but the styryl ring of S-SO points toward the 3'-end of the template (i.e. facing the oncoming polymerase) (22). Thus, a perturbation in the DNA that is triggered by an S-SO adduct would be expected to be detected by the polymerase before a similar distortion created by an R-SO lesion occupying the same position in the template.
|
Since significant termination occurs at the +1 as well as the +3
position, it is also possible that modification of Trp266
may also result in "local" side-chain movements. In this context, the model structure (15, 16), as well as the published crystal structures of RT-DNA complexes (12, 13), indicate that there are van
der Waals contact between Trp266 and Tyr232.
Tyr232 is part of the 
12-13 loop that is observed to
interact with the DNA backbone between the first two nucleotides of the
primer strand. This motif has been termed the "primer grip" (20)
and alteration of Trp232 by mutagenesis has demonstrated
the functional importance of this residue (32, 33). Thus, reducing the
size of the side chain at residue 266 could result in a change in the
position of Tyr232 (i.e. primer grip) resulting
in altered interactions near the primer 3'-terminus.
Substitutions of Trp266 with an aromatic side chain (W266F, W266Y) were not particularly deleterious to RT function. This is not surprising, given the conservative nature of the mutation. However, Trp266 substitutions by aliphatic side chains both decreased enzyme processivity and increased adduct-directed termination. These findings are consistent with the strong correlation observed between buried apolar surface area, DNA binding affinity, and frameshift fidelity (16). In addition, molecular modeling of the charged alternate side chains at residue 266 (glutamate and arginine) indicate that these side chains can form hydrogen bonds with surrounding residues. In the case of the glutamate substitution, the carboxylate side chain is within hydrogen bonding distance to the Gln269 side-chain amide, which is also part of the MGBT. Alanine substitution for this residue (Q269A) diminishes the DNA binding properties of this mutant enzyme (15). Repulsive charge-charge interactions between the carboxylate of the glutamate side chain and the negatively charged phosphates in the DNA backbone may also contribute to the inability of this mutant to polymerize efficiently when the lesion, particularly S-SO, is positioned in the template-primer stem. Moreover, there is a significant loss of van der Waals interactions upon the glutamate substitution for Trp266 (16). In contrast, arginine, like tyrosine, has the ability to form a hydrogen bond. In the model structure, one of the nitrogens of the guanidinium group is within hydrogen bonding distance to N3 of a template purine. The model is supported by the data comparing adduct-directed termination by the W266R and W266E RT mutants. The W266R mutant was perhaps the least detrimental, aside from the conservative aromatic side-chain substitutions, to adduct bypass, whereas the W266E mutation was by far the most detrimental due to the combined effects of a loss in processivity and enhanced termination.
It is important to note that even the wild-type enzyme shows a small increase in termination at both the +1 and +3 positions after bypassing the R-SO lesion. However, the non-aromatic side-chain substitutions at residue 266 caused a disproportionate increase in relative termination at +1 and +3. In other words, when the termination probabilities (Fig. 3) are corrected for the intrinsic processivity of each mutant (Fig. 4) and then compared with the relative termination probability exhibited by wild-type enzyme, termination at the +1 and +3 positions are increased significantly for each mutant (Table I). Indeed, the +3 position, without exception, exhibits the greatest normalized relative termination probability for templates with the R-SO lesion. Thus, the vertical scan at Trp266 results in RT mutants that consistently terminate synthesis at the +1 and +3 sites, even though the normalized relative termination probability through most other sites within the 10 nucleotide window was largely unaffected. A similar conclusion can be drawn from the S-SO normalized relative termination probabilities (Table I).
A priori, it is perhaps surprising that the adenine
N6-SO adducts direct RT termination. The SO
lesions used in this study are known to lie in the major groove without
causing a significant perturbation to the local DNA structure (21, 22).
In contrast, DNA binding by RT is predominantly through the DNA minor
groove and RT is unable to bypass R- or S-SO
lesions situated in the DNA minor groove (34). However, it is important
to note that the sites of termination (positions +1 and +3 in the DNA
template) correspond to the major groove adduct being positioned in the template-primer stem in the region of the RT-induced DNA bend. Although
the structural significance of the DNA bend during enzyme catalysis is
unclear, one possibility is that the MGBT residues displace the spine
of water molecules along the minor groove, resulting in a widening of
the minor groove and narrowing of the major groove. This would favor a
structural transition in the DNA to be more A-like. However, if a
monocyclic adduct, such as SO, is "sandwiched" in the major groove,
groove narrowing may be restricted. Thus, one explanation for the
adduct-induced termination of DNA synthesis by the Trp266
mutants is that the SO lesion causes the DNA to become less flexible and more resistant to structural transitions, such as groove widening or narrowing. In this context, it has been demonstrated that RT has low
processivity on rigid DNA (35, 36). This adduct-imposed rigidity in the
DNA helix, coupled with weak mutant RT DNA binding, should result in
greater termination by the mutant polymerases, whereas the wild-type
RT, capable of much stronger binding to DNA, does not terminate
synthesis at efficiently when the adduct is positioned near
Gly262 or Trp266.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Amanda McCullough for preparation of the figures and careful reading of the manuscript. We also thank Rosemary Martinez for careful preparation of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grants ES05355 and ES06766 (to R. S. L.) and National Institutes of Health Grants ES06492 and ES06839 (to S.H.W.) and by grants (to T. A. K. and S. H. W.) from the National Institutes of Health Intramural AIDS Targeted Antiviral Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Ambion, Inc., 2130 Woodward St., Austin, TX 78744.
** Holder of the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. To whom correspondence should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.
Published, JBC Papers in Press, March 9, 2000, DOI 10.1074/jbc.M000279200
2 It should be noted that weak RT binding near the end of SO-adducted templates does not alter the interpretation or conclusions of our previous work (23). The consequences of a lower DNA binding affinity in the vicinity of the adduct would be fully manifested in the control (i.e. unadducted template-primer).
3 A "vertical scan" describes the site-directed mutagenesis of a single protein residue to several alternate amino acids. In contrast, a "horizontal scan" represents mutagenesis of a series of adjacent protein residues to the same amino acid.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; SO, styrene oxide; MGBT, minor groove binding track.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Kuriyan, J., and O'Donnell, M. (1993) J. Mol. Biol. 234, 915-925 |
| 2. | Stillman, B. (1994) Cell 78, 725-728 |
| 3. | Kunkel, T. A., Patel, S. S., and Johnson, K. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6830-6834 |
| 4. | Roberts, J. D., Bebenek, K., and Kunkel, T. A. (1988) Science 242, 1171-1173 |
| 5. | Williams, K. J., Loeb, L. A., and Fry, M. (1990) J. Biol. Chem. 265, 18682-18689 |
| 6. | Preston, B. D., Poiesz, B. J., and Loeb, L. A. (1988) Science 242, 1168-1171 |
| 7. | Ji, X., Klarmann, G. J., and Preston, B. D. (1996) Biochemistry 35, 132-143 |
| 8. | Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C., and Harrison, S. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1222-1226 |
| 9. | Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790 |
| 10. | Smerdon, S. J., Jager, J., Wang, J., Kohlstaedt, L. A., Chirino, A. J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3911-3915 |
| 11. | Ding, J., Das, K., Tantillo, C., Zhang, W., Clark, A. D., Jr., Jessen, S., Lu, X., Hsiou, Y., Jacobo-Molina, A., Andries, K., Pauwels, R., Moereels, H., Koymans, L., Janssen, P. A. J., Smith, R., Koepke, M. K., Michejda, C., Hughes, S. H., and Arnold, E. (1995) Structure 3, 365-379 |
| 12. | Ding, J., Das, K., Hsiou, Y., Sarafianos, S. G., Clark, A. D., Jr., Jacobo-Molina, A., Tantillo, C., Hughes, S. H., and Arnold, E. (1998) J. Mol. Biol. 284, 1095-1111 |
| 13. | Huang, H., Chopra, R., Verdine, G. L., and Harrison, S. C. (1998) Science 282, 1669-1675 |
| 14. | Patel, P. H., Jacobo-Molina, A., Ding, J. P., Tantillo, C., Clark, A. D., Jr., Raag, R., Nanni, R. G., Hughes, S. H., and Arnold, E. (1995) Biochemistry 34, 5351-5363 |
| 15. | Bebenek, K., Beard, W. A., Darden, T. A., Li, L., Prasad, R., Luxon, B. A., Gorenstein, D. G., Wilson, S. H., and Kunkel, T. A. (1997) Nat. Struct. Biol. 3, 194-197 |
| 16. | Beard, W. A., Bebenek, K., Darden, T. A., Li, L., Prasad, R., Kunkel, T. A., and Wilson, S. H. (1998) J. Biol. Chem. 273, 30435-30442 |
| 17. | Beard, W. A., Stahl, S. J., Kim, H.-R., Bebenek, K., Kumar, A., Strub, M.-P., Becerra, S. P., Kunkel, T. A., and Wilson, S. H. (1994) J. Biol. Chem. 269, 28091-28097 |
| 18. | Bebenek, K., Beard, W. A., Casas-Finet, J. R., Kim, H.-R., Darden, T. A., Wilson, S. H., and Kunkel, T. A. (1995) J. Biol. Chem. 270, 19516-19523 |
| 19. | Beard, W. A., Minnick, D. T., Wade, C. L., Prasad, R., Won, R. L., Kumar, A., Kunkel, T. A., and Wilson, S. H. (1996) J. Biol. Chem. 271, 12213-12220 |
| 20. | Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324 |
| 21. | Feng, B., Zhou, L., Passarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036 |
| 22. | Feng, B., Voehler, M., Zhou, L., Passarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 7316-7329 |
| 23. | Latham, G. J., and Lloyd, R. S. (1994) J. Biol. Chem. 269, 28527-28530 |
| 24. | Harris, C. M., Zhou, L., Strand, E. A., and Harris, T. M. (1991) J. Am. Chem. Soc. 113, 4328-4329 |
| 25. | Sawaya, M. R., Prasad, P., Wilson, S. H., Kraut, J., and Pelletier, H. (1997) Biochemistry 36, 11205-11215 |
| 26. | Doublié, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. (1998) Nature 391, 251-258 |
| 27. | Kiefer, J. R., Mao, C., Braman, J. C., and Beese, L. S. (1998) Nature 391, 304-307 |
| 28. | Li, Y., Korolev, S., and Waksman, G. (1998) EMBO J. 17, 7514-7525 |
| 29. | Abbotts, J., SenGupta, D. N., Zon, G., and Wilson, S. H. (1988) J. Biol. Chem. 263, 15094-15103 |
| 30. | Wöhrl, B. M., Tantillo, C., Arnold, E., and Le Grice, S. F. J. (1995) Biochemistry 34, 5343-5350 |
| 31. | Latham, G. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (1995) Chem. Res. Toxicol. 8, 422-430 |
| 32. | Ghosh, M., Jacques, P. S., Rodgers, D. W., Ottman, M., Darlix, J. L., and Le Grice, S. F. J. (1996) Biochemistry 35, 8553-8562 |
| 33. | Powell, M. D., Ghosh, M., Jacques, P. S., Howard, K. J., Le Grice, S. F. J., and Levin, J. G. (1997) J. Biol. Chem. 272, 13262-13269 |
| 34. | Forgacs, E., Latham, G., Beard, W. A., Prasad, R., Bebenek, K., Kunkel, T. A., Wilson, S. H., and Lloyd, R. S. (1997) J. Biol. Chem. 272, 8525-8530 |
| 35. | Lavigne, M., Roux, P., Buc, H., and Schaeffer, F. (1997) J. Mol. Biol. 266, 507-524 |
| 36. | Lavigne, M., and Buc, H. (1999) J. Mol. Biol. 285, 977-995 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Xu, L. Oum, L. S. Beese, N. E. Geacintov, and S. Broyde Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase Nucleic Acids Res., July 26, 2007; 35(13): 4275 - 4288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dash, T. S. Fisher, V. R. Prasad, and S. F. J. Le Grice Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference J. Biol. Chem., September 22, 2006; 281(38): 27873 - 27881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kaushik and S. Kukreti Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human {beta}-globin gene cluster Nucleic Acids Res., July 19, 2006; 34(12): 3511 - 3522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Dash, J. P. Marino, and S. F. J. Le Grice Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference J. Biol. Chem., February 3, 2006; 281(5): 2773 - 2783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bibillo, D. Lener, A. Tewari, and S. F. J. Le Grice Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer J. Biol. Chem., August 26, 2005; 280(34): 30282 - 30290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Yi-Brunozzi and S. F. J. Le Grice Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (-)-DNA Template and (+)-RNA Primer J. Biol. Chem., May 20, 2005; 280(20): 20154 - 20162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Skasko, K. K. Weiss, H. M. Reynolds, V. Jamburuthugoda, K. Lee, and B. Kim Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases J. Biol. Chem., April 1, 2005; 280(13): 12190 - 12200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Dash, H.-Y. Yi-Brunozzi, and S. F. J. Le Grice Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template J. Biol. Chem., August 27, 2004; 279(35): 37095 - 37102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Cannistraro and J.-S. Taylor DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase {eta} J. Biol. Chem., April 30, 2004; 279(18): 18288 - 18295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Rausch, J. Qu, H. Y. Yi-Brunozzi, E. T. Kool, and S. F. J. Le Grice Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection PNAS, September 30, 2003; 100(20): 11279 - 11284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lener, M. Kvaratskhelia, and S. F. J. Le Grice Nonpolar Thymine Isosteres in the Ty3 Polypurine Tract DNA Template Modulate Processing and Provide a Model for Its Recognition by Ty3 Reverse Transcriptase J. Biol. Chem., July 11, 2003; 278(29): 26526 - 26532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. Fisher, T. Darden, and V. R. Prasad Mutations Proximal to the Minor Groove-Binding Track of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Differentially Affect Utilization of RNA versus DNA as Template J. Virol., May 15, 2003; 77(10): 5837 - 5845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lavigne, L. Polomack, and H. Buc DNA Synthesis by HIV-1 Reverse Transcriptase at the Central Termination Site. A KINETIC STUDY J. Biol. Chem., August 10, 2001; 276(33): 31429 - 31438. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||
