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J. Biol. Chem., Vol. 275, Issue 36, 28033-28038, September 8, 2000
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Active Site
Modulate Single-base Deletion Error Rates*
§,,¶
From the Laboratory of Molecular Genetics
and ¶ Laboratory of Structural Biology, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
Received for publication, April 24, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The structures of open and closed conformations
of DNA polymerase One source of the nucleotide deletion and addition mutations often
associated with human diseases is inaccurate DNA replication. Single-base deletions are among the most common replication errors (1).
The intermediates for deletions and additions were initially suggested
to result from slippage of the two DNA strands (2). When this occurs in
a homopolymeric sequence, misaligned template-primers may be stabilized
by correct base pairs (Fig.
1A). As the run length
increases, there is a concomitant increase in the number of correct
base pairs that can stabilize the misalignment. This is
illustrated in Fig. 1A for runs of 4, 6, and 8 template
thymidine residues. There is also an increase in the distance between
the 3' terminus and the extra base and an increase in the number of possible intermediates that can form; only one intermediate is shown
for each run length. These features predict that the single-base deletion error rate during DNA synthesis will increase as the length of
homopolymeric runs increases. Previous studies have shown that the
error rate of several DNA polymerases does indeed increase with
increasing run length (reviewed in Ref. 3), such that single-base
deletion rates can substantially exceed single-base substitution error
rates. The relationship between deletion rate and run length implies
that an extra template nucleotide in a misaligned duplex
template-primer resides some distance from the polymerase active site,
and that deletion error rates may be affected by changes distal to the
polymerase active site. This is supported by studies of HIV-1 reverse
transcriptase (RT)1 (4) and
Klenow fragment polymerase (5). These studies have shown that the rates
of single-base deletions and additions in homopolymeric sequences are
modulated by polymerase interactions with the DNA minor groove three to
five base pairs upstream of the active site.
(pol ) suggests that the rate of
single-nucleotide deletions during synthesis may be modulated by
interactions in the DNA minor groove that align the templating base
with the incoming dNTP. To test this hypothesis, we measured the
single-base deletion error rates of wild-type pol and lysine and
alanine mutants of Arg283, whose side chain interacts
with the minor groove edge of the templating nucleotide at the active
site. The error rates of both mutant enzymes are increased >100-fold
relative to wild-type pol . Template engineering experiments
performed to distinguish among three possible models for deletion
formation suggest that most deletions in repetitive sequences by pol
initiate by strand slippage. However, pol also generates
deletions by a different mechanism that is strongly enhanced by the
substitutions at Arg283. Analysis of error specificity
suggests that this mechanism involves nucleotide misinsertion followed
by primer relocation, creating a misaligned intermediate. The structure
of pol bound to non-gapped DNA also indicates that the templating
nucleotide and its downstream neighbor are out of register in the open
conformation and this could facilitate misalignment (dNTP or primer
terminus) with the next template base.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Three models for initiation of misaligned
intermediates for single-nucleotide deletions. For description,
see "Introduction."
Several distinct events may initiate formation of single-base deletion intermediates. Slippage of the template and primer strands has been suggested to occur during dissociation and/or reassociation of the DNA polymerase with the DNA (6, 7). This idea is supported by a correlation between the processivity of polymerization and single-base deletion and addition error rates (reviewed in Ref. 3). For those polymerases with intrinsic exonuclease activity, misalignments may form (8) or perhaps be realigned (9) during movement of the primer strand between the polymerase and the exonuclease active sites. Frameshift intermediates can also be corrected by exonucleolytic proofreading (9), albeit sometimes with lower efficiency that for base substitutions (10-12).
DNA polymerases also delete non-iterated template nucleotides during DNA synthesis. As an alternative to strand slippage, deletions of either iterated or non-iterated nucleotides may be initiated by nucleotide misinsertion (13). Misinsertion is generally rare because the active site of a DNA polymerase is designed to accept geometrically equivalent Watson-Crick base pairs and to reject base pairs differing from this geometry (see Ref. 3 for a recent review). Occasional failure of geometric selection can result in a base substitution if a mismatched terminus is extended to leave a mismatched base pair in duplex DNA. However, misinsertion immediately followed by relocation of the primer strand can result in a misaligned template-primer containing an unpaired template-strand nucleotide adjacent to a correct terminal base pair that facilitates further polymerization (Fig. 1B). The idea that misinsertion happens first, with the mismatched product then initiating a deletion mutation, is supported by several studies of DNA polymerases copying undamaged or damaged DNA. Polymerase structure-function studies indicate that nucleotide insertion fidelity depends on DNA polymerase interactions in the DNA minor groove in the active site (reviewed in Ref. 3). Here we provide a further analysis of the misinsertion-primer relocation model by determining if altered DNA polymerase interactions with the DNA minor groove in the active site also influence single-base deletion error rates.
We also consider a third model (Fig. 1C) for the origin of
single-base deletion intermediates. This model is suggested by comparison of the position of template strand nucleotides in the crystal structure of DNA polymerase (pol ) in different
conformations. The crystal structures of several DNA polymerases with
and without substrates indicate that they are either in an
"open" or a "closed" conformation (reviewed in Ref. 14). In
the closed conformation, DNA polymerases are poised for catalysis with
the base of the incoming nucleoside triphosphate hydrogen bonded to the
templating base. For example, the crystal structures of the closed
ternary pol ·DNA·ddCTP complexes (15, 16) indicate that the
templating guanine is correctly aligned and paired with a complementary
incoming ddCTP. An important feature of the closed structure of pol bound to a one-nucleotide DNA gap is that the next template base to be
copied (i.e. one base 5' to the templating base, designated n+1 in Fig. 2, panels
A and B) is moved out of the DNA helix axis (Fig.
2A). The nucleotide is stabilized by a histidine side chain in the amino-terminal lyase domain (not shown) of pol (see Ref. 17,
for a review). This displacement gives polymerase side chains access to
the minor groove at the growing end of the duplex, allowing correct
alignment that depends on Arg283, located on 
-helix N
(Fig. 2A) in the carboxyl-terminal subdomain of pol . In
a closed conformation, the side chain of Arg283 (purple
ball-and-stick in Fig. 2A) makes van der Waals contacts with
the minor groove edge of the templating base and can form a hydrogen
bond with the sugar of the n
1 template nucleotide. In contrast, in
the open conformation (green in Fig. 2), the side chain of
Arg283 does not interact with DNA (16, 18, 19). Thus, when
pol is bound to template-primer DNA and is in an open conformation, Arg283 cannot interact with the minor groove edge of the
template nucleotide and the n+1 nucleotide is not stabilized by an
aromatic side chain. In this case, the N3 atom of the template guanine
(green "n" in Fig. 2B) is moved
3.0 Å relative to its location in the closed complex (purple
"n," Fig. 2B). This difference in location is similar to the 3.4 Å difference between the position of the N3 of the
templating guanine in the closed complex compared with the position of
the adjacent template base (n+1) of the open complex (Fig.
2B). This comparison suggests that as -helix N rotates to
a closed position, the incoming nucleotide (purple ddCTP in Fig. 2B) could on occasion pair with the n+1 base,
generating a single-base deletion intermediate (Fig.
1C).
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To test these ideas, here we compare the single-base deletion error
rate of wild-type human pol to those of mutants in which the
Arg283 side chain has been changed to either lysine or
alanine. These amino acid replacements alter interactions between pol
and the DNA minor groove such that the R283K and R283A mutants have
strongly increased misinsertion rates (20, 21) and strongly reduced base substitution fidelity (18), but distinctly different base substitution specificities (22). This provides an opportunity to
examine the importance to frameshift fidelity of DNA minor groove
interactions at the active site. Moreover, the error specificity data
can be interpreted within the context of the three models shown in Fig.
1. As discussed below, we attempt to distinguish between these models
through the use of specifically designed templates that permit
detection of single-nucleotide deletions during synthesis to fill
gapped DNA substrates similar to those used by pol during base
excision repair.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials--
The sources of all materials, including
recombinant human pol derivatives, and the preparation of gapped
substrates have been described (10, 22, 23).
DNA Polymerase Reactions-- DNA synthesis reactions to fill gapped substrates and product analysis to determine error rates were performed as described (22).
Fidelity Assays--
Double stranded circular DNA substrates
contained 6-nucleotide single-stranded template gaps in the coding
sequence of the lacZ -complementation sequence of M13mp2
(22). These sequences are in the +1 reading frame, resulting in a
colorless M13 plaque phenotype. When the products of gap-filling DNA
synthesis are introduced into an Escherichia coli
-complementation strain and plated on indicator plates, polymerase
errors that restore the correct reading frame are seen as blue
revertants. The target sequences are located near the amino terminus of
the LacZ gene that has a promiscuous amino acid composition,
such that concomitant base substitutions (see below) do not limit the
ability to detect frameshift errors as blue revertants.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Deletion of Reiterated Nucleotides by Wild-type Pol --
In an
attempt to distinguish between the three models for deletion errors
illustrated in Fig. 1, we first determined the single-base deletion
error rates for wild-type pol during copying of runs of three to
eight template thymidines located within single-stranded gaps of >300
nucleotides (10). The results (Table I,
A) indicate that the deletion error rate increases with increasing run
length. This relationship has also been observed with other DNA
polymerases (3) and is predicted by the model wherein misaligned
intermediates in repetitive sequences are stabilized by correct base
pairing, as in Fig. 1A. In considering how misalignments
might be initiated, note that all the substrates used in this analysis
(Table I, A) contained a guanine as the 5'-template base flanking the
run. In this case, single-base deletions initiated by misinsertion as
is illustrated in Fig. 1B, requires misinsertion of dCMP
opposite the 5'-most T in the run followed by pairing of this dCMP with the flanking guanine. However, as the length of the run increases beyond four thymidines, the rate of single-base deletions (Table I, A)
exceeds the previously measured misinsertion and stable misincorporation rates for T·dCMP mispairs (Table I, B and C). This
implies that the majority of single-base deletions generated by
wild-type pol in homopolymeric sequences need not be initiated by
misinsertion.
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Modulation of Deletion Error Rates by Minor Groove Interactions in
the Active Site--
Next we tested the hypothesis that frameshift
errors are modulated by polymerase interactions in the DNA minor groove
that align the template base with the incoming dNTP. We compared the single-base deletion error rate of wild-type pol to those of R283K
and R283A mutants that have altered interactions with the minor groove
in the active site. These experiments were conducted with substrates
containing gaps of only 6 nucleotides, since the catalytic efficiency
of the R283K and R283A mutant polymerases is reduced (18). Thus, they
will not fill gaps of >300 nucleotides but readily filled one to five
nucleotide gaps such as those typically encountered during base
excision repair (18, 22). DNA synthesis by wild-type pol to copy a
5'-GTTTTA-3' template sequence within a 6-nucleotide gap generated
products with a revertant frequency of 37 × 10
4
(Table II). This value is consistent with
previous studies (24, 25) indicating that pol has lower frameshift
fidelity than any of the eukaryotic DNA polymerases except pol 
(26). Synthesis by the R283K and R283A mutants generated revertant
frequencies that were 16- and 38-fold higher, respectively, than
for wild-type pol (Table II). This suggests that the ability of
pol to prevent single-base deletions is modulated by the
Arg283 side chain. Sequence analysis of 32 LacZ
revertants generated by wild-type pol showed that 31 had lost one
of the T residues in the TTTT run (Fig.
3), yielding an error rate of 150 × 105 for wild type pol (i.e. one deletion
for every 670 template thymidines copied). All revertants generated by
the mutant polymerases were missing one thymidine (Fig. 3). One
revertant from the R283K reaction with the 5'-GTTTTA-3' substrate
contained a T deletion in combination with a T 
G transversion (Fig.
3). Six of 30 revertants from the R283A reaction contained a T deletion
in combination with a single-base substitution, and one contained a T
deletion in combination with a tandem double-base substitution (Fig.
3). The appearance of these base substitution errors in combination with the deletions is not unexpected, since the R283K and R283A mutant
DNA polymerases have reduced base substitution fidelity (18,
22).
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Deletion of Non-iterated Nucleotides by Wild-type and Mutant Pol
--
The above studies involve a repetitive template sequence.
Previous studies have shown that pol also generates deletions of
non-iterated nucleotides; these comprise 4-7% of all errors generated
by wild-type pol when copying undamaged DNA templates (25, 27, 28).
In contrast to the situation at repetitive sequences, these
non-iterated single-base deletions would seem less likely to be
initiated by strand slippage. This is because a one-nucleotide slip at
a non-iterated sequence would yield a template-primer containing both a
terminal mismatch and an unpaired template-strand nucleotide
(e.g. Fig. 1A, last intermediate shown). To
examine possible alternatives to the slippage model, we next measured
the single-base deletion error rates of wild-type pol and the R283K
and R283A mutants when copying a non-iterated template sequence in a
6-nucleotide gap (Table II). The base substitution specificity of the
mutant polymerases was already known for a TGA codon in a 5-nucleotide
gap (22). We therefore attempted to distinguish between the
misinsertion-primer relocation model (Fig. 1B) and the dNTP
misalignment model (Fig. 1C) by measuring deletion rates
using a template that also contained a TGA sequence, 5'-cagTGA-3'.
Deletion of a single T, G, or A will yield blue plaques, whereas
deletion of any of the first three nucleotides (i.e. cag)
generates nonsense codons in the correct reading frame and therefore
does not yield a blue plaque.
DNA synthesis by the wild-type enzyme generated products with a
revertant frequency of 2.2 × 104 (Table II), and 11 of 12 revertants subjected to sequence analysis had lost one nucleotide
(Fig. 3). This yields an error rate of 11 × 105, a
value 13-fold lower than for deletions by wild-type pol in the TTTT
run (Table II). This difference between a TTTT run and a non-iterated
sequence is consistent with the strand slippage model (Fig.
1A) and the relationship between run length and error rate
shown above (Table II).
We then determined the single-base deletion error rates for synthesis
by the R283K and R283A mutants. These mutant polymerases generated DNA
products with revertant frequencies that were elevated by 100- and
190-fold, respectively, compared with the revertant frequency observed
with wild-type pol (Table II). When 46 LacZ revertants
were analyzed by DNA sequence analysis, all were found to contain
single-base deletions (Fig. 3), including seven that also contained a
single-base substitution. Thus, the R283K and R283A mutants are strong
mutator polymerases for loss of single, non-iterated nucleotides. This
further supports the general hypothesis that interactions of the
Arg283 side chain with the minor groove edge of the
template strand modulate the rate of single-base deletions generated by
pol .
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This study of the single base deletion fidelity of human pol suggests three major conclusions. First, replacement of the Arg283 side chain with either lysine or alanine strongly
increases error rates (Table II), suggesting that the DNA minor groove
interactions in the polymerase active site provided by
Arg283 are important for proper substrate alignment that
prevents formation of deletion intermediates. Second, the correlation
between increasing deletion error rate and increasing length of a
homonucleotide run (Tables I and II), and the fact that the single-base
deletion error rates can greatly exceed misincorporation rates (Table
II), strongly support the strand slippage hypothesis for deletions in
homonucleotide runs generated by wild-type pol (Fig.
1A). Third, wild-type pol also deletes non-iterated
nucleotides, and the error rate is increased by 100-fold or more upon
replacement of the Arg283 side chain (Table II). This
implies that some deletions likely result from a non-slippage mechanism
that is modulated by DNA minor groove interactions at the active site.
We consider two possible models, misinsertion-primer relocation (Fig.
1B) and dNTP misalignment (Fig. 1C).
In an attempt to distinguish between these two models, we used the
following logic. If single-base deletions are initiated by
misinsertion, then the deletion rate should not exceed the known rate
for misincorporations that could realign to form a deletion
intermediate (Fig. 1B). For example, the rate of loss of the
T in the 5'-cagTGA-3' sequence should not exceed the rate at which dCMP
is misinserted opposite the T. This is because this misinsertion is
needed to pair with the adjacent template G in order to generate the
deletion intermediate (Fig. 1B). On the other hand, if
deletions are initiated by dCTP pairing with the adjacent template
guanine (Fig. 1C), then misincorporation of dCMP opposite T
is not necessary. In this case, the deletion error rate could, but need
not necessarily, exceed the base substitution error rate. With this
logic in mind, we used the data in Table II and Fig. 3 to calculate the
rates at which wild-type pol and the R283K and R283A mutants
deleted a T, G, or A nucleotide from the non-iterated sequence. We then
compared these rates to the known base substitution error rates of
these same polymerases at the TGA codon in a five-nucleotide gap (22).
The results (Table III) reveal that base
substitution error rates exceed deletion rates for all three
nucleotides with wild-type pol , for one of three nucleotides (loss
of A) with R283K pol and for two of three nucleotides (loss of G or
A) with R283A pol . In other words, in these situations misinsertion
rates are more than sufficient to explain the deletions by the model in
Fig. 1B. However, the rate at which the R283K mutant deletes
T or G and the rate at which the R283A mutant deletes T are similar to
the base substitution error rates. Some of these deletions may be
initiated via pairing of the incoming dNTP with the next base, as
illustrated in Fig. 1C and suggested by structural
considerations (see below).
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Superimposing the structures of the open conformation of pol bound
to non-gapped template-primer (18) with the closed ternary complex of
pol bound to one-nucleotide gapped DNA (16) indicates that the
template nucleotides (n and n+1) of the open conformation are not in
register relative to those in the closed form (Fig. 2,
panels A and B). Similarly,
superimposing this open conformation with the closed ternary complex of
pol bound to non-gapped DNA (15) results in the same observation
(not shown). The "vicinity" of the n+1 templating nucleotide in the
open conformation with the templating nucleotide (i.e. n) in
the closed form suggests that there are opportunities for the incoming
nucleoside triphosphate to base pair with the n+1 template base (dNTP
misalignment, Fig. 1C). Additionally, if an incorrect
nucleotide is inserted opposite the templating nucleotide
(i.e. n) and the misinserted nucleotide is complementary to
the n+1 templating nucleotide, then the vicinity of the n+1 template
base to the dNTP-binding site could facilitate primer relocation
following misinsertion (Fig. 1B). As illustrated by
comparing the last intermediates shown in Panels
B and C of Fig. 1, an important distinction
between the misinsertion-primer relocation model and the dNTP
misalignment model for single-base deletions is whether the
misalignment occurs before or after phosphodiester bond formation.
Making this distinction will require approaches beyond measuring
deletion errors among the completed products of DNA synthesis,
including examination of intermediate steps in the process. Relevant to
this point are studies of the rate at which pol inserts nucleotides
when copying DNA templates containing abasic sites (29) or
propanodeoxyguanosine adducts (30). In these circumstances, catalytic
efficiencies were greatest for insertion of nucleotides that were
complementary to the n+1 template base. On that basis, it was suggested
that pol generates base substitutions at sites of DNA damage by
dNTP misalignment. In like manner, dNTP misalignment could
theoretically explain some of the base substitutions generated by pol
(25, 27, 28) or by E. coli DNA Pol III (31, 32) when
copying undamaged DNA.
The balance among the deletion models shown in Fig. 1 likely depends on
both the template sequence and the DNA polymerase. Pol I family (9, 10,
33, 34), pol family (10, 35, 36) and RT family polymerases (37) and
human DNA polymerase (26) all generate single-nucleotide deletions,
but at quite different rates. Some of these are deletions of
non-iterated nucleotides that occur at rates consistent with the
misinsertion-primer relocation model (Fig. 1B). However,
structural considerations of frameshift fidelity differ among the
polymerases. For example, in contrast to the situation with pol (Fig. 2B), the templating nucleotide in the open complex of
HIV-1 RT bound to DNA (38) is located in a position similar to that
seen in the closed ternary complex poised for catalysis (39). In other
words, the templating base in the open complex of HIV-1 RT is in
register. This is illustrated by comparing the position of the
templating base n in Fig. 2, panels B and
C. This difference between pol and HIV-1 RT may relate
to differences in the nature of the subdomain movements that occur
following dNTP binding. For example, the template strand in the pol complex may be minimally constrained until the dNTP binds and helix N
rotates to a closed conformation. This motion allows the
Arg283 side chain to contact the minor groove edge of the
template strand (Fig. 2A), thus aligning it for correct
pairing. However, the opposite symmetry holds for HIV-1 RT,
i.e. the axis for rotation to a closed conformation is on
the other side of the binding pocket for the nascent base pair (Fig.
2C). This may allow stabilizing contacts between the enzyme
and the templating strand even in the open conformation, with closure
of the 3-4 loop of RT eventually correctly positioning the
incoming dNTP. The axis of rotation to a closed conformation is
similarly located on the templating-nucleotide side of the binding
pocket in pol I family DNA polymerases (reviewed in Ref. 3). Also, in
the ternary closed complex of T7 DNA polymerase (40), HIV-1 RT (39),
and pol bound to a one-nucleotide DNA gap (16), the n+1 nucleotide
is flipped out of the DNA helix and stacks with an aromatic amino acid
side chain. Thus, depending on yet to be determined conformational
dynamics as these polymerases move from open to closed conformations
following dNTP binding, the incoming dNTP may not have the opportunity
to pair with the n+1 nucleotide. Differences in the symmetry of
subdomain closure, in template nucleotide register and in template
nucleotide flipping, may explain why the insertion specificity of
Klenow fragment polymerase (a homolog of T7 pol) and HIV-1 RT do not
suggest that the incoming dNTP has paired with the n+1 nucleotide when
these enzymes copy damaged templates (30, 41). Flipping of the n+1
nucleotide may or may not modulate the single-nucleotide deletion error
rate of pol . The n+1 nucleotide is displaced and stacked with
His34 in a closed ternary complex of pol bound to DNA
containing a single-nucleotide gap (16). However, no such nucleotide
displacement was observed when the downstream DNA was single-stranded
(15, 18), as is the case for the gapped substrates used in this study. In this case, the amino-terminal 8-kDa domain (i.e.
His34) is not observed to be interacting with the DNA.
The strong frameshift mutator phenotypes of the R283K and R283A mutants
(Table II) imply that prevention of single-base deletions by wild-type
pol is modulated by side chain interactions with the DNA minor
groove at the active site. This may explain the elevated rates of
single-nucleotide deletions observed with two other pol mutants,
Y265C (27) and F272L (28). Both Tyr265 and
Phe272 occupy positions in the -helix M that rotates as
helix N opens and closes. Since -helix N contains
Arg283, substitutions of other side chains for
Tyr265 and Phe272 could indirectly alter the
ability of the Arg283 side chain to correctly align the
template strand.
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ACKNOWLEDGEMENTS |
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We thank Katarzyna Bebenek for many thoughtful discussions and Youri Pavlov and Anna Bebenek for critical comments on the manuscript.
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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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: R&D Pathogen Safety, Bayer Corp., 1200 New Hope Rd., Raleigh, NC 27610.
To whom correspondence should be addressed. Tel.:
919-541-2644; Fax: 919-541-7613; E-mail: kunkel@niehs.nih.gov.
Published, JBC Papers in Press, June 12, 2000, DOI 10.1074/jbc.M003462200
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ABBREVIATIONS |
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The abbreviations used are:
RT, reverse
transcriptase;
pol , DNA polymerase ;
pol, polymerase;
dNTP, 2'-deoxynucleoside 5'-triphosphate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) and a base
substitution is indicated by an underlined letter. The
number of times each sequence was detected and the number of revertants
sequenced is noted (N). Loss of the (underlined)
C, A, or G in the 5'-cagTGA-3' sequence is not detectable because each
of these deletions yields a LacZ coding sequence containing
a nonsense codon.