(Received for publication, December 26, 1996, and in revised form, February 19, 1997)
From We screened 67 mutants in the O-helix of
Thermus aquaticus (Taq) DNA polymerase I (pol
I) for altered fidelity of DNA synthesis. These mutants were obtained
(Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc.
Natl. Acad. Sci. U. S. A. 93, 9670-9675) by substituting an
oligonucleotide containing random sequences for codons 659-671, and
selecting for complementation of a growth defect in Escherichia
coli caused by temperature-sensitive host pol I. Thirteen mutants
decreased fidelity in a screen that employed primer extension reactions
lacking one of four complementary deoxynucleoside triphosphates
(dNTPs). Three mutants were purified and exhibited 29-68% of
wild-type specific activity. Homogeneous polymerases A661E, A661P, and
T664R extended primers further than the wild-type, synthesizing past
template nucleotides for which the complementary dNTP was absent. The
data indicate that both misinsertion of incorrect nucleotides and
extension of mispaired primer termini were increased. In a
lacZ Thermus aquaticus (Taq) DNA polymerase I
(pol I)1 is used extensively in the
amplification of DNA by the polymerase chain reaction. Based on its
primary sequence, crystal structure, and catalytic mechanism,
Taq pol I is classified in the same family as
Escherichia coli pol I (1-3). The amino acid sequences of
Taq pol I and E. coli pol I are 38% homologous,
both enzymes have 5 The O-helix in E. coli pol I is constituted of 15 amino
acids which form part of the DNA binding cleft (8). Crystallographic, biochemical, and mutagenesis studies indicate that four conserved residues in the O-helix which face toward the cleft interact with either the incoming dNTP or the template-primer. Considerable evidence
indicates that in E. coli pol I, Arg-754 and Lys-758 interact with the phosphate groups in the incoming dNTP, that Phe-762
interacts with the deoxyribose moiety in the dNTP, and that Tyr-766
binds to the template-primer (9-11). The nearly identical structures
of the polymerase domains of Taq pol I and E. coli pol I (6, 7) suggest that the corresponding amino acids in
Taq pol I have analogous functions. By using random sequence mutagenesis together with genetic complementation, we recently observed
that the O-helix of Taq pol I contains four amino acids which are immutable (or nearly immutable) and are apparently essential for catalysis in vivo (12). These residues, Arg-659,
Lys-663, Phe-667, and Tyr-671, correspond to the four above mentioned
residues in E. coli pol I. Importantly, Phe-667 in
Taq pol I and the corresponding Phe-762 in E. coli pol I are critical for substrate discrimination (13).
Substitution of Tyr for Phe results in increased incorporation of
dideoxynucleoside triphosphates. Analysis of the effects of this and
other mutations in the O-helix on the incorporation of non-complementary nucleotides may be important to understanding substrate recognition by DNA polymerases.
The fidelity of DNA polymerases is central to the accurate transmission
of genetic information. This fidelity is conferred primarily by the
accuracy of polymerization, with an additional contribution from
proofreading for enzymes which possess an editing 3 The fidelity of polymerization depends on the ability of the polymerase
site to discriminate correct from incorrect substrates, i.e.
complementary from non-complementary nucleoside triphosphates (19) and
matched from mismatched primer termini (20). A molecular understanding
of polymerization fidelity will require identification of the amino
acids which participate in substrate discrimination and elucidation of
the role(s) played by each. As recently pointed out, lack of mutants
with altered accuracy, particularly for DNA polymerases whose
three-dimensional structure is known, is a limiting factor in obtaining
a molecular description of fidelity (9). Some accuracy mutants have
been obtained by site-directed mutagenesis (9). However, this approach
does not readily permit examination of large numbers of amino acid
replacements and frequently yields mutants with markedly reduced
catalytic efficiency.
In the present work, we illustrate a different approach to obtaining
fidelity mutants of Taq pol I. We have previously used random sequence mutagenesis together with genetic complementation to
create a collection of catalytically active mutants with amino acid
replacements in the O-helix (12), and we have now screened some of
these mutants for reduced fidelity to examine the effects of the
mutations on accuracy. The importance of the O-helix in interactions
with incoming dNTPs and with the template-primer suggests that amino
acid substitutions within this segment that alter fidelity are likely
to do so by altering these interactions. Moreover, because our mutant
selection depends on complementation of a temperature-sensitive
phenotype in vivo, Taq pol I derivatives with
near wild type activity are obtained (21), and the effects of mutation
on fidelity are therefore observed in the absence of major impairments
of catalysis.
-Wild-type Taq pol I and its mutant
derivatives were selected by genetic complementation as previously
reported (12). E. coli DH5 A single colony of E. coli DH5 Purification was carried out
according to Lawyer et al. (22) with modifications. A single
colony of E. coli DH5 The lysate was centrifuged at 15,000 rpm (Sorvall, SA-600 rotor) for 15 min, and the supernatant solution was incubated at 72 °C for 20 min.
Insoluble material was removed by centrifugation. Ammonium sulfate (0.2 M) and Polymin P (0.6%) were added and the suspension was
held on ice for 1 h. After removal of the precipitate by
centrifugation and filtration through a Costar 8310 filter, the
filtrate was applied to a 3 × 8-cm phenyl-Sepharose HP (Pharmacia Biotech Inc.) column equilibrated with buffer A containing 0.2 M ammonium sulfate and 0.01% Triton X-100. The column was
washed with the same buffer (300 ml) and activity was eluted with
buffer B (TE buffer containing 0.01% Triton X-100 and 50 mM KCl). The eluate (100 ml) was dialyzed overnight against
4 liters of buffer B and loaded onto a 0.8 × 8-cm
heparin-Sepharose CL-6B (Pharmacia Biotech Inc.) column equilibrated
with buffer B. After washing with buffer B (50 ml), activity was eluted
in a 30-ml linear gradient of 50-500 mM KCl in TE buffer
containing 0.01% Triton X-100. Active fractions were collected,
dialyzed against 50 mM Tris-HCl (pH 8.0) containing 50 mM KCl and 50% glycerol, and stored at Enzyme was incubated at 72 °C for 5 min
in 50 mM Tris-HCl (pH 8.0), 2 mM
MgCl2, 100 µM each dATP, dGTP, dCTP, and
dTTP, 0.2 µCi of [3H]dATP, and 200 µg/ml activated
calf thymus DNA. Incorporation of radioactivity into an acid-insoluble
product was measured according to Battula and Loeb (23). One unit
represents incorporation of 10 nmol of dNMP in 1 h, corresponding
to 0.1 unit as defined by Perkin-Elmer.
The 14-mer primer 5 The
non-coding strand of the lacZ We have previously generated a
large collection of functional mutants of Taq pol I (12).
Extracts of E. coli expressing these mutated Taq
pol I were used to screen for mutations that alter fidelity. The
mutants were created by substituting random nucleotides for the 13 codons specifying amino acids 659-671 in the O-helix of plasmid-borne
Taq pol I. Functional mutations were then selected based on
their ability to complement the growth defect of an E. coli
strain carrying a temperature-sensitive host pol I (12). Sequence
analysis of the selected, functional mutants revealed that 4 of the 13 mutagenized amino acids, Arg-659, Lys-663, Phe-667, and Tyr-671,
tolerated no substitutions or only a few conservative substitutions, in
accord with biochemical and crystallographic evidence that Arg-659 and
Lys-663 participate in the formation of a metal-dNTP binding pocket
(10, 11). The nine remaining residues were tolerant of several or many
replacements. To explore the contribution of individual amino acids in
the O-helix to replicational fidelity, we screened functional mutants
in a primer elongation assay to identify substitutions that affect
ability to utilize non-complementary dNTPs.
We screened 67 of the 75 active mutants previously sequenced, including
all 38 with single amino acid substitutions (described in Fig.
3A of Ref. 12). Plasmids encoding the mutant polymerases were cloned, purified, and grown in E. coli, and host cells
were analyzed for expression of Taq pol I by measuring the
activity of crude extracts (see "Experimental Procedures").
E. coli DNA polymerases and nucleases were inactivated by
heating at 72 °C for 20 min. The ability of heat-treated extracts to
elongate primers in the absence of a complete complement of four dNTPs
was then determined using a set of five reactions. One reaction
contained all four complementary triphosphates while each of the others lacked a different dNTP ("minus conditions"). Elongation in the minus reactions is limited by the rate of misincorporation at template
positions complementary to the missing dNTP.
Fig. 1 illustrates representative results obtained for
wild-type Taq pol I and for mutants with elongation patterns
that differed from wild-type. In the presence of all four dNTPs, every
extract examined extended more than 90% of the hybridized primer to a product of length similar to that of the template. In the minus reactions, wild-type Taq pol I (lanes 2-6)
extended 48-60% of the primer up to, but not opposite, the first
template position complementary to the missing dNTP. The remaining
primer was terminated opposite the missing dNTP, presumably by
incorporation of a single non-complementary nucleotide, or was
terminated further downstream, presumably by extension of the mispaired
primer terminus. A variety of elongation patterns was observed for the
67 mutants. Thirteen mutants extended more of the primer and/or
synthesized a greater proportion of longer products than the wild-type
enzyme in three or four of the minus reactions (e.g. mutant
2 (lanes 12-16) which formed full-length products in
reactions lacking dGTP (lane 15) or TTP (lane
16)). This increased extension presumably reflects increased
incorporation and/or extension of non-complementary nucleotides. Other
mutants (e.g. mutant 5, lanes 27-31) extended less of the primer or synthesized shorter products than the wild-type enzyme. In several cases, different amino acid substitutions at the
same position either increased or decreased extension in comparable minus reactions.
Fig. 2 contains a compilation of amino acid replacements
in the 13 mutants that displayed increased extension in at least three
of the minus reactions. With the exception of Gly-668, one or more
substitutions that putatively reduce the accuracy of DNA synthesis were
observed for each of the 9 non-conserved amino acids. Eleven mutants
harbored substitutions at either Ala-661 or Thr-664, including several
single mutants. This initial screen with crude extracts suggested that
a large number of changes are permitted in the O-helix that do not
reduce the ability of Taq pol I to complement the growth
defect of recA718 pol A12 (12). Many of the substitutions in
the O-helix that do not reduce the ability of Taq pol I to
carry out functional complementation reduce the fidelity of DNA
synthesis in vitro.
To demonstrate that the
reduction in fidelity exhibited by crude extracts is due to the mutant
Taq pol I, we purified the wild-type enzyme as well as the
polymerase from the three single mutants Ala-661
Primer extension
assays were carried out with the homogenous mutant polymerases. Fig.
4A shows reactions incubated for 5 min at
42 °C. Wild-type Taq pol I extended most of the primer to
one nucleotide before the template position opposite the missing
complementary dNTP. Only about 30% of the primers were elongated
further. In reactions containing equivalent activity, the mutant
polymerases A661E, T664R, and A661P extended a larger proportion of the
primers past the sites where the wild-type polymerase ceased synthesis. The control enzyme I665T yielded an elongation pattern similar to that
of the wild-type enzyme. Elongation reactions with the three
polymerases were also carried out for 60 min (Fig. 4B). Again, A661E and T664R synthesized a greater proportion of longer products than obtained with the wild-type and I665T polymerases. Notably, A661E, T664R, and A661P synthesized longer products in 5 min
(Fig. 4A) than the wild-type did in 60 min (Fig.
4B).
Primer extension by purified Taq
DNA polymerases. Primer elongation was catalyzed by purified
wild-type Taq pol I or one of the mutant derivatives A661E,
A661P, T664R, or I665T. A, incubation was for 5 min at
45 °C in reactions containing all four complementary dNTPs and in reactions lacking one dNTP, as indicated. B,
incubation was for 60 min at 45 °C in reactions containing all four
complementary dNTPs and in reactions lacking one dNTPs as indicated.
C, incubation was at 45 °C for the times indicated in the
absence of dCTP. Open and filled triangles
represent the positions of the primer and the fully extended product,
respectively. The sequence of the incorporated complementary
nucleotides is given at the right. Only the primer was
loaded in lanes 1 and 7.
To further document the reduced fidelity illustrated in Fig. 4,
A and B, we carried out a time course of primer
elongation. Fig. 4C shows results for the minus dCTP
reaction catalyzed by the wild-type and T664R polymerases. Wild-type
Taq pol I extended 9% of the primers past the first
deoxyguanosine template residue within the 60-min incubation period,
but elongation past the second deoxyguanosine was not detected
(lane 6). In the same interval, T664R extended 93% of the
primer past the first template deoxyguanosine, and elongation proceeded
past as many as five template deoxyguanosines (lane 12).
Importantly, a comparable proportion of primers was extended at all
time points, despite the striking difference in the length of the
products. These time course data indicate that greater elongation
reflects increased ability to utilize non-complementary substrates and
primer termini, rather than a putative difference in the amount of
activity present. Such a difference might be hypothesized, because
activity of the individual polymerases was based on assays
utilizing a gapped DNA template-primer (i.e. activated calf
thymus DNA).
Interestingly, very little primer was extended to a length greater than
21 nucleotides in any of the minus dATP reactions (Fig. 4). Cessation
of elongation may be due to the run of two Ts in the template at
positions corresponding to a 21- and 22-mer. Similarly, extension may
have ceased at the template Gs corresponding to 33- and 34-mer products
in minus dCTP reactions, and at template As corresponding to 25- and
26-mer products in minus dTTP reactions. Apparently, extension of
primers terminating with two non-complementary nucleotides is much less
efficient than extension of primers terminating with a single
mismatched nucleotide. These results suggest that extension of
mismatched primer termini is catalyzed by a
template-dependent mechanism characteristic of DNA
polymerases, rather than by a template-independent mechanism such
as that of terminal deoxynucleotidyl transferase.
To quantitate the fidelity of DNA
synthesis by the purified polymerases, we measured the frequency of
mutations produced by copying a biologically active template in
vitro (25). The target sequence was the lacZ
Mutation frequency in the lacZ Mutation frequencies were measured after in vitro copying of
M13mp2 DNA by purified mutant and wild type Taq pol Is. Both white and pale blue plaques were scored as mutants. Each mutant plaque
was verified by replating with an equal number of wild-type M13mp2. In
this experiment, the mutation frequency observed for uncopied DNA (no
enzyme control) was 3.5 × 10 The Joseph Gottstein Memorial Cancer Research
Laboratory,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
forward mutation assay, A661E and T664R yielded
mutation frequencies at least 7- and 25-fold greater, respectively,
than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA
synthesis.
-3
exonuclease domains (4, 5), and the crystal
structures of the polymerase domains are nearly identical (5, 6). The
structure of the vestigial 3
-5
exonuclease domain of Taq
pol I is very different, however; unlike E. coli pol I,
purified Taq pol I exhibits no proofreading activity
(7).
-5
exonuclease
(14). In the case of Taq pol I, which lacks proofreading
activity, the error rate during a single round of replication has been
estimated as one misincorporation for every 9000 nucleotides
polymerized (15). This estimate is in approximate agreement with
studies on the product of polymerase chain reactions obtained after
multiple rounds of DNA synthesis in some (16) but not all studies (17).
The frequency of misincorporation by Taq pol I is greater
than that of the Klenow fragment of E. coli pol I, even when
assayed in 1 mM dNTP (conditions that substantially reduce
the Klenow-associated 3
-5
exonucleolytic activity). This comparison
implies that despite the similarity in structure of the two enzymes,
there are subtle differences that affect fidelity. Interestingly, the
fidelity of Taq pol I is markedly influenced by reaction
conditions; both base substitution and frameshift error rates of less
than 1 × 10
5 are observed at pH 5-6 (18).
Polymerase Sources, Strains, and
Oligonucleotides
[F
,
80dlacZ
M15,
(lacZYA-argF)U169, deoR, recA1, endA1, phoA, hsdR17(rk
mk+),
supE44,
, thi-1, gyrA96, relA1] was
used to express Taq pol I protein. E. coli MC1061
[hsdR, mcrR, araD,
(139
araABC
leu),
7679lacX74, galU, galK, rspL,
thi
] was used for transfection in the forward
mutation assay, and transfectants were plated on the indicator strain
E. coli CSH50 [
(pro-Blac)/F
+raD36,thi
,
ara
,proAB,
lacIqZ
M15].
Oligonucleotides were synthesized and purified by Operon Technologies
Inc. (Alameda, CA).
carrying wild-type or mutant Taq pol I was
inoculated into 40 ml of 2xYT (16 g/liter tryptone, 10 g/liter yeast
extract, 5 g/liter NaCl, pH 7.3) containing 30 mg/liter
chloramphenicol. After incubation at 37 °C overnight with vigorous
shaking, an equal amount of fresh medium with 0.5 mM
isopropyl-
-D-thiogalactoside was added, and incubation
was continued for 4 h. Cells were harvested, washed once with TE
buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and
suspended in 100 µl of buffer A (50 mM Tris-HCl, pH 8.0, 2.4 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 0.5 mg/liter leupeptin, 1 mM EDTA, 250 mM KCl). Bacteria were lysed by incubating with lysozyme
(0.2 mg/ml) at 0 °C for 2 h. The lysate was centrifuged at
15,000 rpm (Sorvall, SA-600 rotor) for 15 min, and the supernatant
solution was incubated at 72 °C for 20 min. Insoluble material was
removed by centrifugation.
carrying wild-type or mutant
Taq pol I was inoculated into 10 ml of 2xYT. Two ml of the
inoculum was immediately added to each of 5 bottles containing 1 liter
of 2xYT with 30 mg/liter chloramphenicol. After overnight incubation at
37 °C with vigorous shaking, 1 liter of 2xYT containing 30 mg/liter
chloramphenicol and 0.5 mM
isopropyl-
-D-thiogalactoside was added, and incubation
was continued for 4 h. Cells were harvested, washed once with TE
buffer, and suspended in 100 ml of buffer A. Bacteria were lysed by
incubating with lysozyme (0.2 mg/ml) at 0 °C for 2 h and then
sonicating on ice for 45 s by using a micro-tip probe (Sonifier,
Branson Sonic Power, Danbury, CT).
80 °C.
-CGCGCCGAATTCCC
was 32P-labeled at the 5
end by incubation with
[
-32P]ATP and T4 polynucleotide kinase and annealed to
an equimolar amount of the template 46-mer
5
-GCGCGGAAGCTTGGCTGCAGAATATTGCTAGCGGGAATTCGGCGCG. Crude extracts
containing 0.3-1 unit of mutant or wild-type Taq pol I were
incubated at 45 °C for 60 min in 50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 50 mM KCl, 20 µM each dATP, dGTP, dCTP, and TTP, and 1.4 ng of the
annealed template-primer. A set of four additional reactions, each
lacking a different dNTP, was carried out for each polymerase. Purified
enzyme (1 unit) was incubated for the times indicated under the same
conditions as for crude extracts. After electrophoresis in a 14%
polyacrylamide gel containing 8 M urea, reaction products
were analyzed by autoradiography. Extension was quantified by using an
NIH imaging program.2
gene contained in 200 ng of
gapped M13mp2 DNA (24) was copied by using 5 units of wild-type or
mutant Taq pol I in a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl2,
20 µM each dNTP, and 50 mM KCl. After
incubation at 72 °C for 5 min, the DNA was transfected and the
plaques were scored (15). The nucleotide sequence of mutants was
determined by using a Thermo SequenaseTM cycle sequencing
kit (Amersham LIFE SCIENCE, Cleveland, OH).
Screening for Fidelity Mutants
Fig. 1.
Screening for altered fidelity of Taq
pol I mutants in a primer extension assay. Heat-treated
extracts of E. coli expressing wild-type Taq pol
I or one of its mutant derivatives were used to catalyze primer
elongation in the absence of a full set of complementary dNTPs. For
each pol I, 32P-labeled primer was extended in a control
reaction with all four dNTP and in a set of four additional reactions
in which one of the dNTPs was omitted, as indicated in the figure.
Elongation patterns for seven extracts are shown to exemplify results
of the screen, including wild-type Taq pol I (lanes
2-6) and a catalytically non-functional derivative (lanes
32-36) in which the O-helix was replaced with another sequence
(12). M1, A661E, I665T; M2, A661P, N666I; M3, T664N, L670I; M4, F667Y;
M5, R660K. The non-functional mutant was displayed in a separate
portion of the gel and spliced with the others. Open and
filled triangles represent the positions of the primer and
the fully extended product, respectively. Positions of nucleotides
complementary to the template are shown at the right.
[View Larger Version of this Image (70K GIF file)]
Fig. 2.
Low fidelity mutants of Taq pol I
identified in the primer extension screen. Amino acid replacements
are listed for mutants that elongated primers to a greater extent than
wild-type Taq pol I in at least three of the "minus"
reactions in the primer extension screen (see Fig. 1). Amino acid
substitutions at positions Arg-659 through Tyr-671 are shown below the
wild-type (WT) sequence. Mutant designations at the left are
the same as described previously (12). Note that Arg-659, Lys-663,
Phe-667, and Tyr-671, for which no replacements are listed, were
previously found to be immutable or nearly immutable (12).
[View Larger Version of this Image (8K GIF file)]
Glu (A661E),
Ala-661
Pro (A661P), and Thr-664
Arg (T664R). As a control, we
also purified enzyme from the single mutant Ile-665
Thr (I665T),
which the screen suggested would have no alteration in fidelity. As
shown in Fig. 3, each polymerase was purified to
apparent homogeneity and had mobility corresponding to the expected
molecular weight of 99,000. The specific activities of the purified
enzymes, determined in assays with activated calf thymus DNA, were
66,000, 45,000, 19,000, 23,000, and 30,000 units/mg protein for the
wild-type, A661E, A661P, T664R, and I665T, respectively. That the
mutated enzymes retain at least 29% of wild-type activity in
vitro is in accord with their ability to complement the growth
defect caused in E. coli by temperature-sensitive host pol
I, and ensures that analysis of fidelity will not be complicated by
major impairments of catalytic efficiency.
Fig. 3.
SDS-polyacrylamide gel electrophoresis of
purified Taq DNA polymerases. Purified wild-type or
mutant pol I (0.5 µg) was heated at 72 °C for 10 min in buffer
containing 1% SDS, 50 mM Tris-HCl (pH 6.8), 10% glycerol,
1% 2-mercaptoethanol. Samples were subjected to electrophoresis in a
7.5% SDS-polyacrylamide gel (53) and the gel was stained with
Coomassie Brilliant Blue R. The positions of size markers are indicated
at the left.
[View Larger Version of this Image (82K GIF file)]
Fig. 4.
[View Larger Version of this Image (45K GIF file)]
gene
located within a single-stranded region in gapped circular
double-stranded M13mp2 DNA (26). The gapped segment was filled by
synthesis with the wild-type or mutant enzymes. The double-stranded
circular product was transfected into E. coli, and the
mutation frequency was determined by scoring white and pale blue mutant
plaques. A comparison of the specific activities and mutation
frequencies of the purified enzymes is presented in Table
I. After synthesis by wild-type Taq pol I,
the mutation frequency was not greater than that of the uncopied
control. Synthesis by A661E and T664R gave rise to mutation frequencies
more than 7- and 25-fold greater, respectively, than that of the
wild-type polymerase.
forward mutation assay
3, greater than that of DNA
copied with the wild-type, and therefore not subtracted from any of the
mutation frequencies.
Taq pol I
Specific
activity
Plaques scored
Mutation frequency
Total
Mutant
units/mg
×10
3
WT
66,000
8,637
22
2.5
A661E
45,000
6,782
116
17.1
T664R
23,000
5,148
324
62.9
A sample of independent, randomly chosen mutants produced by T664R was
characterized by DNA sequence analysis. Both base substitutions and
frameshifts were found throughout the targeted lacZ gene and its regulatory sequence. Of the 64 independent plaques, 57 had
mutations in the target. Other mutations presumably occurred outside
the target region. Some had more than one base substitution and a total
of 66 mutations were observed (Fig. 5). Among them, 61 were base substitutions. Transitions (38/61) were more frequent than
transversions (23/61). T
C transitions accounted for 31 of 61 base
substitutions, while T
A (9/61), A
T (8/61), and G
A
(5/61) substitutions were less frequent. This base substitution spectrum is essentially the same as that reported for wild-type Taq pol I (27). From these data, the base substitution
fidelity of T664R can be calculated according to Tindall and Kunkel
(27) as 8.6 × 10
4 or 1 error per 1200 nucleotides. On the basis of the five frameshift mutants detected, the
frameshift error can be calculated as 4.9 × 10
5 or
1 error per 20,000 nucleotides.
We have utilized random sequence mutagenesis and a genetic
complementation system to obtain a large series of catalytically hardy
mutants in the O-helix of Taq pol I (12). In the present work, we screened preparations of 67 of the mutant enzymes for alterations in the fidelity of DNA synthesis by measuring elongation of
DNA primers in reactions lacking one of four complementary dNTPs.
Thirteen of the mutants displayed reduced fidelity compared with that
of the wild-type polymerase in this primer extension assay. Three low
fidelity enzymes were purified to homogeneity, found to have specific
activities that were at least 29% of wild-type, and re-examined in the
primer extension assay. Relative to the wild-type enzyme, the purified
mutant enzymes extended a greater proportion of primers to a position
opposite that of a template residue for which there was no
complementary dNTP, indicating that they discriminate less strictly
between a correct and an incorrect incoming nucleotide (19, 28). In
addition, the mutant enzymes synthesized longer products than the
wild-type polymerase by utilizing nascent primers with
non-complementary 3-termini, indicating that they also
discriminate less stringently between matched and mismatched primer
termini. Concomitant increases in misincorporation and extension of
mispaired termini has also been observed in human
immunodeficiency virus type-1 reverse transcriptase (29).
We also assessed the fidelity of the purified mutant enzymes in the
M13mp2 forward mutation assay (30). This assay has several important
advantages for the measurement of in vitro misincorporation. First, one can quantitate mutation frequency and identify the types of
mutations that are produced (Table I and Fig. 5). Second, synthesis can
be carried out with equal concentrations of the four dNTPs, whereas
misincorporation in the primer extension assay occurs in the absence of
one of the nucleotide substrates (Figs. 1 and 4). Third, one can
examine the sequence dependence of mutations. DNA polymerases such as
E. coli pol I (31), pol (32, 33), pol
(30), pols
and
(35), and human immunodeficiency virus type 1 reverse
transcriptase (36) produce mutations non-randomly when copying DNA
templates, producing a disproportionate number of mutations at "hot
spots" that are unique to each polymerase. We observed at least a 7- and 25-fold increase for mutants A661E and T664R, respectively,
compared with the wild-type enzyme. Although the relative
misincorporation, T664R > A661E > wild-type, was the same
in the forward mutation and primer extension experiments, the base
substitution specificity appeared to differ. The primer extension
experiments indicated that approximately equal amounts of primer were
extended past the target nucleotides by T664R in each of the four
reactions lacking a different dNTP. Thus, the A, C, G, and T target
nucleotides may have about equal probability of substitution (Fig.
4A, lanes 13-16). In contrast, half of the base
substitutions produced by T664R in the forward mutation assay were T
C transitions (Fig. 5). This difference may be attributable to
differences in the sequence context of the target nucleotides in the
two assays.
Most work on the fidelity of DNA polymerases has focused on the
contribution of proofreading by the 3-5
exonuclease. Fewer studies
have aimed at understanding the basis of discrimination between
complementary and non-complementary nucleotide substrates. Such studies
are most readily interpretable for polymerases that lack a proofreading
exonuclease, because mutation in the polymerase domain can cause
differences in partitioning between states active for polymerization or
exonucleolytic proofreading (37). Studies on the fidelity of mutant DNA
polymerases that lack editing exonuclease activity have been limited.
D1002N and T1003S in the highly conserved region I of human DNA
polymerase
have been shown to display 70- and 40-fold higher
insertional fidelity in Mn2+-catalyzed reactions,
respectively, than that of the wild-type enzyme, as well as reduced
Mn2+-catalyzed extension of mismatched termini (38). G262A
and W266A in helix H of the thumb domain of human immunodeficiency
virus type-1 reverse transcriptase displayed reduced processivity and increased template-primer slippage errors (39). In addition, Y766S in
the O-helix of exonuclease-deficient E. coli pol I Klenow fragment displayed reduced insertional fidelity (40); since this
mutation also decreased DNA binding affinity (10) it would appear that
Tyr-766 may influence dNTP discrimination via an interaction with the
template. Because the fidelity of DNA synthesis has been examined in
just a few mutant enzymes, it is possible that a wide variety of other
mutations within the polymerase site may also alter fidelity.
Most of the substitutions that reduced fidelity in the present study of
the O-helix in Taq pol I were found in the N-terminal part
of the helix, and many of these substitutions clustered on either side
of the essential (10, 11, 41, 42) and non-mutable Lys-663 (12) (Figs. 2
and 6). The most frequent substitutions among our low
fidelity mutants occurred at Ala-661 and Thr-664 (Fig. 2). These amino
acids are not on the face of the helix that interacts with the incoming
dNTP, as judged from the crystal structure of E. coli pol I
(8), nor are they essential for catalytic activity in vivo,
based on the ability of other amino acids to replace them (12). In
fact, we have reported that six different amino acids could replace
either Ala-661 or Thr-664 and yield active DNA polymerases, in the
absence of mutations at other positions that could compensate (12). A
variety of amino acids are also found in the corresponding positions in
other DNA polymerases within the pol I family. Ser, Asn, Lys, or Glu is
found at the position corresponding to Ala-661, and Ala, Val, Lys, or
Ile is found at the position corresponding to Thr- 664 (3). Thus, many
substitutions are tolerated adjacent to the essential Lys-663 and
Arg-659 in Taq pol I and homologous residues in other DNA polymerases.
Among the 13 low fidelity mutants we identified (Fig. 2), only two contain substitutions in the five C-terminal amino acids of the O-helix (Phe-667 through Tyr-671). This segment interacts with the terminal region of the template-primer duplex as judged from the crystal structure of Taq pol I in the presence of a blunt-ended oligonucleotide (43). The double-stranded oligonucleotide could represent the product of the polymerase reaction. Moreover the two substitutions we observed occurred in the presence of other replacements that could alter fidelity.
We offer two explanations for how the diverse substitutions we identified in the O-helix could reduce the fidelity of DNA synthesis. First, these substitutions might alter the local conformation around the catalytic pocket and thereby reposition critical residues that interact with either the template-primer or the incoming dNTPs. Among the substitutions in the 13 low fidelity Taq pol I mutants were five prolines, which disrupt helical structure, and three glycines, which could relax the conformation around critical residues at the active site.
A second explanation for the reduced fidelity of our mutants involves a
pyrophosphate error prevention mechanism (44, 45). It might have been
predicted that amino acid substitutions adjacent to the Phe and Tyr
(Fig. 6) would alter the fidelity of DNA synthesis since they interact
with the template-primer binding site (38). However, most of the
substitutions that alter fidelity are located upstream from these
residues in proximity to Lys-663. The assignment of individual amino
acid residues that interact with specific atoms in the incoming dNTPs
is tentative, at best (46). It is based primarily on inferences from
kinetic studies (45), binding studies with binary complexes (44), and
cross-linking studies with wild-type and mutant DNA polymerases (11).
Nevertheless, evidence suggests that both Lys-663 and Arg-659, and the
homologous residues in other DNA polymerases, interact with the -
and
-phosphates in the incoming dNTPs and could thus facilitate
removal of pyrophosphate during catalysis (44). The incoming dNTP may
initially interact with the carboxylate region of the palm domain (46,
47). The carboxylates could anchor a pair of divalent metal ions to
facilitate deprotonation of the 3
-hydroxyl terminus of the primer
strand (46). As judged from the structure of Taq pol I with
a double-stranded oligonucleotide, Lys-663 and Arg-659 in the O-helix
in the fingers domain are distant from the carboxylate residues in the
palm region and thus a conformational change may be required to
facilitate their interaction with the
- and
- phosphates in the
incoming dNTP. Support for the involvement of Lys-663 and/or Arg-659 in pyrophosphate release is largely indirect. The corresponding Arg-754 and Lys-758 in E. coli pol I have been shown to interact
with phosphate residues in the incoming dNTP (8, 9). Substitution of
Ala at Lys-758 in E. coli pol I reduced
kcat 330-3,500-fold with
poly(dA)·(dT)15 as template-primer, while substitution of Ala at Arg-754 reduced kcat 19-30-fold (10,
48). These data suggest that Arg-754 and Lys-758 in E. coli
pol I are involved in catalysis but they do not establish a direct
interaction with the dNTP or involvement in pyrophosphate release. More
direct evidence stems from crystallographic data that showed that the Klenow fragment is complexed with pyrophosphate near the conserved Lys
and Arg residues (42). Thus, our low fidelity mutants containing substitutions near Lys-663 or Arg-659 may exhibit attenuated
pyrophosphate release. A two-step chemical mechanism for pyrophosphate
release that effects the fidelity of DNA synthesis has been suggested. The first step involves binding of the substrate via the
-phosphoryl group and the second step involves verification of complementary base
pair interaction by coordination with the
-phosphoryl group (44).
This error preventing step follows binding of the substrate and
precedes primer elongation. Enhancement of misincorporation by the
addition of pyrophosphate has been demonstrated for DNA polymerases
(49) and has been attributed to a kinetic proofreading step in a
multi-step proofreading mechanism (45).
The essentiality of individual amino acids in DNA polymerases has been inferred from sequence comparisons (1) that identify evolutionarily conserved and presumably critical residues. Chemical modification, site-specific mutagenesis, and kinetic studies follow, and provide quantitative data on which to assess detailed catalytic function. We have taken a different approach to evaluating the role of amino acids in the O-helix of Taq pol I. We constructed a library of active mutants by using random mutagenesis and genetic selection and identified immutable/essential residues (12). We then screened these active mutants for alterations in the fidelity of DNA synthesis. Our results suggest that many substitutions within the polymerase site can alter fidelity while preserving near wild-type activity. The mutants we have identified may be of value in ascertaining the mechanisms of substrate discrimination by DNA polymerases. In addition, they may have utility in applied molecular evolution (50) by increasing mutagenesis during amplification of genes by the polymerase chain reaction (51, 52).
We thank Bella S. Charurat and Karl Rose for their excellent technical assistance. We are grateful to Ann Blank and Premal Patel for helpful discussions.