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INTRODUCTION |
Efficient and accurate synthesis of DNA, during replication and
repair, is essential to the integrity of any genome. Template-directed synthesis requires that a polymerase select the appropriate
deoxynucleotide triphosphate (dNTP) and exclude incorrect bases. The
interaction between a polymerase and substrates must therefore be
highly specific, yet flexible, in order to maintain sequence fidelity.
The smallest of the eukaryotic enzymes that accomplish this reaction is
DNA polymerase
(pol
),1 a
mammalian polymerase which fills short gaps in DNA (1). pol
has
been implicated in base excision repair (2, 3) and meiosis (4). pol
is highly suitable for structure-function studies of the molecular
mechanism of DNA synthesis due to the availability of information on
the polymerase-DNA-substrate ternary complex (5, 6).
DNA synthesis by pol
can be compromised by the nucleoside analog
drug AZT, which closely resembles a normal substrate. AZT-triphosphate (AZT-TP) presents a normal thymine moiety which may form a Watson-Crick base pair with adenosine, but has a modified sugar ring that results in
chain termination. pol
incorporates AZT into DNA in
vitro (7) implying that pol
is not able to distinguish
perfectly between the drug and the natural nucleotide substrate. This
susceptibility of pol
makes AZT resistance a useful probe of
enzyme-substrate interactions involved in DNA synthesis.
The molecular basis for polymerase substrate specificity, and
specifically AZT discrimination, is not well understood. The clinical
problem of AZT-resistant HIV has been attributed to mutations in HIV
reverse transcriptase. However, the mutant reverse transcriptase enzymes have exhibited little or no change in AZT-TP incorporation in vitro, despite their ability to confer AZT resistance
in vivo.(8) The absence of an apparent in vitro
phenotype makes these enzymes troublesome subjects for mechanistic
studies of substrate specificity.
In order to investigate substrate specificity and drug resistance in
pol
, we have developed a system for the identification of
AZT-resistant forms of pol
, using in vivo
complementation of polymerase-deficient Escherichia coli.
The recA718polA12 E. coli strain carries a
temperature-sensitive mutation in the pol A gene,
rendering DNA polymerase I (pol I) inactive above 37 °C (9).
Expression of rat pol
in these cells restores their ability to
conduct DNA replication and repair at a nonpermissive temperature,
indicating that pol
is able to substitute for E. coli
pol I (10). Because growth of these cells at a nonpermissive temperature depends on the activity of pol
, any inhibitor of pol
is lethal to the cells. At 37 °C in the presence of drug, cells
expressing drug-sensitive pol
will die, while the cells expressing
drug-resistant mutants will survive. The survival of a cell in the
presence of AZT depends on the ability of pol
to distinguish
between deoxynucleotide triphosphates (dNTPs) and very similar
molecules. This creates selective pressure for mutant polymerases with
strict substrate specificity.
In vivo selection is a powerful strategy for the
identification of drug-resistant mutants. The ability to screen large
numbers of clones makes it possible to seek drug resistance mutations among a library of randomly mutated clones. We report here a selection for AZT resistance mutations in pol
. We show that AZT can prevent wild type pol
from complementing the pol I defect, allowing selection for drug-resistant mutants. We have exploited this system to
select clones expressing drug-resistant pol
variants. We have
identified AZT-resistant mutants carrying single amino acid changes in
locations distributed around the enzyme.
We describe two mutant enzymes identified by this selection which carry
nonconservative substitutions in the palm domain causing dramatic
increases in AZT resistance in vivo. The drug resistance of
these mutant enzymes correlates with a drop in the steady state turnover number for AZT-TP incorporation relative to dTTP
incorporation. Both enzymes carry a single amino acid substitution that
is not near the active site or nucleotide binding pocket, suggesting that substrate specificity is influenced by residues distant from the
active site.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Media--
The strain used for isolating
AZT-resistant mutants is known as SC18-12 and has the genotype
recA718 polA12 uvrA155 trpE65 lon-11 sul A1 (11). The strain
BL21 DE3 was used for protein expression and has genotype F
ompT hsdSB(rB-mB
) gal dcm (DE3).
Nutrient agar (NA) was Difco nutrient agar with 5 g/liter NaCl.
Nutrient Broth (NB) and LB broth were prepared according to manufacturer's instructions (Difco).
AZT Survival of SC18-12 Cells Expressing pol
--
The
ability of SC18-12 cells expressing WT pol
to survive a range of
AZT concentrations was measured in order to identify an AZT
concentration which would kill the cells when they depend on pol
but not when pol I is active. SC18-12 cells were transformed by
electroporation with the IPTG-inducible expression vector p
2, a
derivative of pHSG576 (12) containing the pol
cDNA.
Transformants were allowed to recover at 30 °C for 2 h.
Transformation efficiency was determined by plating serially diluted
cells on NA 30 °C. Cells were kept overnight at 4 °C (to allow
transformation efficiency plates to grow and be counted) then plated at
approximately 400 colony forming units/plate on NA with chloramphenicol
(30 µg/ml) and tetracycline (12.5 µg/ml), 1 mM IPTG,
and varying concentrations of AZT (Sigma). Duplicate plates were
incubated at 30 and 37 °C. The number of colonies formed on each
drug concentration was divided by the number formed in the absence of
drug to give the surviving fraction.
Construction of a Mutated pol
cDNA Library--
In order
to generate a pool of mutant pol
enzymes, the rat pol
cDNA
was amplified, with primers designed to anneal immediately upstream and
downstream of the gene, under mutagenic polymerase chain reaction
conditions expected to produce mostly single base changes (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 50 µM dNTPs, 30 pmol of
each primer, 5 units of Taq polymerase cycle parameters: 1 min, 94 °C; 2 min, 55 °C; 3 min, 72 °C for 30 cycles) (13).
The mutated polymerase chain reaction product was subcloned into the
p
2 vector.
Selection of AZT-resistant Clones--
Drug-resistant mutants
were selected out of the pool of random mutations on the basis of their
ability to grow in the presence of AZT. SC18-12 cells were transformed
by electroporation with the mutated pol
cDNA library.
Transformants were allowed to recover at 30 °C for 2 h.
Transformation efficiency was determined on NA at 30 °C. Cells were
diluted in saline and plated at approximately 400 colony forming
units/plate on NA with antibiotics, IPTG, and 600 nM AZT
(Sigma), and incubated at 37 °C for 2 days. Three equivalent plates
were incubated at 30 °C (permissive temperature) to determine the
total number of colony forming units screened.
The surviving colonies were isolated on NA with antibiotics. The p
2
plasmid was isolated from each of these strains and sequenced to
identify mutations in the pol
cDNA.
Rotary Streak Assay of AZT Survival--
In order to confirm
that AZT resistance was due to the p
2 plasmid, rather than a
cellular mutation, mutant plasmids were electroporated back into
SC18-12 cells and assayed for the ability to grow in the presence of
AZT. Strains expressing wild type or mutant pol
were grown
overnight at 30 °C in NB without shaking and streaked radially onto
rich medium with antibiotics, 1 mM IPTG, and 600 nM AZT where indicated. Plates were incubated for 2 days at
37 °C. SC18-12 cells expressing WT pol
were not able to grow
enough to form rotary streaks in these conditions, so those mutants
that did form streaks were considered AZT resistant.
AZT Survival of Drug-resistant Strains--
The AZT resistance
phenotype of the confirmed mutants was quantified by plating on
increasing concentrations of AZT. Plasmids identified as carrying drug
resistance mutations within the pol
cDNA were
electrotransformed into SC18-12 cells. Single colonies were grown
overnight in NB at 30 °C, diluted in saline, and plated on NA with
antibiotics, IPTG, and varying concentrations of AZT. Plates were
incubated at 37 °C for 2 days. The number of colonies formed on each
drug concentration was divided by the number formed in the absence of
drug to give the surviving fraction.
Expression and Purification of Mutant Enzymes--
The portion
of the each mutant pol
cDNA containing the mutation was
subcloned into the pHis-
vector, which expresses pol
as a fusion
protein with a His tag at the N terminus. The enzymes were purified
using Ni2+-charged His-bind resin from Novagen or Ni-NTA
resin from Qiagen according to the manufacturers instructions. The
proteins were expressed in BL21 DE3 cells, which were grown to
OD600 = 0.5 and then induced overnight at room temperature
with 1 mM IPTG. Cells were harvested by centrifugation and
resuspended in 20 mM Tris, pH 7.9, 500 mM NaCl,
5 mM imidazole, pepstatin, chymostatin, and antipain (0.1 mg/ml), leupeptin (0.5 mg/ml), and aprotitin (1 mg/ml), and lysed by
sonication. Extracts were cleared by ultracentrifugation, and then
loaded onto a column containing approximately 1 ml of Ni2+
charged resin/100 ml of starting culture. The column was washed with 60 mM imidazole and protein was eluted with 1 M
imidazole in 0.5 M NaCl. Protein was concentrated if
necessary in a Centricon 30 and stored at
80 °C in 50 mM Tris, pH 7.5, 1 mM EDTA, 100 mM
NaCl, 15% glycerol, and protease inhibitors as above. We estimate the
preparation to be at least 90% homogenous (data not shown).
In steady-state kinetic assays measuring incorporation of dNTPs (see
below) the His-tagged pol
enzyme and the wild type (untagged) have
identical kinetic constants Km(dNTP), Kcat, and Km(DNA) when
compared side by side in incorporation assays (data not shown).
Therefore, we employed the His-tagged enzyme in biochemical assays.
Incorporation Assay--
The activity of each enzyme was
measured by incorporation of [
-32P]dATP into activated
calf thymus DNA. Reaction conditions were 50 mM Tris, pH
8.4, 20 mM MgCl2, 100 mM NaCl, 200 µg/ml bovine serum albumin, 200 µM dithiothreitol, 50 µM dNTPs, and 1 mM DNA. Reactions were
incubated at 37 °C for 15 min and then stopped with EDTA. Reactions
were spotted onto GFA filters, which were washed twice in 22.5 mg/ml
NaPPi, 8.5% concentrated perchloric acid, twice in 22.5 mg/ml NaPPi, 8% concentrated hydrochloric acid, and then
once in 95% EtOH. Filters were dried and counted in a scintillation counter.
Primer Extension Assay--
The ability of each enzyme to
incorporate dTTP and AZT-TP into a primer template was tested using
annealed 16-mer and 45-mer of the following sequence:
5'-AACCAAGAGCATACGA and
ATGTTGGTTCTCGTATGCTACCAATCGCAACTTGGATATAACAAC-5'. The site of incorporation is underlined. This sequence was chosen because pol
had previously been demonstrated to incorporate AZT-TP
into this primer template (14). The oligos were synthesized at the Yale
Department of Pathology facility. The primer oligo was gel purified by
standard methods. Both oligos were radiolabeled at the 5' end by
standard methods using T4 polynucleotide kinase. The oligos were
annealed at a primer:template molar ratio of 1:1.2 in 50 mM
Tris, pH 8.0, 250 mM NaCl, 50 mM
MgCl2. Complete annealing of primer was confirmed on a 20%
acrylamide native minigel.
Reactions were conducted at 37 °C in 50 mM Tris, pH 8.0, 10 mM MgCl2, 20 mM NaCl, 2 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 2.5%
glycerol, and 200 nM 32P-end-labeled
primer-template. The reactions were preincubated at 37 °C for 1 min,
started by the addition of substrate, incubated for the indicated
reaction time, and stopped by the addition of 0.5 M EDTA.
Samples were analyzed on a 20% Sequel NE (American Bioanalytical) gel.
Bands were quantified by PhosphorImager analysis to measure product
formation as a function of time. The kinetic constants Km and Kcat were derived by
fitting the Michaelis-Menten equation to plots of the results (velocity
versus substrate concentration) using Sigmaplot
curve-fitting software. The results of three independent determinations
were averaged.
DNA Binding Assay--
The dissociation constant
KD (DNA) was measured using a gel mobility shift
assay (15, 16). 16-mer and 45-mer oligos were prepared and annealed as
described above. Fifteen protein concentrations ranging from 4 µM to 0.25 nM, expected to bracket the
KD, were incubated with O.1 nM DNA in buffer containing 10 mM Tris, pH 7.5, 6 mM
MgCl2, 100 mM NaCl, 10% glycerol, and 0.1%
Nonidet P-40. After a 10-min incubation at 20 °C, samples were
loaded onto a 6% acrylamide nondenaturing gel with the current running
at 300 V. After loading, voltage was reduced to 150 V, and the gel was
run for 1 h. Fractions bound were determined by PhosphorImage
quantitation of the gel. The dissociation constant
KD was derived from Sigmaplot fitting of the
fraction bound versus protein concentration with the
equation: Y = [(m1 *
x)/(x + KD)] + m3, where m1 is a scaling
factor and m3 is the apparent minimum
Y value (15).
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RESULTS |
AZT Prevents Heterologous Complementation by pol
--
WT rat
pol
is capable of substituting for the E. coli enzyme
DNA polymerase I in recA718polA12 cells, restoring the
cells' ability to grow on rich medium at 37 °C (10). We examined
whether AZT would interfere with functional complementation of the
polymerase defect of recA718polA12 cells by pol
. A
representative rotary streak assay is shown in Fig.
1A. On the top row are
plates grown at 30 °C, a permissive temperature at which pol I
is active. The SC18-12 cells grow well at 30 °C with or without
expression of WT pol
. The addition of 600 nM AZT to the
medium (upper right) resulted in streaks that were somewhat
sparse and translucent in appearance, but did not prevent the streak
from growing out to the edge of the plate, where the cells are least
dense. At 37 °C (bottom row, Fig. 1A), SC18-12
cells are unable to form a rotary streak unless they express WT pol
. In the presence of AZT (bottom right), SC18-12 cells
expressing WT pol
were unable to form a streak, demonstrating that
pol
was unable to complement the growth defect of SC18-12 cells in
the presence of AZT. These results indicate that AZT is toxic to the
cells when they are dependent on pol
activity. At 30 °C, when
pol I is functional, SC18-12 cells are not prevented by AZT from
forming a rotary streak (top right), demonstrating that the
drug at this concentration is not lethal to the cells when they can
rely on bacterial polymerases and do not require pol
.

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Fig. 1.
A, rotary streak assay. SC18-12 cells or
SC18-12 cells expressing WT pol were grown on NA at 30 or 37 °C
in the presence of antibiotics and IPTG, and with 600 nM
AZT where indicated. Cells were streaked by drawing an inoculating loop
slowly across the radius of the plate while spinning it on a plating
wheel, creating a cell density gradient from the center to the
perimeter of the plate. Typical results are shown. B, AZT
survival curve. SC-18 cells expressing WT pol were grown on NA at
30 ( ) or 37 °C ( ) in the presence of antibiotics and IPTG, and
varying concentrations of AZT. The number of colonies formed on each
drug concentration was divided by the number formed in the absence of
drug to give the surviving fraction. Results are representative of
three independent experiments.
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In order to identify the range of AZT concentrations that would affect
pol
but not bacterial polymerases, we measured the ability of the
cells to grow on increasing concentrations of AZT (Fig. 1B).
The surviving fraction (ratio of colonies formed on a given drug
concentration to colonies formed in the absence of drug) drops off
sharply at 400-500 nM AZT. Those colonies that do form in
the presence of AZT (at all concentrations measured) are usually
smaller than normal, and appear translucent rather than opaque white,
and irregularly shaped rather than round. The presence of 600 nM AZT in the medium is sufficient to kill virtually all
pol
dependent growth, without affecting colony formation at
30 °C. Therefore, we conclude that drug-resistant pol
mutants can be identified by selecting for ability to complement the
recA718polA12 growth defect in the presence of 600 nM AZT.
Selection of Drug-resistant Mutants--
We selected AZT-resistant
mutants from a pool of plasmids carrying the randomly mutated pol
cDNA (see "Experimental Procedures"). Of approximately 66,000 mutant colonies screened, 171 grew at 37 °C on nutrient agar
containing 600 nM AZT. When the pol
expressing plasmids
were rescued from these colonies and sequenced, the majority were found
to have more than one mutation, and were put aside. In order to confirm
that the AZT-resistant phenotype resulted from expression of mutant pol
, rather than a cellular mutation, we transformed each of the mutant
plasmids that carried a single mutation back into SC18-12 cells. Of
those that carried only one predicted amino acid substitution, nine
were consistently able to confer resistance to AZT on
recA718polA12 cells when re-transformed into these cells.
For further characterization, we chose two highly drug-resistant
mutants, D246V and R253M.
Location of Mutations in the Protein Structure--
The pol
protein is composed of two domains: the 8-kDa domain, which contains
the N terminus of the protein and has deoxyribophosphatase activity
(17), and the 31-kDa domain, which can perform the incorporation
reaction alone. The 31-kDa domain can be further subdivided into
fingers, palm, and thumb domains by analogy to the shape of a hand. As
in other polymerases, the active site is defined by two
Mg2+ ions which participate in the catalytic reaction;
these atoms are held in place by three conserved acidic residues (5,
18, 19). In the case of pol
, the Mg2+ ions are
coordinated by the residues Asp-190, Asp-192, and Asp-256, located in
the palm domain.
We mapped the altered amino acids to the pol
crystal structure (5,
6); both carry nonconservative substitutions located in a loop
connecting two
strands within the palm domain (Fig. 2). Neither residue appears to contact the
DNA or incoming nucleotide. Residue Arg-253 is at least 12 Å, and
Asp-246 is more than 30 Å, from the active site Mg2+ ions
and bound nucleotide. Replacement of the Arg residue at position 253 with Met (using the program Whatif) does not introduce any obvious
steric clash with neighboring side chains, but does eliminate a
hydrogen bond with Glu-154 and another with the backbone at Asp-226.
The side chain of Asp-246 is solvent exposed in this structure. Both
Arg-253 and Asp-246 are conserved in the rat, human, and
Xenopus pol
enzymes (22), while most residues in the
240-253 loop are not, which is consistent with the suggestion that
these residues have an important role in pol
function.

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Fig. 2.
Ribbons diagram of pol ternary complex with DNA and incoming dNTP substrate.
Coordinates from Sawaya et al. (6). Primer-template DNA
contains a single base gap. Mg2+ ions are shown as
gray spheres. Asp-246 and Arg-253 side chains are shown in
black.
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AZT-resistant Heterologous Complementation by D246V and R253M
Mutant Enzymes--
To characterize more fully the AZT resistance
phenotype of mutant enzymes, we tested their ability to substitute for
pol I in the presence and absence of AZT. The D246V and R253M mutant enzymes were able to complement the growth defect of SC18-12 cells as
well as wild type in the absence of drug (data not shown). In the
presence of AZT, cells expressing pol
with the mutation D246V or
R253M were substantially more drug resistant than cells expressing WT
pol
. Fig. 3A shows typical
results of a rotary streak assay of SC18-12 cells expressing WT and
mutant pol
. The D246V substitution conferred a moderate improvement
in ability to grow on AZT, forming a partial streak. The R253M mutation
allowed the cells to form a streak on AZT extending out to the
perimeter of the plate. For a quantitative measure of AZT resistance,
we determined the surviving fraction of cells expressing WT and mutant pol
when plated on increasing concentrations of AZT. Both pol
R253M and D246V enabled SC18-12 cells to form colonies on AZT just as
readily as on no drug, up to the drug concentration that is tolerated
by the bacterial polymerases (Fig. 3B). In addition, the
colonies formed by cells expressing pol
R253M appeared round and
opaque, and otherwise hearty, unlike the colonies formed by cells
depending on pol
WT or D246V, which were irregularly shaped and
translucent.

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Fig. 3.
A, rotary streak assay. SC18-12 cells
expressing pol WT (left), D246V (middle), or
R253M (right) were grown and streaked as described in the
legend to Fig. 2 on NA at 37 °C in the presence of antibiotics,
IPTG, and 600 nM AZT. B, AZT survival curve.
SC-18 cells expressing pol WT ( ), D246V ( ), or R253M ( )
were grown on NA at 37 °C in the presence of antibiotics and IPTG,
and varying concentrations of AZT. The number of colonies formed on
each drug concentration was divided by the number formed in the absence
of drug to give the surviving fraction. Results are representative of
three independent experiments.
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Mutants That Complement pol I Defect in the Presence of AZT Exhibit
Reduced AZT-TP Incorporation in Vitro--
To determine whether the
D246V and R253M enzymes are resistant to AZT in vitro, we
purified the mutant proteins as described under "Experimental
Procedures." We found that both enzymes were as active as wild type,
as measured by their ability to incorporate dNTPs into activated DNA
(data not shown).
We used a steady-state primer extension assay to ask whether the
in vivo drug-resistant phenotypes of the D246V and R253M mutants were due to increased specificity for dTTP over AZT-TP. All
three pol
enzymes did extend the primer by one AZT-TP molecule, although the reaction was much slower than dTTP incorporation (Fig.
4A). In steady state conditions, the AZT-TP
incorporation reaction reaches saturation in about 35 min, while dTTP
incorporation saturates after 2 min. We measured the reaction rate
(incorporation as a function of time during the linear phase of the
reaction, Fig. 4B) at eight substrate concentrations for
each enzyme to determine the steady state constants for AZT-TP and dTTP
incorporation by WT and mutant pol
enzymes. Fig. 4C
shows typical Michaelis-Menten plots from which Km
and Kcat are derived. The WT pol
enzyme has
a 10-fold higher Km and 10-fold lower
Kcat value for AZT-TP incorporation as compared
with dTTP (Table I). The catalytic
efficiency Kcat/Km is
therefore nearly 2 orders of magnitude lower for AZT-TP incorporation
than dTTP. This substantial difference in catalytic efficiency explains
why pol
preferentially incorporates dTTP.

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Fig. 4.
A, primer extension assay. WT and mutant
pol enzymes were incubated with AZT-TP and primer template DNA as
described under "Experimental Procedures." In the assays shown, the
reactions contained 200 nM DNA and 0.5 mM
substrate. Enzyme concentration was 2.5 nM for AZTTP
incorporation or 0.1 nM for dTTP incorporation. Longer
reaction times were used for pol R253M because of the inefficiency
of the AZT-TP incorporation reaction. B, rates of AZT-TP and
dTTP incorporation. Time points were collected every 5 min during the
linear portion of the curve and quantified by PhosphorImager analysis
of the gel. Steady state rate for each substrate concentration was
derived from the slope of the plot of product formation
versus time. The plots shown represent WT pol incorporation of AZT-TP (left) at concentrations of 66 µM ( ), 99 µM ( ), 149 µM
( ), 224 µM ( ), 335 µM ( ), 503 µM( ), 754 µM ( ), 1.1 mM
( ) or dTTP (right) at concentrations of 8 µM ( ), 16 µM ( ), 31 µM
( ), 63 µM ( ), 125 µM ( ), 250 µM ( ), 500 µM ( ), 1 mM
( ). C, plots of reaction rate versus substrate
concentration for AZT-TP and dTTP incorporation by WT and mutant pol
. The steady-state constants Km and
Kcat were derived by fitting the
Michaelis-Menten equation to these plots.
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Table I
Steady-state kinetic constants of WT and AZT-resistant pol enzymes
Km and Kcat were derived from
Michaelis-Menten plots of primer extension assays.
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The Km (AZT-TP) of both mutants was only slightly
higher than that of WT. The most significant change in these reactions was in Kcat (AZT-TP), which decreased almost an
order of magnitude for D246V and 3-fold for R253M compared with WT.
This produced a decrease in catalytic efficiency
(Kcat/Km) of AZT-TP one order
of magnitude below WT for D246V, and 3.5-fold for R253M. The
steady-state constants of the D246V mutant enzyme for dTTP incorporation were essentially the same as wild type (Table I), implying that the reaction with natural substrate was unaffected by the
mutation. While R253M was equally competent at dTTP incorporation as
measured by catalytic efficiency, it exhibited a 7-fold higher Km and 4-fold higher Kcat.
For this enzyme, the incorporation of dTTP is not compromised, but
apparently proceeds via a kinetically altered pathway.
The in vivo phenotypes, where R253M is the strongest cannot
be explained by the kinetics of the AZT reaction alone. The
drug-resistance phenotype seems to correlate well with the ratio of
turnover numbers Kcat(dTTP)/Kcat(AZTTP).
This ratio is increased from 11.5 for wild type to 52.2 in the
moderately resistant D246V and 116 in the strong phenotype mutant R253M.
We performed a gel mobility shift assay to determine whether the mutant
enzymes had an altered affinity for primer-template DNA (Fig.
5). The KD for DNA of WT
pol
was 15.4 ± 5.7 nM, compared with 3.0 ± 1.1 nM for D246V and 12.7 ± 2.1 nM for R253M. Therefore, the R253M enzyme appears to interact with
primer-template DNA with an affinity similar to that of WT pol
,
while the D246V enzyme may bind slightly tighter.

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Fig. 5.
Gel mobility shift assay. End-labeled
16:45 DNA at 0.1 nM was incubated with indicated protein
concentrations as described under "Experimental Procedures." The
gel shown is an assay of the R253M protein and is representative of
three independent determinations. Fraction bound was determined by
PhosphorImage quantitation of the gel. The dissociation constant
KD was derived from Sigmaplot fitting of the
fraction bound versus protein concentration as described
under "Experimental Procedures."
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 |
DISCUSSION |
A Genetic Selection for AZT-resistant pol
Mutants--
We
found in this study that the addition of AZT to plating medium
prevented WT pol
from functionally substituting for E. coli pol I. The E. coli cells are able to survive in
the presence of the drug when they are not depending on pol
,
demonstrating that the lethality of AZT results from its interaction
with pol
. This evidence that pol
incorporates AZT-TP in
physiological conditions confirms the findings of Bouayadi et
al. (14) that expression of pol
-sensitized mammalian cells in
culture to AZT. Although incorporation of AZT-TP by pol
is
inefficient compared with pol
(7) and HIV-reverse transcriptase, it
clearly can be lethal to cells, which may be clinically important when
AZT is used therapeutically.
We exploited the AZT sensitivity exhibited by SC18-12 cells when
depending on pol
to select AZT-resistant mutants of pol
. This
approach allowed us to identify drug-resistant mutants within a large
pool containing random mutations. These pol
mutants present an
opportunity to examine the biochemical mechanisms of AZT resistance.
The two mutants characterized, D246V and R253M, discriminate more
effectively than WT pol
against AZT-TP. Both mutants incorporate AZT-TP less efficiently than WT, but are still proficient in dTTP incorporation. These pol
mutants are the first AZT-resistant polymerases to demonstrate significantly reduced incorporation of
AZT-TP in steady state. The drug-resistance phenotype of pol
D246V
is explained by the 10-fold reduction in catalytic efficiency of AZT-TP
incorporation. In the absence of any changes to the dTTP reaction, this
enzyme is presumably 10 times better than WT at discriminating against
AZT. The R253M mutant, however, probably owes its drug resistance to
the combination of moderately less efficient AZT-TP incorporation and a
simultaneous perturbation of the dTTP incorporation reaction. The
increase in Km and Kcat for
dTTP implies the reaction pathway is altered, possibly by a change in
rate-limiting step, while remaining about equally efficient. The
Km values for D246V were similar to WT, and for
R253M the value of Km(dTTP) increased so that Km(dTTP) is similar to
Km(AZT-TP), implying that the difference in
Km for dTTP and AZT-TP does not determine the
enzyme's ability to discriminate between the two substrates and
exclude AZT-TP. While steady-state Km is not
necessarily equal to the substrate binding constant, these results are
consistent with a scenario in which substrate binding affinity does not
control drug resistance. For the three enzymes examined here, AZT
resistance in vivo increases with the ratio Kcat(dTTP)/Kcat(AZTTP),
in agreement with the suggestion that turnover number is the critical parameter.
The 5-fold decrease in KD(DNA) exhibited by D246V
represents a modest increase in DNA binding affinity. The difference is
small, but raises the possibility that tighter binding to
primer-template DNA increases discrimination against AZT-TP, possibly
by tightening the sterics of the binding pocket so as to exclude a
larger than normal substrate.
The identification of these mutants implies that single amino acid
substitutions in pol
are capable of improving its discrimination against AZT-TP as a substrate. The retained efficiency of dTTP incorporation implies that the improved specificity has occurred at
little or no cost to the enzyme.
Molecular Mechanisms of Substrate Specificity--
In theory, the
most straightforward mechanism for a mutation to affect AZT
incorporation would be a mutation that discouraged AZT-TP binding
through a change in a residue that contacts incoming dNTP molecules, or
the protrusion of a new side chain into the binding pocket. This would
lead to the prediction that AZT resistance mutations would arise in the
amino acids that surround the bound nucleotide in the enzyme-substrate
complex. However, the D246V and R253M mutations are located on a loop
in the palm domain at a distance from the nucleotide binding pocket
which makes it highly implausible that they participate directly in
nucleotide binding. These mutations demonstrate that substrate
selection is influenced by amino acids which are not in direct contact
with the substrate. Their effect on nucleotide interactions could be
explained by a more subtle mechanism, in which disruption of the side
chain interactions that hold the palm domain together has an indirect impact on the geometry of the active site, or influences the dynamic abilities of the protein.
Any changes in the palm domain caused by the D246V and R253M mutations
could affect the geometry of the active site by altering the enzyme's
contacts with the Mg2+ ions, the primer-template DNA, or
the incoming substrate itself. Crystal structures have indicated that
pol
positions nucleotide substrates through interactions with all
three components of the molecule: the base moiety pairs with the
template base, the phosphate groups coordinate the catalytic
Mg2+ ions, and the ribose ring contacts the protein side
chains and backbone (5, 21). In crystal soaking experiments, Pelletier et al. (23) found that AZT-TP could enter the nucleotide
binding pocket, but that steric clash of the azide group with the
protein backbone at residues 271-274 forced the nucleotide into a
distorted conformation in which not only the sugar ring but the base
and the phosphates are positioned incorrectly. Therefore, although only
the sugar ring of AZT-TP differs from the natural substrate, a change
in the contacts with the other groups of the molecule could exacerbate
the already poor fit of AZT-TP in the binding pocket.
Pol
is known to undergo conformational changes during the binding
and catalysis cycle (24, 6). These conformations, or the transitions
between them, may require stabilization by various side chain
interactions. The
-sheet surface of the palm domain includes
residues that contact the DNA and nucleotide substrates, and the three
essential Asp residues that coordinate the Mg2+ ions as
well. Immediately adjacent to Arg-253 lies a potentially crucial
interaction between Asp-256, one of the catalytic aspartates, and
Arg-254. In the crystal structure of pol
bound to gapped DNA alone
(1bpx), this interaction includes two apparent hydrogen bonds between
the amino and carboxyl groups. In the "closed" structure, in which
pol
is bound to DNA and ddCTP (1bpy), the bidentate interaction is
reduced to a single H-bond and Arg-254 forms a new interaction with the
backbone at Ile-255. Moreover, in the structure of pol
bound to a
non-gapped primer-template (1bpf), Arg-254 H-bonds instead to the 3'
end of the primer. The different contacts with catalytically essential
moieties formed by the Arg-254 side chain in different binding states
suggests that Arg-254 may be involved in the stabilization of the
different conformations of the enzyme or the transitions between them.
This role would be analogous to that proposed by Sawaya et
al. (6) for Arg-258, which also interacts with one of the
catalytic Asp residues.
Arg-253 forms hydrogen bonds with the side chain of Glu-154 and the
backbone at Asp-226 (5, 21), making it part of an intricate network of
H-bonds and hydrophobic contacts that hold the palm domain together. A
possible explanation for the R253M phenotype is that the elimination of
the these H-bonds, combined with the steric and electrostatic effect of
the introduction of a Met residue, displaces the neighboring Arg-254
residue. This could then disturb catalytic geometry by influencing the
positioning of the primer terminus, or the Asp-256 side chain. In
addition, if the Arg-254 side chain normally cycles during the various
binding events between various contacts with the primer, protein
backbone, and Asp-256, then a change in the position of this residue
could alter the kinetics of the conformational changes.
The effect of the D246V mutation is more difficult to rationalize.
Asp-246, as a charged residue, may also participate in palm domain
intramolecular interactions, although its role is not apparent from the
crystal structures. The 240-253 loop sticks out from the palm domain
in the crystal structure so that the side chains are mostly solvent
exposed, and Asp-246 is located at the tip of the loop. One possible
explanation is that the tip of the loop is flexible, and does not
assume a physiologically relevant position in crystallization
conditions. If the loop moved a bit in either direction, Asp-246 and
the surrounding residues could be closely associated with either the
palm or N-terminal domains, possibly forming a hinge-like region.
Mutation of the hinge between the palm and C-terminal domains has been
found to reduce fidelity by interfering with conformational changes
during incorporation, demonstrating the importance of the dynamic
properties of the enzyme (25, 26).
Another intriguing, although speculative, possibility is that the loop
is the site of protein-protein interactions. The loop could be the site
of regulatory contacts, such as the interaction with AP endonuclease
during BER (27) or the binding of XRCC1 which prevents pol
from
engaging in strand displacement (28). In that case, the loop could be
seen as a molecular switch affecting the enzyme's catalytic and
binding capacities, and the mutation could cause the switch to
"stick." A switch trapped in a on, off, or halfway position could
freeze the enzyme in an inappropriate conformation. A distortion of
this nature would explain why such a mutation could affect pol
function even in E. coli, where the normal protein
regulators are probably absent, and in vitro.
These mutants demonstrate that changes in residues at some distance
from the nucleotide binding pocket can have a profound impact on
substrate selection by pol
. Similarly, while some clinical isolates
of AZT-resistant reverse transcriptase mutants have been found to carry
mutations in the nucleotide binding pocket, others are mutated in
residues not believed to interact with nucleotide substrates (29-31).
Therefore, remote control of substrate choice by amino acids distant
from the binding pocket may be a general property of DNA polymerases.
These intramolecular interactions may be responsible for much
polymerase substrate specificity, including sequence fidelity.
Implications for Therapeutic Use of AZT--
That pol
is able
to incorporate lethal amounts of AZT in bacterial cells raises the
possibility that pol
contributes to the genotoxic effects of AZT.
Cells in culture have been shown to up-regulate pol
activity and
down-regulate other polymerases in response to methotrexate and during
induced apoptosis (32, 33). This indicates that tumors subjected to
DNA-damaging drugs may increase pol
activity in an attempt to
repair their DNA, while shutting down DNA synthesis by other
polymerases. If AZT or other drugs commonly used in combination with
AZT trigger this response, pol
may be the most active native
polymerase in many cells during antiviral therapy, and the ability of
these cells to survive AZT treatment with intact genomes could depend
on the ability of pol
to avoid incorporating AZT into DNA.
Therefore the genotoxicity of antiviral therapy may be affected by pol
substrate specificity.