(Received for publication, March 8, 1996)
From the
In the crystal structure of a substrate complex, the side chains
of residues Asn, Tyr
, and Arg
of DNA polymerase
are within hydrogen bonding distance to
the bases of the incoming deoxynucleoside 5`-triphosphate (dNTP), the
terminal primer nucleotide, and the templating nucleotide, respectively
(Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut,
J.(1994) Science 264, 1891-1903). We have altered these
side chains through individual site-directed mutagenesis. Each mutant
protein was expressed in Escherichia coli and was soluble. The
mutant enzymes were purified and characterized to probe their role in
nucleotide discrimination and catalysis. A reversion assay was
developed on a short (5 nucleotide) gapped DNA substrate containing an
opal codon to assess the effect of the amino acid substitutions on
fidelity. Substitution of the tyrosine at position 271 with
phenylalanine or histidine did not influence catalytic efficiency (k
/K
) or fidelity.
The hydrogen bonding potential between the side chain of
Asn
and the incoming nucleotide was removed by replacing
this residue with alanine or leucine. Although catalytic efficiency was
reduced as much as 17-fold for these mutants, fidelity was not. In
contrast, both catalytic efficiency and fidelity decreased dramatically
for all mutants of Arg
(Ala > Leu > Lys). The
fidelity and catalytic efficiency of the alanine mutant of
Arg
decreased 160- and 5000-fold, respectively, relative
to wild-type enzyme. Sequence analyses of the mutant DNA resulting from
short gap-filling synthesis indicated that the types of base
substitution errors produced by the wild-type and R283A mutant were
similar and indicated misincorporations resulting in frequent
T
dGTP and A
dGTP mispairing. With R283A, a dGMP was
incorporated opposite a template thymidine as often as the correct
nucleotide. The x-ray crystallographic structure of the alanine mutant
of Arg
verified the loss of the mutated side chain. Our
results indicate that specific interactions between DNA polymerase
and the template base, but not hydrogen bonding to the
incoming dNTP or terminal primer nucleotide, are required for both high
catalytic efficiency and nucleotide discrimination.
Accurate DNA synthesis during replication and DNA repair is
crucial in maintaining genomic integrity. Although DNA polymerases play
a central role in these essential processes, the fundamental mechanism
by which they select the correct deoxynucleoside 5`-triphosphate (dNTP) ()from a pool of structurally similar compounds and
substrates to accomplish rapid and efficient polymerization is poorly
understood. Vertebrate DNA polymerase
(
-pol) has been
suggested to play a role in both DNA
repair(1, 2, 3, 4, 5) and
replication(6, 7, 8) . The x-ray crystal
structures of rat and human
-pol in complex with substrates have
suggested a detailed model of the chemical mechanism for the
nucleotidyl transfer reaction and also have suggested several
protein/substrate interactions that may play a role in nucleotide
discrimination(9, 10, 11, 12) .
Additionally, these structures allow us to experimentally test
model-derived predictions about the role(s) of individual amino acids.
DNA and RNA polymerases, for which the structure has been
determined, have been described by analogy to the anatomical features
of a hand as consisting of fingers, palm, and thumb
subdomains(13) . Conserved carboxylates, which bind
catalytically essential divalent metal ions, are found in the palm
subdomains of these polymerases. The dNTP binding site of -pol is
formed by the DNA template base, the 3`-terminal nucleotide of the
primer strand, and the palm and thumb subdomains of the
polymerase(10) . Only three amino acid residues of the thumb
subdomain have side chains that are within hydrogen bonding distance to
the nucleotide bases within this binding pocket. These hydrogen bond
donors are indiscriminate in that they bond to the O2 of pyrimidines or
the N3 of purines in the DNA minor groove(14) . The structure
of the
-pol ternary complex reveals a single hydrogen bond between
the base of the incoming ddCTP and Asn
; Tyr
and Arg
are also within hydrogen bonding distance
to the O2 and N3 atoms of the terminal primer and templating base,
respectively (Fig. 1). To assess the role of these interactions
in nucleotide selection and incorporation, we replaced
Tyr
, Asn
, and Arg
with
alternate residues by site-directed mutagenesis to remove and/or alter
each interaction.
Figure 1:
Ribbon drawing of the dNTP
binding pocket of rat DNA polymerase . The side chains of residues
Asn
, Tyr
, and Arg
of DNA
polymerase
are within hydrogen bonding distance (dashed
lines) to the bases of the incoming dideoxynucleoside triphosphate (ddCTP), the terminal primer nucleotide, and the templating
nucleotide, respectively(10) . These residues can hydrogen bond
indiscriminately to the O2 of pyrimidines or the N3 of purines in the
DNA minor groove. They are part of two
-helices, M and N, which
are interrupted by a cis-peptide bond between residues
Gly
-Ser
. The observed distances from the
side chain hydrogen bond donor and O2 or N3 are 2.7, 3.0, and 3.2
Å for Tyr
, Asn
, and
Arg
, respectively(10) . Also indicated are active
site carboxylate side chains (Asp
, Asp
,
Asp
) which coordinate two Mg
ions in
the palm subdomain. This figure was made with
MOLSCRIPT(30) .
Figure 3:
Short gap fidelity assay. A, experimental outline for the short gap fidelity assay as described
under ``Experimental Procedures.'' B, mutation
frequencies for the products synthesized by the wild-type and mutant
-polymerases. The background reversion frequency for the assay was
0.001%. Frequencies are shown as the mean and standard deviation of
at least two independent determinations.
Expression constructs of human -pol were prepared:
Tyr
was replaced with either histidine or phenylalanine;
Asn
was replaced with either alanine or leucine; and
Arg
was replaced with either alanine, leucine, or lysine.
Each altered human
-pol gene was expressed in E. coli and
the recombinant enzymes were soluble in the crude cell extracts.
Following purification, SDS-PAGE analysis indicated that the mutant
-pol polypeptides had the same apparent molecular weight as the
wild-type enzyme and were greater than 99% homogeneous (data not
shown).
To compare the catalytic efficiency of the wild-type and
mutant enzymes, the steady-state kinetics on a simple TP system,
poly(dA)-p(dT)
with dTTP as the incoming nucleotide, were
analyzed (Fig. 2). Whereas the catalytic activity of the
Tyr
and Asn
mutants were not significantly
influenced (i.e. <10-fold), k
of the
lysine, alanine, and leucine mutants of Arg
were
decreased greater than 20-, 150-, and 600-fold, respectively (Fig. 2A). In contrast, the K
for
T
P was increased with the R283L mutant (17-fold), and the K
for dTTP was increased to the greatest extent
with the R283A mutant (29-fold) (Fig. 2A). Catalytic
efficiency, as measured by the ratio of k
and K
, was not influenced by the
histidine substitution at Tyr
, while the phenylalanine
mutant displayed a modest (2-fold) decrease (Fig. 2B).
Since the phenylalanine substitution had only a small effect on
catalytic efficiency, substrate interactions with Tyr
appears to offer very little transition state stabilization.
Elimination of the hydrogen bond between the incoming dNTP and the
Asn
side chain with an alanine or leucine substitution
decreased catalytic efficiency further, but again only modestly
(
10-fold). In this case, catalytic efficiency was dependent solely
on the apparent dNTP binding affinity, (
)since k
of each mutant was similar to wild-type
enzyme. The most dramatic decrease in catalytic efficiency was observed
for the mutants of Arg
(Ala > Leu > Lys). A
5000-fold decrease in efficiency was observed for the alanine mutant,
whereas catalytic efficiency of the lysine mutant, which could
potentially hydrogen bond to the template base, was decreased over
100-fold.
Figure 2:
Steady-state kinetic parameters for
wild-type and mutant -pol. Assays were performed as described
under ``Experimental Procedures.'' The substrate
concentrations were varied from at least 0.3 to 3
K
under saturating concentrations of the
other (i.e. >4
K
).
Initial velocities were fitted to the Michaelis equation by nonlinear
least squares methods. The results represent the mean and S.E. of at
least two independent determinations. A, the k
, K
, and K
values for the mutant
enzymes relative to wild-type are presented in the left, center, and right panels, respectively. The
corresponding values for k
, K
, and K
with wild-type enzyme
are 0.8 ± 0.1 s
, 120 ± 30 nM,
and 8.6 ± 1.3 µM, respectively. B, the
catalytic efficiency (k
/K
)
relative to wild-type enzyme is presented in order of decreasing
efficiency. The corresponding value for k
/K
with wild-type enzyme is 8.8 ± 1.6
10
M
s
.
In vivo, -pol is involved in short gap DNA
repair(1, 2, 3, 5) . DNA polymerase
is an ideal polymerase to examine ``intrinsic'' base
substitution fidelity, because it lacks an associated 3`
5`
proofreading exonuclease. In vitro, pol-
fills these
short gaps (
6 nucleotides) processively, whereas longer gaps are
filled distributively(21) . The fidelity of
-pol-dependent
long gap DNA synthesis (i.e. >100 nucleotides) had
previously been examined on undamaged(22, 23) and
damaged DNA templates(24, 25) . To determine the
fidelity of wild-type
-pol on a physiologically relevant DNA
substrate and to assess the effect of the amino acid substitutions on
fidelity, a reversion assay was developed on a short (5 nucleotide)
gapped DNA substrate containing an opal codon (Fig. 3A). This codon is within the non-essential lacZ
gene of bacteriophage M13mp2. Polymerase errors that
restore
-complementation activity yield a blue or light blue
plaque phenotype. This assay can detect eight different base
substitution errors.
The result of in vitro gap filling
synthesis by wild-type -pol and the mutants described above on the
reversion of the opal codon is shown in Fig. 3B.
Wild-type
-pol produced one revertant per 370 filled gaps
(reversion frequency of 27
10
). Whereas
deletion of the hydrogen bond donor at Tyr
did not alter
the reversion frequency, alanine substitution at Asn
significantly reduced it signifying an apparent increase in
fidelity. This apparent increase in fidelity could reflect a reduced
misinsertion rate or a reduced ability to extend mispairs, since both
must occur to score a mutant. In contrast, alteration of the
Arg
side chain, which interacts with the templating base,
dramatically lowered fidelity, as demonstrated by the strong increases
in reversion frequency (Fig. 3B).
Sequence analyses
of the DNA of lacZ mutants resulting from short gap
filling synthesis indicated that the types of base substitution errors
produced by the wild-type and R283A mutant were similar (Table 1). However, the frequency of each type of error was much
greater for the R283A mutant. The base substitution errors observed in
the polymerization products of both enzymes reflected misincorporations
resulting in relatively frequent T
dGTP and A
dGTP mispairs.
Seven of the eight mispairs detected by this reversion assay were
observed in the products of wild-type enzyme and the strong mutator
mutant R283A. For the mutant polymerase, a dGMP was incorporated
opposite a template thymidine nearly 46% of the time, whereas the
correct nucleotide was incorporated only 48% of the time. Additionally,
sequence analysis often detected two misincorporations by both
wild-type and R283A polymerases within the 5-nucleotide gap. These
misincorporations were, in many instances, consecutive, and in one
case, three consecutive misincorporations were observed. Consecutive
misincorporations had not been observed previously in the forward
mutation assay employing a long single-stranded
template(22, 23) . This suggests that a difference may
exist between the fidelity of
-pol during short processive gap
filling as compared with distributive DNA synthesis on large gaps.
Processive short gap filling synthesis is modulated by the binding of
the amino-terminal 8-kDa domain to the downstream 5`-phosphate group in
gapped DNA(26) .
To understand the structural basis for the
lower catalytic efficiency and fidelity of the alanine mutant of
Arg, we determined the x-ray crystal structure of this
mutant in complex with substrates (Fig. 4). In contrast to the
rat
-pol ternary complex, where the thumb subdomain is in a closed
conformation (10) , the human mutant
-pol ternary complex
crystallized in a different crystal packing form with the thumb in the
open conformation (space group P2
2
2). Hence,
Ala
is moved over 12 Å away from the template. This
open conformer had been observed previously when the wild-type human
enzyme is bound to the blunt end of duplex DNA(11) . We have
been unable to crystallize the mutant with the thumb in the closed
conformation. There is electron density corresponding to the incoming
thymidine moiety of ddTTP at the primer 3` terminus; however, the ddTTP
had not reacted with the primer and the 5`-triphosphate is disordered
and not visible. A F
- F
difference Fourier map
reveals a negative peak enveloping the Arg
side chain
consistent with mutation to alanine (Fig. 4). No other
significant changes were detected between wild-type and mutant enzymes.
Superimposition of the palm subdomains of the rat ternary closed
complex (space group P6
) with the mutant, or wild-type,
open ternary complex reveals that the N3 position of the templating
adenine in the open complex is 3.1 Å from where it is observed in
the closed complex. Superimposition of 97 C
s from the palm
subdomains of the rat ternary closed complex (PDB file 2bpf, see (10) ) and the ternary open complex of wild-type or R283A human
-pol gave a root mean square deviation of 0.75 Å. The
terminal primer sugars in the open and closed complexes are very near
one another,
C3` = 0.8 Å, whereas the equivalent
sugar positions for the templating base and incoming dNTP are displaced
3.8 and 1.2 Å, respectively. Since dNTPs can bind to the enzyme
in the open conformation(12) , closing of the thumb not only
positions the dNTP but positions the templating base for efficient
nucleotide incorporation.
Figure 4:
Structural comparison of wild-type and
alanine mutant of Arg of human
-pol complexed with
DNA and ddTTP. The structure of the ternary substrate complex of the
wild-type and R283A mutant were determined as described under
``Experimental Procedures.'' A F
- F
difference Fourier
map reveals a negative peak enveloping the Arg
side chain
consistent with mutation to alanine. No other significant changes were
detected in the structure. The DNA, helices M and N
(Arg
), and active site carboxylates (Asp
,
Asp
, Asp
) of the wild-type enzyme are
superimposed on the difference map. Although the thymidine moiety of
ddTTP is observed, the 5`-triphosphate is disordered. In contrast to
the rat
-pol ternary complex where the thumb subdomain is in a
closed conformation (Fig. 1)(10) , the human enzyme
(wild-type and mutant) crystallized with the thumb in the open
conformation. Therefore, Ala
is moved over 12 Å
away from where it is observed in the closed ternary
complex.
The fingers and thumb subdomains are
structurally diverse among the different classes of polymerases, and
except for -pol, the dNTP binding site is not clearly defined.
Therefore, the functional role of each subdomain may be unique to each
class of polymerase, and care must be taken in extrapolating the
present results to the thumb subdomain of other DNA
polymerases(27) .
In summary, fidelity assays coupled with
kinetic and structural evaluation of the alanine mutant of Arg indicate that this residue plays a central role in nucleotide
discrimination by correctly positioning and stabilizing the templating
base for efficient nucleotide incorporation. Although the guanidinium
group of Arg
is within hydrogen bonding distance to N3 of
the template guanine, the hydrogen bond geometry is unfavorable.
Therefore, correct van der Waal's interactions may also be
important at this site. This is consistent with the low catalytic
efficiency and reduced fidelity exhibited by the lysine mutant of
Arg
, which would be expected to preserve hydrogen bonding
to the templating base. Our results support the hypothesis that
discrimination and catalytic efficiency are modulated by polymerase
interactions near the templating base and are sensitive to precise
Watson-Crick base pairing by possibly ``sensing'' C1`
distances and bond angle geometry(28, 29) . In
contrast, alteration of direct interactions with the incoming dNTP
decreased dNTP binding affinity but not fidelity. Thus, the coupling
between catalytic efficiency and discrimination is residue-specific.
Our results indicate that we can modulate discrimination and catalytic
efficiency based upon ternary complex crystal structures, and
site-directed mutagenesis will be a productive avenue for future
analysis of polymerase structure-function relationships.