(Received for publication, September 27, 1994)
From the
We have employed site-directed mutagenesis to identify those
amino acid residues that interact with the deoxynucleoside triphosphate
(dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary
complex. Earlier structural, mutagenesis, and labeling studies have
suggested that the incoming dNTP molecule contacts a region on one side
of the polymerase cleft, primarily involving residues within the
so-called ``fingers'' subdomain. We have made mutations in
residues seen to be close to the dNTP in the crystal structure of the
Klenow fragment-dNTP binary complex and have examined their kinetic
parameters, particularly K. The results are
consistent with the notion that there are significant differences
between the dNTP interactions in the binary and ternary complexes,
although some contacts may be present in both. When dTTP is the
incoming nucleotide, the side chains of Arg
and
Phe
make the largest contributions to binding;
measurement of K
suggests
that Arg
contacts the
- or
-phosphate of the
dNTP. With dGTP, the contribution of Arg
remains the
same, but the additional interactions are provided by both Lys
and Phe
, suggesting that the binding of the
incoming dNTP is not identical under all circumstances. Mutations in
Arg
and Lys
also cause a substantial
decrease in the rate of polymerase-catalyzed incorporation, and sulfur
elemental effect measurements indicate that loss of Arg
(and perhaps also Lys
) slows the rate of the
chemical step of the reaction. Mutations of Arg
,
His
, and Tyr
affect the binding of DNA,
suggesting that these mutations, whose effect on dNTP binding is small,
may influence dNTP binding indirectly via the positioning of the DNA
template-primer.
The Klenow fragment of Escherichia coli DNA polymerase
I contains the polymerase and 3`-5` (proofreading) exonuclease active
sites of the parent molecule. Structural and biochemical studies have
revealed that these two catalytic sites are located on two distinct
structural domains(1, 2) . Klenow fragment was the
first DNA synthesizing enzyme with a known three-dimensional
structure(1) . Extensive mutagenesis and kinetic studies have
combined with this structural data to provide a wealth of information
on Klenow fragment, making it an ideal model to study the molecular
mechanism of template-directed DNA synthesis (e.g.(3, 4, 5, 6, 7, 8) ;
reviewed in (9) ). Protein sequence alignments (10) and
examination of the four known polymerase structures, Klenow
fragment(1) , human immunodeficiency virus-1 reverse
transcriptase(11) , T7 RNA polymerase (12) , and DNA
polymerase (13, 14) , suggest that even distantly
related polymerases may have analogous active sites with a small number
of crucial side chains in common. As a consequence, structure-function
studies pursued on a simple enzyme system such as Klenow fragment may
well be applicable to other more complex replication enzymes.
The
molecular mechanism of the 3`-5` exonuclease reaction has been well
characterized thanks to the structural data for complexes of Klenow
fragment with deoxynucleoside monophosphate and with single-stranded
DNA bound in the editing mode(1, 15, 16) ,
and to extensive mutagenesis studies(3, 17) . However,
since the three-dimensional structure of the catalytically competent
ternary complex for the polymerase reaction (Klenow fragment-duplex
DNA-dNTP) ()is not known, the reaction mechanism at the
polymerase active site is not as fully understood, although a plausible
mechanism has gained widespread acceptance(18) . Comparison of
the four known polymerase structures shows that the polymerase domain
in each case contains a deep cleft, whose overall shape has been
compared to that of a half-open right hand(11) , with the
-sheet that forms the base of the cleft described as the
``palm,'' and the two subdomains that form the walls of the
cleft described as ``fingers'' and ``thumb'' (Fig. 1). Protein sequence alignments (10) and
mutagenesis data (reviewed in (9) ) indicate that the
polymerase active site is located primarily on the palm subdomain. The
most important active site residues appear to be a group of two or
(more commonly) three carboxylates: Asp
,
Asp
, and Glu
in Klenow fragment. It has
been proposed that these carboxylates act to coordinate a pair of
divalent metal ions which then catalyze the phosphoryl transfer by a
mechanism analogous to that used at the 3`-5` exonuclease
site(9, 18) ; this mechanism is convincingly supported
by the recent structural data for mammalian DNA polymerase
(13, 19) . Kinetic studies have shown that, prior
to the phosphoryl transfer, the ternary complex must undergo a
nonchemical transformation, described as a conformational
change(6, 8) ; the precise nature of this step is
unclear at present.
Figure 1:
Positions of amino acid residues that
were mutated. The -carbon backbone of Klenow fragment (1, 9) is shown schematically with
-helices
represented by spiral ribbons and
-strands by arrows. The 3`-5` exonuclease domain, in a slightly darker
shade of gray, occupies the foreground. The polymerase domain is viewed
down the large cleft, with the palm subdomain at the base, the fingers
to the left and the thumb to the right. With the exception of
Asp
and Asp
, which serve to mark the
polymerase active site, the side chains that are illustrated correspond
to those mutated in this study.
To understand fully the polymerase reaction
catalyzed by Klenow fragment, the amino acid side chains that make
contact with the primer-template and the incoming dNTP in the ternary
complex must also be identified, and the role (if any) of these contact
residues as the reaction proceeds must be determined. A variety of
structural, chemical cross-linking, and mutagenesis studies have
addressed the location of the dNTP binding site. Although these studies
are generally in agreement in indicating a dNTP binding region that is
largely on the fingers subdomain, encompassing the exposed face of
Helix O (residues Arg to Tyr
) and
neighboring portions closer to the palm subdomain (Fig. 1), none
of the experimental approaches allows an unambiguous interpretation of
the data. The crystallographic studies (20) and the majority of
the chemical cross-linking experiments were carried out on the binary
Klenow fragment-dNTP complex; however, this complex is not
catalytically competent since there is a requirement for the binding of
the template-primer before dNTP can bind
productively(21, 22) . Clearly, data obtained from
studies of the binary complex will be of value in identifying
catalytically relevant interactions only to the extent that particular
interactions may be common to both binary and ternary complexes. Beese et al. (20) have made the entirely reasonable
suggestion that the phosphate positions in the binary complex (near the
positively-charged side chains of Lys
and
Arg
) are more likely to be relevant to the ternary
complex than are the base and sugar positions. In the binary complex
the deoxyribose was seen to be close to Phe
, while the
position of the base varied in different experiments, presumably due to
the absence of interactions with the opposing template base that would
serve to anchor the dNTP in the enzyme-DNA-dNTP ternary complex.
As
indicated above, most of the chemical cross-linking experiments that
have been reported for Klenow fragment were carried out on binary
complexes, so that, for example, experiments indicating that the
nucleotide base is close to Tyr or His
are
subject to the same caveat as the structural data, namely that
the position of the base moiety in the binary complex may be
misleading(23, 24) . A more convincing labeling
experiment, using 5`-fluorosulfonylbenzoyl deoxyadenosine as an
affinity label in the presence of primer-template DNA, in order to
mimic the ternary complex, suggested that the dNTP phosphates are close
to the side chain of Arg
(25) . Somewhat harder to
classify is the labeling of Lys
by pyridoxal
phosphate(26) , a reagent claimed to be directed to the dNTP
binding site. In this experiment dNTP, when bound either as a binary or
a ternary complex, protected Lys
from modification. The
pyridoxal phosphate experiment illustrates a general weakness of the
labeling approach, namely that some reagents may show specificity for
certain types of side chains, so that labeling is not necessarily
diagnostic for the closest side chain, but rather for a side chain of a
particular chemical class that is within reasonable proximity to the
reagent.
In the absence of structural data for the ternary complex,
site-directed mutagenesis and measurement of the effect of the
resulting mutations on K offers considerable advantages over the approaches already
described because it probes the enzyme-DNA-dNTP ternary complex. The
weakness of this approach is that it may be difficult to distinguish
between mutations that exert an effect on dNTP binding due to loss of a
dNTP contact residue and those whose effect is indirect via a change in
position of the opposing DNA template base at the dNTP binding site.
Thus far our mutational studies have identified 5 residues
(Asp
, Glu
, Tyr
,
Arg
, and Asn
) whose mutation causes a
weakening in dNTP binding, as measured by an increase in K
(4, 5) . Since none of the effects were
quantitatively large, and since several of these residues (particularly
Tyr
, Arg
, and Asn
) are in a
region of the protein likely to be close to the template
strand(27) , it is possible that our experiments to date have
not identified any important direct contacts to the dNTP molecule in
the ternary complex. To continue this investigation, we describe here a
further series of mutations in residues that point into the polymerase
cleft within the likely dNTP contact region (Fig. 1).
Figure 4:
Measurement of K(DNA) using a gel mobility shift assay. A, the 68-mer hairpin oligonucleotide. This molecule, labeled
at the 5` end with
P, was employed as the substrate in the
mobility shift assay. B, gel assay of the binding of the Q708A
mutant protein to the 68-mer, carried out as described under
``Experimental Procedures.'' The protein concentration (in
nM) increases from lane to lane in 2-fold steps from left to
right (not all lanes are labeled). U indicates the mobility of
the unbound 68-mer; A and B indicate the positions of
the two protein-bound species that we have detected. C, a plot
of the binding data obtained for the Q708A (experiment shown in panel B) and R682A mutant proteins. The fraction of the DNA
substrate that is enzyme-bound is plotted as a function of protein
concentration (
, Q708A;
, R682A). By interpolation, K
was calculated to be
1.4 nM for Q708A and 4.7 nM for
R682A.
Additionally, K was determined for wild-type
Klenow fragment and the R682A, H734A, R754A, K758A, and Y766A mutant
derivatives using DNase I footprinting. The experiment was carried out
as described previously(4) , except that the hairpin 68-mer was
used instead of a primed M13 substrate, and the data were quantitated
on a PhosphorImager instead of by densitometry of autoradiographs.
Figure 2: Eadie-Hofstee plots of the steady-state kinetic data for the polymerase reaction catalyzed by wild-type Klenow fragment (A), and the R754A (B), and H734A (C) mutant proteins. In each case, V represents the rate of incorporation of labeled dTTP, and S is the concentration of dTTP. Note that the axis scales are different in the three panels.
The H734A mutant protein was unusual
in that the Eadie-Hofstee plot was not linear, but appeared instead to
be composed of two linear plots (Fig. 2, panel C).
Since this type of plot might be obtained if the mutant protein
preparation were contaminated with a small amount of wild-type Klenow
fragment, we repeated the determination with another preparation of the
H734A protein from a different batch of induced cells. The same
biphasic curve was obtained with the new enzyme preparation, indicating
that this unusual behavior is indeed a property of the H734A mutant
protein. Analysis of the rate data for H734A at relatively low
concentrations of dTTP gave a K of 10 µM; at dTTP concentrations above about 10
µM the measured rates were higher than expected based on a
linear extrapolation, corresponding to an apparent activation by
substrate dNTP. Substrate activation can be explained by the binding of
a second molecule of substrate in such a way that the reaction rate is
enhanced(33) . Analysis of the steeper portion of the
Eadie-Hofstee plot (Fig. 2, panel C) indicated that the
putative second binding site has a low affinity for dTTP (K
of around 300 µM)
and causes the reaction rate to increase about 20-fold (values in parentheses in Table 1).
The steady-state kinetic
constants for dGTP incorporation on a poly(dC) template were determined
for wild-type Klenow fragment and those mutant proteins (H734A, R754A,
K758A, F762A, and Y766A) that showed the largest differences from wild
type in dTTP incorporation. We felt it was important to check the
kinetics with a different dNTP substrate because of a recent study of
the K758A mutant of Klenow fragment (34) that reported
different kinetic constants for dGTP and dTTP incorporation. Our
results showed interesting differences in the response of the mutant
proteins to the two dNTP substrates (Table 1). For most of the
proteins studied, K was higher
than K
; the K758A mutant was the
only one for which K
was
significantly higher than K
. For
the H734A derivative the two K
values were approximately equal but the use of dGTP eliminated
the apparent substrate activation seen with dTTP.
Figure 3:
Pyrophosphorolysis reaction catalyzed by
the K758A mutant of Klenow fragment. Panel A shows the
electrophoretic separation of aliquots of the reaction mixture removed
at the time intervals (in hours) indicated at the top of each lane. The
positions of the substrate, p(dT), and the first three
degradation products are indicated at the right-hand side. Panel B shows the rate plot from the experiment in Panel A; the
number of phosphodiester bonds broken per molecule of starting material
is plotted as a function of time.
As a check
on the validity of the gel mobility shift method, K was also determined by DNase I
footprinting for the wild-type, R682A, H734A, R754A, K758A, and Y766A
proteins. The results are in good agreement with K
values determined from the gel
assay (Table 3), and give us confidence that gel mobility shifts
give a reliable estimate of K
in
this system.
Figure 5: Conservation of the residues mutated in this study in the pol I (or A) family of DNA polymerases. The alignment of the relevant regions of E. coli DNA polymerase I with the DNA polymerases from Streptococcus pneumoniae (Sp), Thermus aquaticus (Tq), Thermus flavus (Tf), bacteriophages T5, T7, SP01, and SP02, and the yeast mitochondrial DNA polymerase (Mip1) is based on that of Braithwaite and Ito(36) . To this alignment have been added the sequences for the DNA polymerases from Thermus thermophilus (Tt)(45) , Deinococcus radiodurans (Dr)(46) , Bacillus caldotenax (Bc)(47) , Mycobacterium tuberculosis (Mtb) (V. Mizrahi, personal communication (GenBank accession no. L11920)), Mycobacterium leprae (Mlep) (D. R. Smith, personal communication (GenBank accession no. U00021)), bacteriophage T3(48) , and mycobacteriophage L5(49) . The residues relevant to this study (which are by no means the only conserved residues within the regions shown) are boxed; dark grey shading indicates identity with the E. coli prototype sequence, and lighter grey indicates a conservative substitution.
To measure dNTP binding, we
first determined K values for the
polymerase reaction for each of the mutant Klenow fragment derivatives,
since K
has previously been shown
to be a valid measure of the binding affinity of dNTP (K
= K
) in the catalytically competent
ternary complex(6, 22, 37) . Substitution of
Arg
and Phe
caused K
to increase by 2 orders of
magnitude. This is by far the largest increase seen for any Klenow
fragment mutation reported to date, and translates into a loss of about
3 kcal mol
in binding energy. (Aside from the
present study, 23 single amino acid mutations comprising substitutions
at 13 different positions in the polymerase domain of Klenow fragment
have been studied in
detail(4, 5, 32, 34, 37, 38) ).
Although measurements of K
for a
mutant protein cannot distinguish between direct effects (due to
removal of a side chain that contacts the dNTP molecule) and indirect
effects (via a protein contact to the template strand), we suspect that
the large increases due to the R754A and F762A mutations reflect direct
contacts of Arg
and Phe
with the incoming
dNTP. Conversely, the smaller changes in K
that resulted from the Y766A
and H734A mutations are suggestive of indirect effects via template
strand contacts, a conclusion that is supported by the observation that
both mutations caused a decrease in DNA binding affinity. The mutations
R682A and Q708A, in residues that contact the dNTP in the crystalline
binary complex(20) , also had extremely modest effects on dNTP
binding, arguing against direct contacts in the ternary complex.
Interactions with the negatively charged phosphate groups of the
dNTP molecule are likely to account for an important subset of the
Klenow fragment-dNTP interactions. Certainly, earlier studies on the
binary complex indicated that the dNTP phosphates are important
determinants of binding specificity(39) . Moreover,
pyrophosphate is a competitive inhibitor of the polymerase reaction,
suggesting that the PP binding site overlaps the dNTP
binding site in the ternary complex(8) . Comparison of the
values of K
and K
(Fig. 6) suggests that
the positively charged Arg
interacts with the
-
and/or
-phosphates of the dNTP, whereas Phe
contacts
some other part of the molecule. Given the hydrophobic nature of the
Phe side chain, a plausible contact would be with the deoxyribose, or
with that portion of the base not involved in contacts with the
opposing template strand.
Figure 6:
Relative K(dTTP) (A) and K
(PPi) (B) values for wild-type
and mutant Klenow fragment derivatives. Values are normalized to the
wild-type value, set at 1.
The comparison between K and K
for selected mutant proteins
showed some interesting differences in the utilization of these two
substrates. Wild-type Klenow fragment bound dGTP 4-fold more tightly
than dTTP in the ternary complex (as measured by the ratio of the two K
values), perhaps reflecting the stronger base
pairing of dGTP with the template. Although the K
values were much higher, the R754A mutant protein gave the same
ratio, indicating that the binding of both dGTP and dTTP is impaired to
a similar extent, as would be expected for a mutation that affects a
phosphate contact remote from the nucleotide base. By contrast, the
other mutant proteins tested gave quite different ratios, ranging from
K758A, which affected dGTP binding more than dTTP binding, to F762A for
which the opposite was observed. Thus it appears that, in the region
where the protein contacts the dNTP base and ribose, the side chains
assume different degrees of importance depending on the exact
circumstances. With the homopolymer DNA substrates used in our assays,
Phe
is the most important contact (in addition to the
phosphate contact at Arg
) when T is the incoming base,
whereas Lys
and Phe
contribute
approximately equally when G is added. Mutation of His
also has a substantial effect on dGTP binding; however, as
indicated above, we suspect that this effect may be mediated via the
DNA template. Presumably the apparent changes in protein contacts for
different incoming dNTPs reflect subtle differences in geometry in the
respective ternary complexes; however, our data do not allow us to
distinguish between several possibilities that may form the basis for
these differences. The polymerase could have a distinct mode of
interaction with each of the four bases; alternatively, it might
distinguish between purines and pyrimidines, or between A-T versus G-C pairings. A more complex but perfectly plausible scenario is
that the geometry of the dNTP interaction is influenced by the local
sequence context at the position of insertion. This latter possibility
is certainly consistent with the report by Eger et al.(37) of two distinct K
(dTTP) values
for wild-type Klenow fragment on non-homopolymeric substrates, both of
which were substantially higher than the K
(dTTP)
reported here for incorporation on poly(dA)-oligo(dT).
We do not have an explanation for the apparent substrate activation shown by the H734A mutant protein at high dTTP concentrations. This type of kinetic behavior is relatively rare (e.g. refs. 33, 40); when observed it has been attributed to a second, weaker substrate binding site whose occupancy leads to an increase in reaction rate. The reaction rates have been measured for wild-type Klenow fragment and for each of the mutant derivatives in this study over the same range of dTTP concentration as in the H734A determination, but none of the proteins (aside from H734A) showed any unusual relationship between reaction rate and dTTP concentration. It is unclear at present why the H734A mutation, which probably removes a DNA contact, should allow this aberrant binding of a second dTTP molecule, or why the effect should be seen with dTTP but not with dGTP.
In addition to
their effect on K, the R754A and
K758A mutations caused a substantial decrease in the rate of the
polymerase reaction. The sulfur elemental effect data imply that loss
of Arg
slows the chemical step of the reaction, while
Lys
may be involved to some extent in both the chemical
step and the preceding conformational change. The roles of the
Arg
and Lys
side chains in accelerating the
polymerase reaction could involve stabilization of the relevant
transition state(s) via interactions that are related to those proposed
with the dNTP ground state. For Arg
, we suggest that the
interaction with the
- or
-phosphate may assist in the
removal of the pyrophosphate leaving group as the chemical step of
catalysis proceeds. For Lys
, it seems that the extent to
which the interaction between this side chain and the dNTP molecule is
manifested in the ground state or in the relevant transition state(s)
varies depending on the identity of the incoming dNTP. When dTTP is the
incoming nucleotide, Lys
stabilizes the transition state
without contributing to ground state binding, since k
is decreased 300-fold while K
is virtually unchanged. Given the positive charge on the Lys side
chain, it is possible that it may interact so as to neutralize the
developing negative charge at the
-phosphate oxygens. Whatever the
nature of the interaction that takes place at Lys
, it is
clear that, when dGTP is the incoming nucleotide, more of the binding
energy is used in the ground state and consequently less is available
for transition state stabilization. This is shown by the observation
that the K758A mutation has a greater effect on K
and a smaller effect on k
when addition of dGTP is compared with
addition of dTTP.
Our data for the Y766A mutation can be
compared with previous studies on the Y766S and Y766F mutations (4, 32) to give a fuller picture of the requirements
at this position. The Ala substitution is slightly more detrimental
than the Ser substitution, while the Y766F protein behaves very
similarly to wild-type Klenow fragment (32) . ()Like
Y766S, but unlike Y766F, the Y766A mutation has a mutator phenotype in vivo (data not shown). Thus, the phenolic ring appears to
be the most important feature of the Tyr
side chain, with
the hydroxyl group playing a significant but secondary role, consistent
with the observation that Tyr (not Phe) is invariably found at this
position in related polymerase sequences (Fig. 5; (10) and (36) ).
Two of the mutations in the present
study, R682A and K758A, have also been investigated recently by Pandey et al.(34, 38) , and our results are largely
in agreement with theirs although we differ on some points of
interpretation. For the K758A mutant protein, Pandey et al.(34) reported essentially identical kinetic constants to
ours, using similar homopolymer substrates, and likewise noted a
difference in the incorporation of dTTP and dGTP. However, we disagree
with their suggestion that Lys may interact with the dNTP
- or
-phosphate or that it plays a specific role in
translocation. First, our observation that the K758A mutation did not
affect pyrophosphate binding argues against an interaction with the
- or
-phosphate. Second, the K758A protein showed a slow rate
of reaction under the single turnover conditions employed in our
elemental effect measurements, indicating a slow step at or preceding
chemistry (see also Footnote 3). Since processivity is determined
simply by the competition between the forward reaction and DNA
dissociation, it is not necessary to postulate, in addition, a slowing
of translocation in order to account for the lower processivity
observed by Pandey et al.(34) , although the data do
not of course rule out another slow step at a later stage in the
reaction pathway. In contrast with the results of Pandey et
al.(34) , we have not observed any particular tendency of
the K758A protein to pause at template A residues. (
)For the
R682A protein Pandey et al.(38) reported a small
increase in K
and a
20-25-fold decrease in k
, whereas we have
found the values to be almost the same as wild type. A possible
explanation for this disagreement is that the homopolymer substrate
used by Pandey et al.(38) in their experiments
apparently contained a DNA primer annealed to an RNA template, whereas
our assays employed the usual DNA-DNA hybrid. As with the K758A
mutation, we do not feel that the data for R682A support a specific
role for this side chain in translocation, as suggested by Pandey et al.(38) , since the observed decrease in
processivity could be simply a consequence of weaker DNA binding.
Instead, we suggest that the rather insignificant effects of the R682A
mutation on the polymerase reaction are probably related to the
decrease in DNA binding affinity caused by this mutation. The lack of
any dramatic effect of the R682A mutation on dNTP binding was somewhat
surprising, given the results of affinity labeling experiments in which
this side chain was labeled by 5`-fluorosulfonylbenzoyl deoxyadenosine
in a template-dependent manner (25) . It is possible that the
Arg
side chain, while not participating in a specific
interaction with the dNTP, may yet be sufficiently close to the
reactive portion of the affinity analog to become labeled, especially
since the reagent is selective for nucleophilic side chains.
Data from a
number of sources (reviewed in (9) ) indicate that the active
site of DNA polymerases is defined by a trio of carboxylate groups
(Asp, Asp
, and Glu
in Klenow
fragment) which are proposed to anchor a pair of divalent metal ions
that carry out catalysis of the phosphoryl transfer reaction (9, 18) . The results of this and our earlier studies (4, 5) allow us to make predictions about some of the
other interactions that take place in the active site. The proposed
reaction mechanism requires the dNTP to bind with its
-phosphate
group positioned between the metal ions, and this expectation has been
confirmed by structural studies of mammalian DNA polymerase
(13, 19) . Our previous elemental effect data (5) suggest that the Sp oxygen at the
-phosphate
is close enough to the Asp
side chain to provide steric
obstruction in the transition state. (
)From the studies
presented here, we propose also that the dNTP
- and/or
-phosphate interacts with Arg
, and the uncharged
portion of the molecule, perhaps the deoxyribose ring, is bound close
to Phe
. As the phosphoryl transfer reaction proceeds,
polar side chains in the active site region (including
Arg
, Lys
, Arg
, and
Gln
, (5) ) may make interactions that stabilize
the transition state and thus facilitate the reaction. Our current data
suggest that Arg
acts to assist in removal of the
pyrophosphate leaving group; we do not have evidence to assign precise
roles to the other three side chains at present. We hope that, in the
future, these predictions can be tested, and the interactions at the
active site defined in more detail, by crystallographic data for a
ternary complex of Klenow fragment with DNA and dNTP substrates.