(Received for publication, May 11, 1995; and in revised form, June 22, 1995)
From the
The function of a lysine residue, Lys, of human
DNA polymerase
located in the third most conserved region and
conserved in all of the
-like polymerases was analyzed by
site-directed mutagenesis. Lys
was mutagenized to Arg,
Ala, or Asn. The mutant enzymes were expressed in insect cells infected
with recombinant baculoviruses and purified to near homogeneity. The
mutant enzymes had specific activities ranging from 8 to 22% of the
wild type. All three Lys
mutants utilized Mn
as metal activator more effectively than the wild type enzyme and
showed an increase in K
values for
deoxynucleoside triphosphate but not k
values in
reactions with either Mg
or Mn
as
the metal activator. Although mutation of the Lys
residue
caused an increase in K
values for
deoxynucleoside triphosphates, mutations of Lys
to Arg,
Ala, or Asn did not alter the mutant enzymes' misinsertion
efficiency in reactions with Mg
as a metal activator
as compared with that of the wild type, suggesting that the base of the
incoming deoxynucleoside triphosphate is not the structural feature
interacting with the Lys
side chain. In reaction with
Mn
as a metal activator, all three Lys
mutants had an improved fidelity for deoxynucleotide misinsertion
compared to wild type. Inhibition studies of the three Lys
mutant derivatives with an inhibitor, structural analogs of
deoxynucleoside triphosphate, and pyrophosphate suggest that the
deoxyribose sugar and
-,
-phosphate groups are not the
structural feature recognized by the Lys
side chain.
Comparison of the mutant enzymes to the wild type enzyme for their
affinities for dCTP
S versus deoxynucleoside triphosphate
suggests that this highly conserved Lys
is involved in
interacting either directly or indirectly with the oxygen moiety of the
-phosphate of the incoming deoxynucleoside triphosphate.
Compilation and alignment of the protein sequences of DNA
polymerases deduced from the nucleotide sequence data have classified
DNA polymerases into three families, family A, B, and C, according to
their similarities to Escherichia coli polymerase I, II, and
III, respectively(1, 2) . The -like DNA
polymerases including the E. coli polII belongs to family B
withpolymerase
as the prototype(3) . The lack of an
intrinsic proofreading nuclease in DNA polymerase
makes this
enzyme an ideal model to study the structure-function relationship of
the active site and serves as a model for all of the
-like DNA
polymerases, particularly in identifying residues in the active site
that are responsible for DNA synthetic
fidelity(4, 5) . We have overproduced functionally
active recombinant human DNA polymerase
catalytic subunit in
insect cells infected with a recombinant baculovirus (6) and
established a one-step immunoaffinity purification protocol to purify
the enzyme to near homogeneity(7) . By site-directed
mutagenesis followed by physical and steady-state kinetic studies,
several highly invariant residues in the catalytic site of human DNA
polymerase
were
analyzed(4, 5, 8, 9) . We have
established that three residues, -Asp-Thr-Asp-, in the most conserved
region of the
-like DNA polymerases (region I, -YGDTDS-) are
involved in metal activator binding. The two aspartate residues,
Asp
and Asp
, like the aspartate residues
in the active site of Klenow and HIV reverse transcriptase forming the
``catalytic triad,'' directly participate in chelating the
metal
ion(8, 9, 10, 11, 12) .
Mutations of Asp
to Asn and Thr
to Ser
also yielded mutant polymerases as metal ion-induced anti-mutators (8) . Thus, residues Asp
, Thr
,
and Asp
in the most conserved region also play critical
roles in the observed metal-induced infidelity in DNA synthesis of
cellular DNA polymerases.
Five highly conserved residues in the
second most conserved region (region II) were also analyzed. Mutation
of a conserved glycine, Gly, appears to affect both
catalysis and substrate dNTP binding, suggesting that this glycine
residue is essential for the maintenance of the overall active site
structure. Mutations of Tyr
altered the affinity of the
mutant enzyme to bind the incoming dNTP. Analysis of the Tyr
mutant enzyme for its DNA synthetic fidelity has shown that the
phenyl ring side chain of Tyr
directly interacts with the
nucleoside base moiety of the incoming dNTP and plays a critical role
in nucleotide misinsertion fidelity of DNA synthesis(5) .
Mutation analyses of the second serine residues in the conserved
region, -SLYPSI-, have revealed that the hydroxyl side chain of this
serine residue, Ser
, directly interacts with the 3`-OH
terminus of the primer and plays an essential role in mispaired primer
extension fidelity of DNA synthesis(4) .
In this report, we
continue to investigate the contributions of those highly conserved
amino acid residues in the catalytic site of -like DNA polymerases
for either substrate binding or for catalysis. We analyzed an invariant
lysine residue in the third most conserved region (region III) that is
conserved from human DNA polymerases
,
, and
to E.
coli polymerase II.
Recombinant baculoviruses
were generated by co-transfection of Sf9 cells with the transfer
plasmids and linear baculovirus DNAs as described(9) . Five
micrograms of transfer plasmid DNA were co-transfected with 1 µg of
linear AcGal viral DNA, and the recombinant viruses were selected
using standard baculovirus techniques.
Figure 1:
Sequence alignment of the third most
conserved region of -like DNA polymerases. Amino acid residues
943-967 of human DNA polymerase
were aligned with amino
acid residues from 18 other
-like DNA polymerases. Gaps are
indicated by dashes, and extensive gaps are indicated by the
number of amino acids contained within the gap. All of the amino acid
sequences are derived from (1) and (2) . The amino
acid sequences of human polymerase
, bovine polymerase
, E. coli DNA polII, Schizosaccharomyces pombe polymerases
, and
are from (27, 28, 29, 30) , respectively.
Conserved residues are boxed. The highly conserved lysine
residue studied in this report is marked by number sign. Two
other highly conserved residues, tyrosine (Y) and glycine (G), are marked with *.
In reactions with Mn as
the metal activator, mutants K950R, K950A, and K950N had 8-, 4-, and
6-fold increases in K
for dNTPs and moderate 3-,
3.5-, and 4.5-fold increases in their k
, as
compared with the wild type, respectively. In reactions with
Mn
, these three mutant enzymes also had comparable
DNA synthetic processivity and K
values for primer
terminus as the wild type enzyme (Table 1). These kinetic
parameters thus render the mutant enzymes with specific activities
comparable with the wild type enzyme in reactions with
Mn
.
Since mutation of Lys to Arg or
Ala but not Asn increased the mutant's affinity (decreased the K
) for primer terminus in reactions with
Mg
as the metal activator, we also investigated the
effect on template interaction when the positively charged side chain
of this lysine residue is replaced by either a larger size charged side
chain or is completely abolished. Inhibition by single-stranded DNA was
compared, and no apparent difference was found between wild type and
all three mutant enzymes (data not shown). This result indicates that
this lysine residue is not involved in template interaction.
Results
of these kinetic parameter studies suggest that mutation of this highly
conserved Lys residue primarily affects the affinity for
the incoming dNTP substrate and does not affect catalysis.
Figure 2:
Metal titration assays for the wild type
and mutant enzymes. Reactions were carried out as standard DNA
synthesis reactions with varying concentrations of MgCl (
--
) or MnCl
(
--
). Titration curves are plotted as
specific activity of enzyme versus metal ion concentration for
each enzyme.
We first tested the effect of an
inhibitor, aphidicolin, on these three Lys mutant
enzymes. Aphidicolin is a general inhibitor for all three major
cellular
-like DNA polymerases,
,
, and
(3) . Aphidicolin acts as a competitive inhibitor of
pyrimidine deoxynucleoside triphosphate, but, structurally, aphidicolin
is not an analog of dNTPs. We have previously proposed a model of how
aphidicolin forms hydrogen bonds with the purine base of the nucleotide
in template in the active site of
-like DNA
polymerases(5) . To test if Lys
plays a role in
the active site in interacting with metal activator(s) or the
dNTP-metal activator complex, we tested the inhibitory effect of
aphidicolin on the three mutant derivatives of Lys
and
compared their 50% inhibition point to that of the wild type reactions
with Mg
as metal activator. The three Lys
mutant enzymes, K950R, K950A, and K950N, were 10, 14, and 33
times more sensitive to aphidicolin inhibition than the wild type
enzyme, respectively (Table 3). These results suggest that
Lys
functions in the active site and aphidicolin affects
the interaction between the Lys
side chain and the dNTP
substrate.
We next compared the three Lys mutant
derivatives to wild type enzyme for their 50% inhibition points by an
analog of dGTP, BuPdGTP, and its
,
-methylene derivative,
BuPdGMPCH
PP (Table 3). All three Lys
mutant derivatives showed higher sensitivity to both of these
compounds than the wild type enzyme. Mutant enzyme K950R had about 3
times higher sensitivity to both BuPdGTP and BuPdGMPCH
PP.
Mutant enzyme K950A and mutant enzyme K950N both showed much higher
sensitivity to the inhibition by BuPdGTP and BuPdGMPCH
PP
than did the K950R (Table 3). These indicate that the Lys
side chain indeed has a role in the active site and is involved
in interacting with the incoming dNTP.
Results of the kinetic and
inhibitor studies suggest that the positively charged side chain of
Lys has a function in the active site and is involved in
interacting with the dNTP-metal activator complex.
In
reactions with Mg, wild type enzyme had 22-fold
higher affinity for dTTP than dCTP
S (Table 5). Mutant enzyme
K950R had 3-fold higher affinity for dCTP
S than dNTP
(K
= 86 ± 46 µM and
K
= 250 ± 43 µM),
but comparable affinity for dCTP
S as the wild type enzyme
(K
= 86 µM ± 46
µM for K950R and K
= 74
± 12 µM for the wild type enzyme). Mutant enzyme
K950A had 5- to 6-fold lower affinity to dCTP
S than the normal
dNTP; mutant enzyme K950N had 13-fold lower affinity for dCTP
S
than dNTP (Table 5). Mutant enzymes K950A and K950N had 6 and 24
times lower affinity (higher K
values) for
dCTP
S than the wild type enzyme. These results indicate that
substitution of the oxygen moiety of the
-phosphate by sulfur
profoundly affects the affinity of the Lys
mutant enzymes
for the
-phosphate analog. This suggests that the Lys
side chain interacts with the
-phosphate group of the
incoming dNTP substrate.
In contrast to the reactions
with Mg as the metal activator, in reactions with
Mn
as metal activator, all three mutant enzymes as
well as the wild type enzyme had lower affinity (higher K
values) for dCTP
S than the wild type
enzyme. Furthermore, the three mutant enzymes and the wild type enzyme
had comparable or equal affinity ratio of dCTP
S to dTTP
(K
/K
) (Table 5).
We then compared the three mutant enzymes to the wild type enzyme
for their catalysis rate (k) in utilizing
dCTP
S versus dNTP as substrate. In reactions with
Mg
, the wild type enzyme had 2.6-fold higher k
in utilizing normal dNTP versus dCTP
S as substrate with a ratio of
k
/k
of 0.38. Mutant
enzyme K950R did not show a significant difference in its k
value when either normal dNTP or dCTP
S
was used as substrate. Mutant enzyme K950A like the wild type enzyme
had an approximately 2-fold higher k
when dNTP
was used as substrate than when dCTP
S was used as substrate
(k
= 0.68 and k
= 0.27). Mutant enzyme K950N, interestingly, had higher k
when dCTP
S was used as substrate versus dNTP (Table 5).
When Mn was
used as the metal activator, the wild type enzyme had identical k
values in utilizing either dCTP or dCTP
S
as a substrate. Mutant enzymes K950R and K950N both had comparable k
values in using dCTP
S or normal dNTP as a
substrate. Mutant enzyme K950A showed a 2-fold lower k
in using dCTP
S as the substrate than with dCTP as the
substrate, like that observed in the Mg
-catalyzed
reaction.
By comparing the affinity (K) and
catalysis (k
) of these mutant enzymes to the
wild type enzyme for utilizing dNTP and dCTP
S as substrate, it is
apparent that substitution of the -P=O by
-P=S in the
-phosphate group of dCTP profoundly
affects the affinity of the enzyme's binding to the incoming dNTP
substrate, but does not significantly affect the rate of catalysis.
These results together with the findings that the three Lys mutant enzymes are able to utilize Mn
as metal
activator strongly suggest that the positively charged side chain of
Lys
either directly or indirectly participates in
interactions with the oxygen moiety of the
-phosphate group of the
incoming dNTPs, either to position the dNTP to interact with the metal
activator or to facilitate the nucleophilic attack by the 3`OH group of
the incoming primer.
We have used the recombinant human DNA polymerase as
the prototypic model for the three principal cellular DNA polymerases
,
, and
to elucidate the functional roles of several
highly invariant amino acid residues in the active site. We altered
several invariant residues by site-directed mutagenesis based on the
rationale that we described(20) . We generated a panel of
mutants that did not have any detectable gross alteration of the
protein structure(4, 5, 9, 20) .
Thus, it is reasonable to assume that the structural alterations
resulting from each mutation are confined to the position of the
mutated side chains. By steady-state kinetic analysis of the mutant
enzymes, we have defined the functions of several residues in the two
most conserved regions (regions I and II) of the active
site(4, 5, 8, 9) . In this report,
we have extended our studies of the active site by investigating the
function of a highly invariant lysine residue in the third most
conserved region (Fig. 1).
Previous study has documented that
the interaction of DNA polymerase with its substrates obeys a
rigidly ordered sequential terreactant mechanism, with template as the
first substrate, followed by primer as the second substrate and dNTP as
the third. Specification of which of the four dNTPs has kinetically
significant binding is determined by the base sequence of the
template(21) . A similar ordered sequential terreactant
mechanism was also proposed by Dahlberg and Benkovic (22) for
the Klenow fragment of E. coli polymerase I. Given the
universal ordered sequential mechanism for both eukaryotic DNA
polymerase
and prokaryotic E. coli polymerase I, and
depending on the base sequence of the template, DNA polymerases have
different modes of interaction with the incoming dNTP. Studies of
Klenow fragment have shown a rate difference in using dTTP versus dGTP(23) . In this study, we did not compare the
difference of either the wild type or the mutant enzymes for their
affinity (K
) in binding each
different incoming dNTP or the difference in binding purine versus pyrimidine deoxyribose triphosphate. It is possible that the side
chain of this highly invariant Lys of
-like DNA polymerases like
that of E. coli polymerase I has a different mode of
interaction with each different incoming dNTP(23) . Here, we
assume in the enzyme where the protein contacts the dNTP, the
Lys
side chain has the same interaction with all four of
the dNTPs in all circumstance. We also only evaluated the side chain
function of Lys
by kinetic analysis in the state of
catalytically competent ternary complex at the point of phosphodiester
bond formation and pyrophosphate release.
Studies of E. coli polymerase I
have proposed that there is an indirect interaction between metal
activator and the deoxyribose of dNTP(24) . It has been
proposed that E. coli polymerase I-Mg complex selectively prefers the C2`-endo conformation of the
deoxyriboside of dNTPs, while the polymerase I-Mn
complex is less selective for this conformation. Thus, in the
Mn
-catalyzed reaction, the deoxyribose freely
equilibrates between the C2`- and C3`-endo conformations of the
deoxyriboside. It is possible that the polymerase
-Mn
complex like that of E. coli polymerase I-Mn
complex, is less selective for C2`-endo conformation of
deoxyriboside and allows the sugar ring to freely equilibrate between
C2`- and C3`-endo conformations. This might enhance the stringency for
the polymerase
-Mn
complex in its specification
for a correct dNTP over an incorrect dNTP resulting in improved
misinsertion fidelity.
Based on structural data together with
mutagenesis studies of Klenow fragment, Joyce and Steitz and co-workers (12, 25) have proposed a possible mechanism for the
polymerase reaction in which two metal ions are involved in mediating
catalysis. In the active site of a DNA polymerase, several carboxylate
side chains, such as the Asp and Asp
of the
Klenow and Asp
and Asp
of human
polymerase
, function to anchor two divalent metal ions
(Mg
) for catalysis. One Mg
promotes
the deprotonation of the 3`-hydroxyl of the primer, while the second
Mg
facilitates the formation of the pentacovalent
transition state at the
-phosphate of the dNTP and the loss of
pyrophosphate. Since mutations of Lys
affect both the
metal activator utilization and affinity for dNTP, the side chain of
Lys
therefore might interact either directly or
indirectly with the second metal ion chelated
-phosphate of the
incoming dNTP.
Since alteration of the deoxyribose in either
araCTP or ddCTP could also affect the orientation of the oxygen group
of -phosphate, we also tested the interactions between the
Lys
side chain and the oxygen group of
-phosphate
with an analog, dCTP
S, in reactions utilizing either
Mg
or Mn
as metal activator. We
compared each enzyme's affinities (K
) and
catalysis (k
) for utilizing dCTP
S versus dNTP as substrate in reactions with either
Mg
or Mn
(Table 5). We also
compared each enzyme's affinity ratio for dCTP
S versus dNTP (K
/K
) with
the wild type enzyme. In reactions with Mg
, mutation
of the Lys
side chain by either replacing it with a
larger charged side chain (K950R) or abolishing the charged side chain
(K950A) had a significant effect on the mutant enzyme's affinity
to dCTP
S versus dNTP substrate. In contrast, replacing
the
-amino side chain of Lys
to Asn (K950N) appears
not to affect the mutant's affinity ratio for dCTP
S versus dNTP. In Mn
-catalyzed reactions, all
three mutants showed comparable affinity ratio,
K
/K
, as the wild type
enzyme. We reason that this difference in metal effect might be due to
the polymerase
-Mn
complex having less selective
preference for C2`-endo deoxyribose. The presence of the C3`-endo form
of dNTP could affect the affinity between the dNTP and the side chain
of Lys
resulting in an observed mild effect on the
affinity of the mutant enzymes for dCTP
S.
In sum, results
presented in this study strongly suggest that the structural feature of
the incoming dNTP recognized by the positively charged Lys side chain is the oxygen moiety of the
-phosphate group.
Our
mutational studies (4, 5, 8, 9) have
identified the functions of side chains of several amino acid residues
localized in the active site of the -like polymerases. The results
have supported a model of the active site of
-like polymerases. In
human polymerase
, Asp
and Asp
, and
Thr
located in an anti-parallel
-sheet (region I)
chelate with the metal activator cation, Mg
, which in
turn chelates to the oxygen moiety of
- and
-phosphate of the
incoming dNTP(8, 9) . The phenyl ring side chain of
Tyr
(in region II) interacts with the nucleotide base of
the incoming dNTP to properly position the incoming dNTP for
Watson-Crick base pairing(5) . The oxygen moiety of the
Ser
hydroxyl side chain in region II forms a hydrogen
bond either directly or indirectly with the 3`-OH terminus of the
primer. The hydrogen bond formation might enhance the oxygen moiety at
the 3`-OH-primer terminus for nucleophilic attack at the
-phosphate of the incoming dNTP(4) . The positively
charged side chain of Lys
located in an
-helix in
region III of human polymerase
interacts either directly or
indirectly with the oxygen group of the
-phosphate of the incoming
dNTP. This interaction of a positively charged side chain will
neutralize the negative charge on the
-phosphate to facilitate
nucleophilic attack of the incoming primer 3`-hydroxy group. This
proposed model of the
-like polymerase active site (Fig. 3)
is based entirely on biochemical data and can only be verified in the
future by crystallographic data of a ternary complex of polymerase
with primer-template and dNTP substrates.
Figure 3:
Model of the -like DNA polymerase
active site residues collaborate for nucleotidyl transfer. Shown are
the proposed functions of residues of human DNA polymerase
studied by site-directed mutagenesis. The three region I residues,
Asp
, Thr
, and Asp
, shown
here bound to the metal-nucleotide complex are adopted from (9) . The phenyl ring of Tyr
in region II that
interacts with the nucleotide base moiety of the incoming dNTP and the
hydroxyl side chain of the Ser
residue that hydrogen
bonds to the 3`-OH terminus of the primer shown here are adopted from (4) . The positive charged side chain of Lys
of
region III is shown here to interact with the oxygen group of the
-phosphate of the incoming dNTP. An X is shown here to
depict the unknown side chain(s) of residue(s) in the active site that
might participate in chelating the Mg
metal
activator.