From the Howard Hughes Medical Institute and Departments of Molecular Biophysics & Biochemistry and Chemistry, Yale University, New Haven, Connecticut 06520-8114
Possibly the earliest enzymatic activity to
appear in evolution was that of the polynucleotide polymerases, the
ability to replicate the genome accurately being a prerequisite for
evolution itself. Thus, one might anticipate that the mechanism by
which all polymerases work would be both simple and universal. Further, these enzymatic scribes must faithfully copy the sequences of the
genome into daughter nucleic acid or the information contained within
would be lost; thus some mechanism of assuring fidelity is required.
Finally, all classes of polynucleotide polymerases must be able to
translocate along the template being copied as synthesis proceeds. The
crystal structures of numerous DNA polymerases from different families
suggest that they all utilize an identical two-metalioncatalyzed
polymerase mechanism but differ extensively in many of their structural features.
From amino acid sequence comparisons (1) as well as crystal structure
analyses (2), the DNA polymerases can be divided into at least five
different families, and representative crystal structures are known for
enzymes in four of these families. Perhaps the best studied of these
families is the DNA polymerase I (pol I)1 or A polymerase family,
which includes the Klenow fragments of Escherichia coli and
a Bacillus DNA polymerase I, Thermus aquaticus DNA polymerase, and the T7 RNA and DNA polymerases, all of whose crystal structures are known (3-11). The second family of
DNA-dependent DNA polymerases is DNA polymerase Independent of their detailed domain structures, all polymerases
whose structures are known presently appear to share a common overall
architectural feature. They have a shape that can be compared with that
of a right hand and have been described as consisting of "thumb,"
"palm," and "fingers" domains (15). The function of the palm
domain appears to be catalysis of the phosphoryl transfer reaction
whereas that of the fingers domain includes important interactions with
the incoming nucleoside triphosphate as well as the template base to
which it is paired. The thumb on the other hand may play a role in
positioning the duplex DNA and in processivity and translocation.
Although the palm domain appears to be homologous among the pol I, pol
Here the functional and structural similarities and differences among
the polymerases of known structure are explored. Of particular interest
are the role of editing in the fidelity of copying, the common
enzymatic mechanism of polymerases, and the manners in which different
domain structures function in the polymerase reaction in analogous ways.
Although the palm domains of the pol I, pol A detailed comparison of the structures from four polymerase families
(Fig. 1) shows that the fingers and
thumbs are different in all four families for which structures are
known (12, 16). (To make a suitable comparison between structural
elements having similar functions, the names of the pol
INTRODUCTION
TOP
INTRODUCTION
Structural Differences among...
Fidelity of Genome Copying
Polymerase Mechanism
Conclusions
REFERENCES
(pol
) or B family DNA polymerase. All eukaryotic replicating DNA
polymerases and the polymerases from phages T4 and RB69 belong to this
family, and a crystal structure of the RB69 polymerase shows some
similarities to the pol I family enzymes and numerous differences (12).
Reverse transcriptases (RT), RNA-dependent RNA polymerases,
and telomerase appear to show some common structural similarities,
whereas the structure of DNA polymerase
shows no structural
relatedness to any of these previous families (13, 14). On the basis of
amino acid sequence comparisons but no crystal structures, it appears
that the bacterial DNA polymerase III enzymes also form a family that is unrelated to the polymerases of known structure (1).
, and RT families, the fingers and thumb domains are different in
all four of these families for which structures are known to date
(16).
Structural Differences among Polymerases
TOP
INTRODUCTION
Structural Differences among...
Fidelity of Genome Copying
Polymerase Mechanism
Conclusions
REFERENCES
, and RT
families are homologous, the fingers and thumb domains are completely different in the structures from all families (16). In the structure of
the DNA polymerase from RB69 five domains are arranged around a central
hole (12). In this enzyme the fingers domain consists largely of two
very long anti-parallel coiled-coil
-helices that extend more than
20 Å out the "back." The thumb domain is seen to be interacting
directly with the exonuclease domain and providing some of the binding
site for the single-stranded exonuclease substrate. After orienting the
palm domains of the RB69 and Klenow fragment enzymes identically, it
becomes clear that their exonuclease domains are located in completely
different places relative to the polymerase active site. Although in
the "standard" orientation the exonuclease domain can be described
as being southeast of the polymerase active site in Klenow fragment,
the corresponding domain of the RB69 polymerase is located northwest of
the polymerase active site. This difference in location of the editing
domain may be in part related to the fact that the RB69 enzyme, like
that from T4 phage, has an exonuclease activity that is 103
times larger than that of the Klenow fragment (17, 18).
thumb and
fingers domains have been switched from the Pelletier et al.
image (19).) Although the structures of the thumb domains are not
homologous, they do exhibit analogous features that consist of largely
parallel or anti-parallel
-helices and in each case at least one
-helix seems to be making important interactions across the minor
groove of the primer-template product. In the case of the pol I family, loops at the top of the thumb also make important and conserved interactions with the DNA backbone (6, 8).
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Fig. 1.
A comparison of primer-template DNA bound to
four DNA polymerases. The complexes shown in A,
B, and D are co-crystal structures, whereas the
complex in C is a homology model (12). These four structures
have been similarly oriented with respect to each other by
superposition of the first two base pairs at the primer terminus. The
primer strand and template strand are labeled. A,
Taq DNA polymerase bound to DNA (6). As with the other three
structures shown in this figure, the DNA stacks against the fingers and
is contacted across the minor groove by the thumb domain. B,
the binary complex of HIV-1 RT and DNA (29). This structure does not
have a nucleotide-binding -helix in the fingers domains. Instead, a
-hairpin probably performs this function. C, the model of
DNA bound to RB69 gp43 (12). A likely DNA-binding
-helix has been
highlighted. It appears that the thumb domain would have to move toward
the primer terminus to bind DNA analogously to the other polymerases.
D, the ternary complex of rat pol
with DNA and
dideoxy-NTP (19). Domain D plays the role of the fingers and presents
an
-helix at the primer terminus. Domain B is analogous to other
polymerase thumb domains and binds the minor groove of the duplex
substrate. This figure is reprinted with permission from Brautigam and
Steitz (16).
Although the fingers domains of all four families are also not
homologous, there are some striking structural analogies among the
families as with the thumbs. In three of the four DNA polymerase families with known structures, the pol I, pol , and pol
families, an
-helix in the fingers domain is positioned at the blunt
end of the primer-template; it contains side chains that are conserved within the families (the B motif) and provides important orienting interactions with the incoming deoxynucleoside triphosphate. In the
case of the reverse transcriptase family, however, it appears that some
of these functions have been taken over by an anti-parallel
-ribbon,
which lies in a similar position, as seen in a recent ternary complex
with the primer-template and deoxynucleoside triphosphate (27).
The perhaps surprising diversity of polymerase structures found in
these families leads one to wonder why the structures of DNA
polymerases turn out to be so diverse when the structures of most
metabolic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase,
are almost identical from microbe to man. One possibility is that the
RT, pol I, and pol families are later evolutionary additions to all
cellular replicating polymerases, which could turn out to be
divergently related to the pol
family polymerases; however,
sequence comparisons do not presently show such a relationship between
the eukaryotic pol
and the prokaryotic pol III DNA polymerases. An
alternative speculation might imagine that a ribozyme DNA polymerase originating in the "RNA world" may have persisted beyond the
divergence of eukaryotes and prokaryotes and was replaced domain by
domain differently.
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Fidelity of Genome Copying |
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The greatest insights into the mechanisms by which polymerases achieve faithful copying of the template come from structural and biochemical studies of the pol I family of polymerases (2, 20). Fidelity arises both from constraints imposed on base pairing at the polymerase active site as well as the editing of mismatched base pairs at a 3'-exonuclease active site. The crystal structure of the Klenow fragment of DNA polymerase I first showed that this enzyme is divided into two domains, one of which catalyzes the polymerase reaction and the second of which has an active site more than 30 Å away from the polymerase active site and catalyzes the 3'-5'-exonuclease reaction (3). The co-crystal structure of Klenow fragment with duplex DNA containing a 3' overhanging tetranucleotide shows that the single-stranded 3' end binds into the exonuclease active site (4). Extensive structural, mutagenic, and biochemical studies of single-stranded substrates bound to the exonuclease active site gave rise to the proposal of a two-metal ion mechanism of phosphoryl transfer (20, 21, 23). When duplex DNA is bound to the homologous T. aquaticus DNA polymerase, which does not contain a functioning exonuclease active site, the 3' end of the primer strand is found to lie in the polymerase active site adjacent to highly conserved carboxylate residues known to be important for the polymerase reaction (6). Likewise in the ternary complex between T7 DNA polymerase, primer-template DNA, and dNTP, the 3' end is in the polymerase active site. Although the duplex portion of substrates lies in the same approximate position adjacent to the thumb whether the primer-template is bound to the enzyme in polymerase mode or in exonuclease mode, the 3' ends are located in active sites that are separated by more than 30 Å.
The mechanism whereby the exonuclease domain exerts its editing
function is proposed (Fig. 2) to involve
a competition between these two active sites for the 3' end of the
primer strand and a rapid shuttling of the primer terminus between them
(2, 4, 21). The 3'-exonuclease active site binds single-stranded DNA whereas the polymerase active site binds duplex DNA with the ratio of
about 1 to 10 for correctly Watson-Crick base-paired duplex DNA (18).
Mismatched base pairs destabilize the duplex DNA and thereby enhance
the binding of the 3' single-stranded DNA to the exonuclease active
site. Furthermore, polymerization is stalled after incorporation of
mismatched base pairs presumably because of misorientation of the
3'-hydroxyl group of the primer terminus onto which the next nucleotide
is to be added. Once again, this stalling of the polymerization
reaction serves to enhance the probability of excision by the
exonuclease activity. The additional role that the polymerase domain
plays in fidelity is considered below.
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Polymerase Mechanism |
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Structural studies as well as sequence comparisons among
polymerases strongly suggest the hypothesis that the phosphoryl
transfer reaction of all polymerases is catalyzed by a two-metal ion
mechanism (Fig. 3) originally proposed
(22, 24) by analogy to the well studied two-metal ion mechanism in the
3'-exonuclease reaction (20-23). The first observation of a polymerase
complex with both primer-template DNA and dNTP·Mg2+ bound
to the polymerase active site that directly showed the structural basis
of a two-metal ion mechanism was a complex with rat pol (19). These
two-metal ions are bound by three carboxylates contained in a domain
that is not homologous to other polymerases (14). A higher resolution
structure (2.1 Å) of human pol
complexed with a gapped DNA
substrate and dideoxy-CTP shows precise detail of the interaction of
two hexacoordinated, partially hydrated Mg2+ ions
interacting with the three phosphates (25). In the homologous "palm" domains of the pol I and RT families these two-metal ions (normally magnesium ions) are observed to bind to the enzyme through two completely conserved carboxylate residues but only in the primer-template complex with the dNTP (8). These metal ions are
separated by just under 4 Å in the ternary complex of T7 DNA polymerase complexed with primer-template DNA and dNTP (8). Metal ion A
interacts with the 3'-hydroxyl of the primer strand and is proposed
(24) to lower the pKa of the hydroxyl, facilitating
its attack on the
-phosphate of the incoming dNTP. Metal ions A and
B are also proposed to stabilize both the structure and charge of the
pentacovalent transition state that occurs during the course of this
reaction. Finally, metal ion B binds to and is proposed to facilitate
the leaving of the
- and
-phosphates. Chemically similar
mechanisms of two-metal ion-catalyzed phosphoryl transfer reactions are
used by many enzymes including ribozymes (26).
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Although amino acid sequence comparisons had suggested that there were
three highly conserved carboxylate-containing residues in the active
sites of all classes of polymerases, comparison of the crystal
structures of Klenow fragment, HIV-1 RT, RB69 pol polymerase, and
the T7 RNA polymerase (12) shows that only two aspartic acid residues
are structurally conserved among these four enzymes. Furthermore, the
crystal structure of T7 DNA polymerase complexed with a primer-template
and a deoxynucleoside triphosphate (8) shows that the two divalent
metal ions are bound by only one aspartic acid residue emanating from
conserved sequence motif A and one aspartate residue emanating from
conserved sequence motif C (residues Asp-705 and Asp-882 of Klenow
fragment). Further, comparison of the pol
and pol I structures
shows that previous sequence alignments of the motif C, which
positioned the first Asp of a DXD sequence on the first Asp
of a DE sequence (1), needs to be modified so that, rather, the second
Asp of the pol
family DXD sequence superimposes on the
first Asp of the RT family DE sequence or the pol I family DD sequence
(1).
In spite of the fingers domains of the pol , RT, pol I, and pol
DNA polymerases all having different evolutionary origins, they share
some similar functional features. The binding of dNTP to the pol
,
RT, and T7 DNA polymerases complexed with primer-template DNA results
in a significant rotation of the fingers domain when compared with the
corresponding binary polymerase complexes with either DNA or dNTP (8,
19, 27). The orientation of the dNTP in the binary complex differs
considerably from its orientation in the ternary complex. Only in the
presence of the next correct dNTP is a ternary complex formed in which
the fingers rotate and the incoming nucleotide makes a base pair with
the template. In those ternary complexes in which the fingers do not
rotate, the dNTP binds as in the dNTP binary complex. All of the
structures together (8, 19, 25, 27) are consistent with the early proposal that dNTP first binds to a primer-template complex with the
polymerase in a non-template-dependent fashion, but after a
rate-limited conformational change (finger rotation?) tight dNTP
binding is template sequence-specific (28). The proposal has been
further made that only with the correct Watson-Crick base pairing
between template and dNTP does the fingers rotation essential for
catalysis occur (25). That is, fidelity for incorporation of the
correct nucleotide at the polymerization step is enhanced by this
catalytically essential, induced fit conformational change, which
detects the presence of a correct base pair.
Furthermore, the four known non-homologous fingers domains present
similar residues to the incoming dNTP for the same functional reasons.
Arg-72 in HIV-1 RT and Lys-522 in T7 DNA pol interact with a
non-bridging oxygen of the -phosphate in a way that would help
stabilize the additional negative charge of the pentacovalent transition state. Furthermore, analogous basic residues from the fingers domains interact with the
-phosphate of dNTP.
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Conclusions |
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From the extensive crystallographic, biochemical, and genetic
studies of polynucleotide polymerases there are several general conclusions that can be drawn about this class of enzymes. First, all
polynucleotide polymerases may use the same two-metal ion mechanism to
catalyze the polymerase phosphoryl transfer reactions (24). It is
perhaps of interest to note that such a mechanism, which involves only
the properties of two correctly positioned divalent metal ions, could
easily be used by an enzyme made entirely of RNA and thus could
function in an all RNA world. Second, the fidelity of DNA synthesis
results from a combination of "enforced" Watson-Crick interactions
at the polymerase active site (8, 19, 25) and competitive editing at
the 3'-exonuclease active site (4, 6, 21). Misincorporated nucleotides
retard further synthesis, destabilize duplex DNA, and enhance binding
to the exonuclease active site. Third, although the catalytically
important palm domains are seen to be homologous in the pol I, RT, and
pol families, the pol
family palm domain is decidedly unrelated (14); likewise, the catalytic domains from the DNA polymerase III and
the multisubunit RNA polymerase families are likely to be different as
judged from amino acid sequence comparisons. Fourth, the thumb and
finger domains are structurally different in all of the polymerase
families for which representative crystal structures are now known,
although analogously positioned secondary structures function in
similar ways.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999.
To whom correspondence should be addressed. Tel.: 203-432-5617;
Fax: 203-432-3282.
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ABBREVIATIONS |
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The abbreviations used are: pol, polymerase; RT, reverse transcriptase(s); HIV, human immunodeficiency virus.
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