DNA Polymerases Require a Template and a Primer

DNA polymerases catalyze the formation of polynucleotide chains through the addition of successive nucleotides
derived from deoxynucleoside triphosphates. The polymerase reaction takes place only in the presence of an appropriate
DNA template. Each incoming nucleoside triphosphate first forms an appropriate base pair with a base in this template.
Only then does the DNA polymerase link the incoming base with the predecessor in the chain. Thus, DNA polymerases
are template-directed enzymes.
DNA polymerases add nucleotides to the 3 end of a polynucleotide chain. The polymerase catalyzes the nucleophilic
attack of the 3 -hydroxyl group terminus of the polynucleotide chain on the -phosphate group of the nucleoside
triphosphate to be added .To initiate this reaction, DNA polymerases require a primer with a free 3 -
hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free singlestranded
DNA template. RNA polymerase, in contrast, can initiate RNA synthesis without a primer .
All DNA Polymerases Have Structural Features in Common
The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be
determined was that of the so-called Klenow fragment of DNA polymerase I from E. coli. This fragment
comprises two main parts of the full enzyme, including the polymerase unit. This unit approximates the shape of a right
hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow
fragment includes a domain with 3 5 exonuclease activity that participates in proofreading and correcting the
polynucleotide product .
DNA polymerases are remarkably similar in overall shape, although they differ substantially in detail. At least five
structural classes have been identified; some of them are clearly homologous, whereas others are probably the
products of convergent evolution. In all cases, the finger and thumb domains wrap around DNA and hold it across the
enzyme's active site, which comprises residues primarily from the palm domain. Furthermore, all the polymerases
catalyze the same polymerase reaction, which is dependent on two metal ions.
Two Bound Metal Ions Participate in the Polymerase Reaction
Like all enzymes with nucleoside triphosphate substrates, DNA polymerases require metal ions for activity. Examination
of the structures of DNA polymerases with bound substrates and substrate analogs reveals the presence of two metal ions
in the active site. One metal ion binds both the deoxynucleoside triphosphate (dNTP) and the 3 -hydroxyl group of the
primer, whereas the other interacts only with the 3 -hydroxyl group .The two metal ions are bridged by
the carboxylate groups of two aspartate residues in the palm domain of the polymerase. These side chains hold the metal
ions in the proper position and orientation. The metal ion bound to the primer activates the 3 -hydroxyl group of the
primer, facilitating its attack on the -phosphate group of the dNTP substrate in the active site. The two metal ions
together help stabilize the negative charge that accumulates on the pentacoordinate transition state. The metal ion
initially bound to dNTP stabilizes the negative charge on the pyrophosphate product.
The Specificity of Replication Is Dictated by Hydrogen Bonding and the
Complementarity of Shape Between Bases
DNA must be replicated with high fidelity. Each base added to the growing chain should with high probability be the
Watson-Crick complement of the base in the corresponding position in the template strand. The binding of the NTP
containing the proper base is favored by the formation of a base pair, which is stabilized by specific hydrogen bonds.
The binding of a noncomplementary base is unlikely, because the interactions are unfavorable. The hydrogen bonds
linking two complementary bases make a significant contribution to the fidelity of DNA replication. However, DNA
polymerases replicate DNA more faithfully than these interactions alone can account for.
The examination of the crystal structures of various DNA polymerases indicated several additional mechanisms by
which replication fidelity is improved. First, residues of the enzyme form hydrogen bonds with the minor-groove side of
the base pair in the active site .In the minor groove, hydrogen-bond acceptors are present in the same
positions for all Watson-Crick base pairs. These interactions act as a "ruler" that measures whether a properly spaced
base pair has formed in the active site. Second, DNA polymerases close down around the incoming NTP .The binding of a nucleoside triphosphate into the active site of a DNA polymerase triggers a conformational change: the
finger domain rotates to form a tight pocket into which only a properly shaped base pair will readily fit. The mutation of
a conserved tyrosine residue at the top of the pocket results in a polymerase that is approximately 40 times as error prone
as the parent polymerase.

Many Polymerases Proofread the Newly Added Bases and Excise Errors
Many polymerases further enhance the fidelity of replication by the use of proofreading mechanisms. As already noted,
the Klenow fragment of E. coli DNA polymerase I includes an exonuclease domain that does not participate in the
polymerization reaction itself. Instead, this domain removes mismatched nucleotides from the 3 end of DNA by
hydrolysis. The exonuclease active site is 35 Å from the polymerase active site, yet it can be reached by the newly
synthesized polynucleotide chain under appropriate conditions. The proofreading mechanism relies on the increased
probability that the end of a growing strand with an incorrectly incorporated nucleotide will leave the polymerase site
and transiently move to the exonuclease site .
How does the enzyme sense whether a newly added base is correct? First, an incorrect base will not pair correctly with
the template strand. Its greater structural fluctuation, permitted by the weaker hydrogen bonding, will frequently bring
the newly synthesized strand to the exonuclease site. Second, after the addition of a new nucleotide, the DNA
translocates by one base pair into the enzyme. The newly formed base pair must be of the proper dimensions to fit into a
tight binding site and participate in hydrogen-bonding interactions in the minor groove similar to those in the
polymerization site itself .Indeed, the duplex DNA within the enzyme adopts an A-form structure,
allowing clear access to the minor groove. If an incorrect base is incorporated, the enzyme stalls, and the pause provides
additional time for the strand to migrate to the exonuclease site. There is a cost to this editing function, however: DNA
polymerase I removes approximately 1 correct nucleotide in 20 by hydrolysis. Although the removal of correct
nucleotides is slightly wasteful energetically, proofreading increases the accuracy of replication by a factor of
approximately 1000.
The Separation of DNA Strands Requires Specific Helicases and ATP
Hydrolysis
For a double-stranded DNA molecule to replicate, the two strands of the double helix must be separated from each other,
at least locally. This separation allows each strand to act as a template on which a new polynucleotide chain can be
assembled. For long double-stranded DNA molecules, the rate of spontaneous strand separation is negligibly low under
physiological conditions. Specific enzymes, termed helicases, utilize the energy of ATP hydrolysis to power strand
separation.
The detailed mechanisms of helicases are still under active investigation. However, the determination of the threedimensional
structures of several helicases has been a source of insight. For example, a bacterial helicase called PcrA
comprises four domains, hereafter referred to as domains A1, A2, B1, and B2 .Domain A1 contains a Ploop
NTPase fold, as was expected from amino acid sequence analysis. This domain participates in ATP binding and
hydrolysis. Domain B1 is homologous to domain A1 but lacks a P-loop. Domains A2 and B2 have unique structures.
From an analysis of a set of helicase crystal structures bound to nucleotide analogs and appropriate double- and singlestranded
DNA molecules, a mechanism for the action of these enzymes was proposed. Domains A1 and
B1 are capable of binding single-stranded DNA. In the absence of bound ATP, both domains are bound to DNA. The
binding of ATP triggers conformational changes in the P-loop and adjacent regions that lead to the closure of the cleft
between these two domains. To achieve this movement, domain A1 releases the DNA and slides along the DNA strand,
moving closer to domain B1. The enzyme then catalyzes the hydrolysis of ATP to form ADP and orthophosphate. On
product release, the cleft between domains A and B springs open. In this state, however, domain A1 has a tighter grip on
the DNA than does domain B1, so the DNA is pulled across domain B1 toward domain A1. The result is the
translocation of the enzyme along the DNA strand in a manner similar to the way in which an inchworm moves. In
regard to PcrA, the enzyme translocates in the 3 5 direction. When the helicase encounters a region of doublestranded
DNA, it continues to move along one strand and displaces the opposite DNA strand as it progresses.
Interactions with specific pockets on the helicase help destabilize the DNA duplex, aided by ATP-induced
conformational changes.
Helicases constitute a large and diverse class of enzymes. Some of these enzymes move in a 5 3 direction,
whereas others unwind RNA rather than DNA and participate in processes such as RNA splicing and the initiation
of mRNA translation. A comparison of the amino acid sequences of hundreds of these enzymes reveals seven regions of
striking conservation .Mapping these regions onto the PcrA structure shows that they line the ATPbinding
site and the cleft between the two domains, consistent with the notion that other helicases undergo
conformational changes analogous to those found in PcrA. However, whereas PcrA appears to function as a monomer,
other members of the helicase class function as oligomers. The hexameric structures of one important group are similar
to that of the F1 component of ATP synthase suggesting potential mechanistic similarities.