Mechanisms of DNA Synthesis

Biochemistry 201
Advanced Molecular Biology
January 19, 2000
Doug Brutlag

Introduction

Today's topic concerns the fundamental mechanism of DNA replication as

exemplified by enzymes from E. coli. Nearly identical mechanisms are found in all
other polymerases, both prokaryotic and eukaryotic, so only the differences will be
mentioned when discussing these other enzymes.

DNA synthetic mechanisms are fundamentally important with respect to the

transfer of genetic information from one generation to the next. They are
particularly important in maintaining this information. They are also involved in
mechanisms for repairing damage to the DNA from external mutagenic agents
between rounds of DNA replication.

Discovery of DNA Polymerase

Dr. Arthur Kornberg and his collaborators discovered the first DNA synthetic
enzyme in the late 50's. They initially found that cell-free extracts from bacteria were
able to incorporate 3H-thymidine into DNA much as could the intact cells, but at a
very reduced rate.

The first event in these extracts was the conversion of the thymidine into
dTTP, and if they used 3H-dTTP the incorporation was much more efficient. Indeed
all four nucleotides were being incorporated into DNA very efficiently.

Using this as an assay for in vitro DNA synthesis they purified the major
DNA synthetic enzyme from the bacterial cells. The reaction that this enzyme, now
known as DNA polymerase I, mediates is:

dATP Mg++
dTTP + DNA → more DNA + PPi → 2Pi
dGTP DNA
dCTP polymerase

The requirement for Mg++ became apparent during the purification of the
enzyme as well as the requirement for DNA in the reaction.

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 DNA Replication
Two Roles for DNA in the DNA Polymerase Reaction

Role of Template

The DNA was found to have

two roles in the synthetic reaction.
The first was that of a template to
direct the order of incorporation of
new nucleotides. This was
demonstrated several ways.

1. First the base composition of

the product was the same as
the template DNA.

2. Second, the frequency of

dinucleotides was the same.

3. But the most convincing

evidence was a series of
synthetic steps that
replicates the complete
genome of ØX174 utilizing
the synthetic capacity of
DNA polymerase I. This
means that all of the genes
present within the 5000
base pair genome of ØX174
were replicated accurately.

The Role of DNA as Primer

The second role that DNA serves in the DNA polymerase reaction is that of a
primer. The newly synthesized DNA is covalently attached to preexisting DNA
chains.

1. Proper deoxynucleoside triphosphate is chosen according to the Watson-

Crick base pairing rules.

2. The 3' hydroxyl group of the primer strand, known as the primer terminus,
attacks the nucleotide splitting off the terminal pyrophosphate group and the
enzyme advances one base pair.

3. DNA chains only grow at the 3' end and the direction of synthesis is
referred to as 5' to 3' growth. We always signify the 3' end of a DNA chain
with an arrowhead.

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 DNA Replication

4. The primer terminus to which the deoxynucleoside triphosphates were

added must be base paired with a single-stranded segment of DNA that was to
serve as template.

5. This requirement for a free 3' hydroxyl primer terminus meant that DNA
polymerase could not utilize many types of DNA normally found in nature.

RNA Polymerase Can Start Chains

One reason that the requirement for a primer terminus seemed so unusual
was that all of these DNA molecules were excellent templates for the enzyme RNA
polymerase. RNA polymerase is responsible for transcribing the genetic information
from DNA into RNA and this enzyme can initiate RNA chains. All DNA
polymerases require a primer terminus.

Another major difficulty with this DNA synthetic mechanism is that the
chains only grow in the 5' to 3' direction. No DNA polymerase has been found that
can extend the 5' ends of DNA chains. This causes a problem with respect to the
replication of the two daughter strands at a replication fork, which of course, have
opposite polarities.

Nearest Neighbor Dinucleotide Analysis

An understanding of this chemical mechanism and its unidirectional mode

of DNA synthesis allowed a chemical demonstration that the two chains of DNA
were antiparallel.

If we assume that the chains were parallel, then we would expect the
frequency of the dinucleotide ApG to be identical to the frequency of the
dinucleotide TpC.

If, on the other hand, the chains were antiparallel, then we would expect the
frequency of ApG to be equal to the frequency of CpT and not necessarily related to
the frequency of TpC:

5'→3' 5'→3'
ApG ApG
TpC TpC
5'→3' 3'←5'

PARALLEL ⇒ AG = TC ANTIPARALLEL ⇒ AG = CT

The frequencies of dinucleotides in nearest neighbor experiments are always

equal to the frequency of the complementary dinucleotide read in the opposite
direction. This is not true for the complementary sequence read in the same
direction, hence the chains of duplex DNA must be anti-parallel.

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 DNA Replication

Exonucleases Associated with DNA Polymerase

Proofreading 3' Exonuclease

The purified DNA polymerase contains two exonuclease activities. The

enzyme contained both a 3' exonuclease activity, which hydrolyzes DNA chains
beginning at the 3' termini and degraded the DNA hydrolytically to
mononucleotides.

The enzyme also possesses a 5'-exonuclease that was capable of hydrolytically

degrading DNA near the 5' end of DNA. This activity released short
oligonucleotides 2 to 10 bases in length as well as mononucleotides from the 5' end
of DNA chains.

The presence of the 3'-exonuclease was curious since the synthetic activity of
DNA polymerase used the 3' terminus as a primer.

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 DNA Replication
It was found that this nuclease would degrade a primer terminus only if the
3'-terminal nucleotide was not base paired with the template. Under conditions of
DNA synthesis the enzyme extended a base paired 3' terminus, but could not extend
a mismatched primer terminus, instead it would degrade it and only begin
extending the 3' end when it was base paired with a template.

These data suggest that the function of the 3' exonuclease is to proofread the
DNA synthetic process and if an incorrect nucleotide has been incorporated, those
mismatches will be detected and removed before further chain elongation occurs.

DNA synthesis is very faithful because every nucleotide is checked twice. The
Watson-Crick base pairing rules select it prior to incorporation into the DNA chain
and the 3' exonuclease check it again prior to being extended.

There is genetic evidence that such an error-correcting exonuclease does

indeed fill this role in vivo.

Strains of T4 with higher than the spontaneous frequencies of mutation are

referred to as mutator strains. In addition, mutants were found which had a lower
than normal level of spontaneous mutation and were referred to as anti-mutators.

DNA polymerases induced by both mutator and anti-mutator strains were

isolated and the relative rate of exonuclease and polymerase activities in the isolated
enzymes was compared. The mutator strains had a markedly reduced level of 3'
exonuclease relative to DNA polymerase activity. On the other hand, the anti-
mutator strains had a much more active 3' exonuclease relative to polymerase.

These data show a direct correlation of the amount of 3'-exonuclease activity

with increased fidelity of replication.

It also shows that the rate of spontaneous mutation is not random. Therefore
the spontaneous mutation frequency is a genetically variable and selectable
characteristic.

The presence of the 3' exonuclease appears to be a common property of all

known bacterial DNA polymerases. Some eukaryotic enzymes have been reported
to be missing such a proofreading function and these same enzymes have been
demonstrated to have a very high frequency of misincorporation in vitro.

Work from Bob Lehman's lab has shown that the major DNA polymerase
isolated from Drosophila has an intrinsic 3' exonuclease but it is masked by the
presence of a 74-kilodalton subunit. Removal of that subunit generates a functional
DNA polymerase that displays an active 3' exonuclease.

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 DNA Replication
Role of the 5' Exonuclease

The role of the 5' exonuclease associated with DNA polymerase I also became
apparent once its mechanism and specificity were determined. The 5' exonuclease
attacks 5' ends of DNA and allows the enzyme to work at nicks in a DNA chain:

3' 5'
→ →
←
This molecule has several primer termini but it has no single-stranded
template.

The 5' exonuclease allows simultaneous degradation of the 5' end at a nick
and extension of the 3' end by the synthetic activity.

This seemingly futile reaction is referred to as nick translation since the nick
is effectively moved along the DNA molecule no net DNA is made.

However, this reaction is good for catalyzing repair of damage to DNA.

Clearly if there were damaged bases in the DNA annealed to the template, this
damage would be degraded and replaced with new nucleotides.

Role of Nick Translation in Repair of DNA

One major form of damage to DNA, caused by UV light, is the dimerization

of adjacent pyrimidines, most frequently, thymine dimers. UV cross-links two
adjacent double bonds to form a cyclobutane ring. If this DNA is irradiated prior to
nick-translation, then it was shown that the DNA polymerase could excise the
thymine dimers and replace them with normal thymines.

The observation that DNA polymerase I can hydrolyze and remove damaged
nucleotides from DNA gave a molecular basis for the pathway of repair known as
excision repair.

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 DNA Replication
In order for damaged bases to removed from DNA by the nick translation
mechanism their clearly has to be a way to specifically introduce nicks near the site
of damage.

In the excision repair pathway, recognition of damaged bases results directly

in the incision of the phosphodiester backbone.

In the pathways to be discussed tomorrow, the recognition of the damaged

base leads first to excision of the base, followed by recognition of the absence of a base
by an enzyme called the apurinic endonuclease.

DNA Ligase Seals Nicks

Subsequent to the nick translation reaction, regardless of the mechanism of

incision we are now left with a nick in the DNA. The enzyme DNA ligase can repair
nicks in DNA.

DNA ligase from E. coli can repair the phosphodiester bond at a nick by using
the energy in the pyrophosphate bond in NAD.

DNA ligase requires the presence of a 3'-hydroxyl group and a 5' phosphoryl
group on the nick in order to seal the nick. DNA ligase catalyzes three reactions in
order to seal a break as shown below.

The intracellular concentration of NAD and ligase are such that most of the
free ligase will be in the charged form.

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 DNA Replication
The ligase then transfers the adenyl group to the 5' phosphoryl group at the
nick. This regenerates a pyrophosphate bond similar to that present in the NAD
itself.

Then the ligase mediates the attack of the free 3' hydroxyl on this
pyrophosphate bond that it just made, causing the release of AMP and leaving the
phosphodiester bond closed.

Many other DNA ligases use a similar mechanism however; not all of them
use NAD for the cofactor. Most eukaryotic ligases utilize ATP directly as the source
of the adenyl group and they release pyrophosphate upon charging rather than
NMN.

The DNA Ligase Reaction is Reversible.

If a supercoiled DNA is incubated in the presence of uncharged DNA ligase

and AMP, the DNA then becomes nicked, at a random site. The AMP is attached
covalently to the 5' phosphoryl at the nick.

At this point the enzyme can continue the backward reaction and transfer the
adenyl group to itself to become a charged AMP-ligase and leave the DNA in a
nicked form, or it can now continue in the forward direction, resealing the DNA
back to a covalently closed circular form.

The presence of the transient nick results in the relaxation of the supercoiled
DNA. Therefore DNA ligase can act like an AMP dependent nicking-closing
enzyme!

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 DNA Replication
Summary of DNA Polymerase Action

Neither intact duplex

circles nor intact duplex linear
DNAs are template primers
for DNA polymerase.

On the other hand a

nicked circle or a nicked linear
are good templates by virtue of
the ability of DNA polymerase
to mediate nick translation.

DNA polymerase can

also extend a 3' primer
terminus will displacing the 5'
end ahead of the growing
chain. This reaction is known
as strand displacement.

In vitro nick translation

is favored at low
temperatures, temperatures
that tend to stabilize the DNA
helix from denaturation,
while strand displacement is
favored at 37°C.

Later, during a synthesis of this type, for reasons that are not completely
understood, the enzyme can switch from copying the template strand to copying the
DNA strand that it is displacing. This reaction is known as template switching.

DNA containing gaps is a good substrate with DNA polymerase first filling in
the gaps, and then catalyzing nick-translation as above.

Even single-stranded DNA can serve as a substrate by virtue of very short

transient base pairing within the DNA. As soon as a limited amount of pairing
occurs and synthesis begins, the newly replicated DNA stabilizes the hairpin
structure and the reaction can continue to the end of the template.

Discovery of Other DNA Polymerases

Because of the specificity of DNA polymerase (its inability to start DNA chains
and its inability to extend 5' termini) several laboratories set out to look for other
DNA synthetic enzymes. Between 1970 and 1972, two other enzymes were found in
E. coli but they all catalyzed the same chemical reaction.

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 DNA Replication

DNA polymerases II and III both utilize 5' deoxynucleoside triphosphates and
both of them extend only 3' primer termini.

DNA
polymerase II is very similar to DNA polymerase I except that it lacks a 5'
exonuclease. It also appears to be unable to displace the 5' strand at a nick and
therefore is completely inert on a nicked DNA template.

This enzyme works best on DNA that contains short single-stranded gaps.

DNA polymerase II does contain a 3' exonuclease which has exactly the same
specificity for mismatched nucleotides as the 3' exonuclease of polymerase I.

DNA polymerase III also has a 3' exonuclease for proof reading and lacks a 5'
exonuclease. It can synthesize DNA at a much greater rate than can either DNA
polymerase I or II, rates approaching those of the replication fork (1-2000 nucleotides
per second).

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 DNA Replication
Mutants that are missing any one of these DNA polymerases have suggested
that both DNA polymerase I and DNA polymerase III are essential for DNA
replication. There are temperature sensitive lethal mutations in both enzymes.

There are other mutations in either DNA polymerase I or DNA polymerase II

which are not lethal and which show an increased sensitivity to mutagenic agents
and an inability to repair damage to DNA. The level of DNA polymerase II in the
cell is also induced over 10 fold in response to damage in the DNA (part of the SOS
response). Thus these enzymes may also play important and redundant roles in
repair of damage to DNA.

One of the main problems with dissecting the functions of the enzymes of
DNA metabolism by genetic studies has long been the redundant nature of all of the
pathways for repair, replication, and recombination. When a cell has a multiplicity
of choices open to it, eliminating one enzyme genetically often has little phenotypic
effect.

REFERENCES

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Beckman, R. A. and Loeb, L. A. (1993). Multi-stage proofreading in DNA replication.

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basis of DNA replication fidelity. Crit Rev Biochem Mol Biol 28(2), 83-126.

Hurwitz, J., Dean, F. B., Kwong, A. D. and Lee, S.-H. (1990). The in vitro Replication
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Kornberg, A. (1988). DNA replication. J. Biol. Chem. 263, 1-4.

Kunkel, T. A. (1992). DNA Replication Fidelity. J. Biol. Chem. 267, 18251-18254.

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 DNA Replication
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