DNA Synthesis

The discovery of the double-helical nature of DNA by Watson & Crick explained how
genetic information could be duplicated and passed on to succeeding generations. The
strands of the double helix can separate and serve as templates for the synthesis of
daughter strands. In conservative replication the two daughter strands would go to one
daughter cell and the two parental strands would go to the other daughter cell. In
semiconservative replication one parental and one daughter strand would go to each of
the daughter cells.

Through experimentation it was determined that DNA replicates via a

semiconservative mechanism. There are three possible mechanisms that can explain
DNA's semiconservative replication.

(a) DNA synthesis starts at a specific place on a chromosome called an origin. In the
first mechanism one daughter strand is initiated at an origin on one parental strand and
the second is initiated at another origin on the opposite parental strand. Thus only one
strand grows from each origin. Some viruses use this type of mechanism.

(b) In the second mechanism replication of both strands is initiated at one origin. The
site at which the two strands are replicated is called the replication fork. Since the fork
moves in one direction from the origin this type of replication is called unidirectional.
Some types of bacteria use this type of mechanism.

(c) In the third mechanism two replication forks are initiated at the origin and as
synthesis proceeds the two forks migrate away from one another. This type of
replication is called bi-directional. Most organisms, including mammals, use bi-
directional replication.
Requirements for DNA Synthesis

There are four basic components required to initiate and propagate DNA synthesis.
They are: substrates, template, primer and enzymes.

Substrates

Four deoxyribonucleotide triphosphates (dNTP's) are required for DNA synthesis

(note the only difference between deoxyribonucleotides and ribonucleotides is the
absence of an OH group at position 2' on the ribose ring). These are dATP, dGTP,
dTTP and dCTP. The high energy phosphate bond between the  and  phosphates is
cleaved and the deoxynucleotide monophosphate is incorporated into the new DNA
strand.

Ribonucleoside triphosphates (NTP's) are also required to initiate and sustain DNA
synthesis. NTP's are used in the synthesis of RNA primers and ATP is used as an
energy source for some of the enzymes needed to initiate and sustain DNA synthesis
at the replication fork. DNA Synthesis, 5' to 3'

The major catalytic step of DNA synthesis is shown below. Notice that DNA
synthesis always occurs in a 5' to 3' direction and that the incoming nucleotide first
base pairs with the template and is then linked to the nucleotide on the primer.
DNA Synthesis is Semi discontinuous

Since all known DNA polymerases can synthesize only in a 5' to 3' direction a
problem arises in trying to replicate the two strands of DNA at the fork.
Notice that the top strand must be discontinuously replicated in short stretches thus
the replication of both parental strands is a semidiscontinuous process. The strand that
is continuously synthesized is called the leading strand while the strand that is
discontinuously synthesized is called the lagging strand.

Leading Strand Synthesis

DNA synthesis requires a primer usually made of RNA. A primase synthesizes the
ribonucleotide primer ranging from 4 to 12 nucleotides in length. DNA polymerase
then incorporates a dNMP onto the 3' end of the primer initiating leading strand
synthesis. Only one primer is required for the initiation and propagation of leading
strand synthesis.

Lagging Strand Synthesis

Lagging strand synthesis is much more complex and involves five steps.

1. As the leading strand is synthesized along the lower parental strand the top parental
strand becomes exposed. The strand is then recognized by a primase which
synthesizes a short RNA primer.
2. DNA polymerase then incorporates a dNMP onto the 3" end of the primer and
initiates lagging strand synthesis. The polymerase extends the primer for about 1,000
nucleotides until it comes in contact with the 5' end of the preceding primer. These
short segments of RNA/DNA are known as Okazaki fragments.

3. When the DNA polymerase encounters the preceding primer it dissociates. The
RNA is then removed by a specialized DNA polymerase or by an enzyme called
RNaseH. Ribonucleotides are then excised one at a time in a 5' to 3' direction. The
RNaseH leaves a phosphate group at the 5' end of the adjoining DNA segment thus
leaving a gap.

4. The gap is filled by a DNA polymerase which uses an Okazaki fragment as a

primer.

5. The 3' hydroxyl group on the 3' nucleotide terminus is then covalently joined, using
DNA ligase, to the free 5' phosphate of the previously made lagging segment.

Structure of DNA

DNA Polymerases
There are many types of DNA polymerases which can excise, fill gaps, proofread,
repair and replicate.

Other Factors Required for DNA Synthesis

Origins: Origins are unique DNA sequences that are recognized by a protein that
builds the replisome. Origins have been found in bacterial, plasmid, viral, yeast and
mitochondrial DNA and have recently been discovered in mammalian DNA. Specific
origins are used for initiating DNA replication in humans. Most origins have a site
that is recognized and bound by an origin-binding protein. When the origin-binding
protein binds to the origin the A + T rich sequence becomes partially denatured
allowing other replication factors known as cis-acting factors to bind and initiate DNA
replication.

Origin-binding Protein: binds and partially denatures the origin DNA while binding
to another enzyme called helicase.

Helicases: unwind double stranded DNA.

Single-stranded DNA Binding Protein (SSB): enhances the activity of the helicase
and prevents the unwound DNA from renaturing.

Primase: synthesize the RNA primers required for initiating leading and lagging
strand synthesis.

DNA Polymerase: recognizes the RNA primers and extends them in the 5' to 3'
direction.

Processivity Factors: help load the polymerase onto the primer-template while
anchoring the polymerase to the DNA.
Topoisomerase: removes the positive supercoils that form as the fork is unwound by
the helicase.

RNaseH: removes RNA portions from Okazaki fragments.

Ligase: seals the nicks after filling in the gaps left by DNA polymerase.

Chemical Inhibitors of DNA Replication

Some types of drugs function by inhibiting DNA replication.

Substrate Analogs: analogs of dNTP's which function as chain terminators can be

incorporated into DNA. These analogs are usually either missing the 3' hydroxyl
group or have a chemical group, other than hydroxyl, in the 3' position.

Cytosine Arabinoside: is an anticancer drug used to treat leukemia.

Azidothymidine (AZT): was used as an anti-HIV drug that, while effective in tissue
culture experiments, proved to be ineffective for treating HIV in humans.

Acyclovir: is an effective anti-herpes virus drug.

Intercalating Agents: are compounds with fused aromatic ring systems that can
wedge (intercalate) between the stacked base pairs of DNA. This disrupts the structure
of the DNA so that the replicative enzymes have difficulty in synthesizing DNA past
the "intercalated" sites. Anthracycline glycosides and Actinomycin D are intercalators
used to treat a variety of cancers.

DNA Damaging Agents: a variety of compounds such as Cisplatin, cause chemical

damage to DNA and are used in the treatment of cancers.

Topoisomerase Inhibitors: Nalidixic acid and Fluoroquinolones are antibiotics used

to inhibit bacterial topoisomerases.

Template

The nucleotide that is to be incorporated into the growing DNA chain is selected by
base pairing with the template strand of the DNA. The template is the DNA strand
that is copied into a complementary strand of DNA.

Primer

The enzyme that synthesizes DNA, DNA polymerase, can only add nucleotides to an
already existing strand or primer of DNA or RNA that is base paired with the
template.

Enzymes

An enzyme, DNA polymerase, is required for the covalent joining of the incoming
nucleotide to the primer. To actually initiate and sustain DNA replication requires
many other proteins and enzymes which assemble into a large complex called a
replisome. It is thought that the DNA is spooled through the replisome and replicated
as it passes through.