DNA Replication

Introduction
When a cell divides, it is important that each daughter cell receives an
identical copy of the DNA. This is accomplished by the process of DNA
replication. The replication of DNA occurs during the synthesis phase, or
S phase, of the cell cycle, before the cell enters mitosis or meiosis. DNA
replication ensures that each resulting cell will have a complete set of
DNA molecules. During DNA replication, the DNA molecule separates
into two strands, and then produces two complementary strands
following the rules of base pairing. Each strand of the double helix serves
as a template for the new strand.

Replication is Semi-Conservative
Recall that Adenine (A) nucleotides pair with Thymine (T) nucleotides,
and Cytosine (C) nucleotides pair with Guanine (G) nucleotides. This
means that the two strands are complementary to each other.
For example:

AGTCATGA

TCAGTACT

Replication is semiconservative because after one round of replication, the original helix is not
conserved; instead, each strand of the helix becomes part of another helix. Each side of the
original “parental” DNA strand becomes a template strand upon which the new complimentary
strands form (Figure 2.0). When two DNA copies are formed, they have an identical sequence of
nucleotide bases and are divided equally into two daughter cells.

Figure 2.0

The semi-conservative model of DNA replication is shown. Gray indicates the original DNA strands, and blue
indicates newly synthesized DNA.
Because eukaryotic genomes are quite complex, DNA replication is a very complicated process
that involves several enzymes and other proteins. DNA replication occurs in three main stages:
initiation, elongation, and termination.

Initiation
During initiation, DNA unwinds and the hydrogen bonds between the two strands are broken.
This process is aided by the enzyme DNA helicase. This enzyme binds to a replication origin on
the DNA and begins unwinding the DNA and breaking the hydrogen bonds between the
complementary base pairs. Replication origins are adenine and thymine rich because A-T base
pairs only contain two hydrogen bonds instead of three hydrogen bonds found in G-C base pairs.
As the two strands of DNA separate, they form a “Y” shaped structure called a replication fork.
Two opposite forks extend away from a replication origin to create a replication bubble.
Replication bubbles form at multiple sites along the DNA molecule, greatly speeding up
replication.

As helicase unwinds the DNA, a twisting tension is created in the double stranded portion of the
DNA (ahead of the helicase). Topoisomerase relieves this tension by working ahead of the
helicase and cutting the sugar phosphate backbones of one or both DNA strands so the double
stranded DNA can untwist and relieve the tension. Once the tension is relieved, the
topoisomerase reforms the phosphodiester bonds to rejoin the sugar phosphate backbones.

At the fork, the newly formed single strands of DNA tend to reanneal (rejoin one another) due to
the hydrogen bonding attraction of their complementary bases. Reannealing is prevented by
single stranded bonding proteins (SSBPs); they bind to the single strands near the fork to prevent
them from rejoining.

Elongation
Elongation can occur continuously toward the replication fork, or discontinuously away from the
replication fork. The strand that grows continuously toward the replication fork is known as the
leading strand and the strand that grows discontinuously away from the replication fork is known
as the lagging strand.

Once the strands have been unwound and separated, DNA polymerase III can begin to build a
new strand. Each new nucleotide that is added to a growing DNA strand is a nucleoside
triphosphate (dATP). Figure 3.0 shows the relationship between a nucleoside triphosphate and
a nucleotide.
Figure 3.0

The Leading Strand

The leading strand is the new strand that grows continuously towards the replication fork. DNA
polymerase reads the template strand in the 3’ to 5’ direction and builds the new strand in the 5’
to 3’ direction (opposite to which it is read) (Figure 4.0). However, DNA polymerase cannot build
a new strand from scratch; it can only build onto a pre-existing strand. Therefore, RNA primase
synthesizes the first nucleotides of the new strand; this is known as a primer sequence (10 to 60
nucleotides long). The resulting segment of RNA primer provides a free 3’ end for DNA
polymerase III to bind to. DNA polymerase III can now assemble the complimentary nucleotides
as it moves along the parental strand in the 5’ to 3’ direction. The helix continues to unwind and
open, allowing the leading strand to grow continuously towards the replication fork. Later, a
different kind of DNA polymerase, DNA polymerase I, will replace the RNA primer sequence with
DNA nucleotides.

Figure 4.0

The leading strand grows continuously towards the replication fork (in the 5' to 3' direction while the lagging
strand grows discontinuously away from the replication fork (in the 3' to 5' direction).
The Lagging Strand
The lagging strand is built in the opposite direction that the helix unwinds. The lagging strand is
the new strand that grows discontinuously away from the replication fork (Figure 4.0). First, RNA
primase adds a section of RNA primer. Then DNA polymerase III begins to synthesize the new
strand of DNA. Before more lagging strand can be built, the helix must continue to unwind. Thus,
the lagging strand is built discontinuously. Once again, RNA primase begins the new strand. The
discontinuous sections are called Okazaki fragments. As in the leading strand, DNA polymerase
I replaces the RNA primer sequence with DNA. Nucleotides. Then DNA ligase joins the sections
of DNA together. Replication continues in this manner along the lagging strand, building in
sections as the helix unwinds. The overall process is depicted in Figure 5.0.

Figure 5.0

Termination
Before DNA replication is terminated, both the leading and lagging strands require “proof
reading”. DNA polymerase III is not perfect and can occasionally insert an incorrect nucleotide.
Though the average rate of error is only one in every million base pairs, there can still be very
harmful consequences if these errors are nor repaired. Exonucleases remove mismatched DNA
nucleotides and replace them with the correct ones.

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in
eukaryotic chromosomes. As you have learned, the DNA polymerase can only add nucleotides in
one direction. In the leading strand, synthesis continues until the end of the chromosome is
reached; however, on the lagging strand there is no place for a primer to be made for the DNA
fragment to be copied at the end of the chromosome. This presents a problem for the cell
because the ends remain unpaired, and over time these ends get progressively shorter as cells
continue to divide. The ends of the linear chromosomes are known as telomeres, which have
repetitive sequences that do not code for a particular gene. Therefore, it is telomeres that are
shortened with each round of DNA replication instead of genes. This essentially means that
telomere shortening is associated with aging.