DNA REPLICATION

The accurate replication of DNA prior to cell division is a basic and crucial function.
In replication, the sequence of parental strands must be duplicated in daughter strands. That is,
one parental helix with two strands must yield two daughter helices with four strands. The two
daughter molecules are then separated during the course of cell division.
Three models of DNA replication are possible:
1. In a conservative model, both strands of the parental duplex would remain intact
(conserved), and new DNA copies would consist of all-new molecules. Both daughter
strands would contain all-new molecules.
2. In a semiconservative model, one strand of the parental duplex remains intact in
daughter strands (semiconserved); a new complementary strand is built rfor each
parental strand consisting of new molecules. Daughter strands would consist of one
parental strand and one newly synthesized strand.
3. In a dispersive model, copies of DNA would consist of mixtures of parental and newly
synthesized strands; that is, the new DNA would be dispersed throughout each strand
of both daughter molecules after replication.

The three models for DNA replication were evaluated in 1958 by Matthew Meselson and Franklin
Stahl. To distinguish between these models, they labeled DNA and then followed the labeled
DNA through two rounds of replication. He concluded that DNA replication is semi-
conservative.
The basic mechanism of DNA replication is semiconservative. At the simplest level, then, DNA
is replicated by opening up a DNA helix and making copies of both strands to produce two
daughter helices, each consisting of one old strand and one new strand.
DNA Replication in Prokaryotes
 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.
 The prokaryotic chromosome is a circular molecule with a less extensive coiling
structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and
highly coiled around proteins.
 Replication requires three things: something to copy, something to do the copying, and
the building blocks to make the copy. The parental DNA molecules serve as a template,
enzymes perform the actions of copying the template, and the building blocks are
nucleoside triphosphates.
 During DNA replication, each of the two strands that make up the double helix serves
as a template from which new strands are copied. The new strand will be complementary
to the parental strand. Each new double strand consists of one parental strand and one
new daughter strand. This is known as semiconservative replication. When two DNA
copies are formed, they have an identical sequence of nucleotide bases and are divided
equally into two daughter cells.
 How does the replication machinery know where to start? It turns out that there are
specific nucleotide sequences called origins of replication where replication begins.

Replication in E. coli initiates at a specific site, origins of replication (called oriC), and ends at
a specific site, the terminus. E. coli has a single origin of replication on its one chromosome, as
do most prokaryotes. The origin of replication is approximately 245 base pairs long and is rich
in AT sequences. This sequence of base pairs is recognized by certain proteins that bind to this
site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the
nitrogenous base pairs. ATP hydrolysis is required for this process because it requires energy.
As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication
forks are formed at the origin of replication and these get extended bi-directionally as replication
proceeds. Single-strand binding proteins coat the single strands of DNA near the replication
fork to prevent the single-stranded DNA from winding back into a double helix.
The next important enzyme is DNA polymerase III, also known as DNA pol III, which adds
nucleotides one by one to the growing DNA chain. The addition of nucleotides requires energy;
this energy is obtained from the nucleotides that have three phosphates attached to them. ATP
structurally is an adenine nucleotide which has three phosphate groups attached; breaking off
the third phosphate releases energy. In addition to ATP, there are also TTP, CTP, and GTP. Each
of these is made up of the corresponding nucleotide with three phosphates attached. When the
bond between the phosphates is broken, the energy released is used to form the phosphodiester
bond between the incoming nucleotide and the existing chain.
In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA
pol III (naming done by order of discovery). DNA pol III is the enzyme required for DNA
synthesis; DNA pol I is used later in the process and DNA pol II is primarily required for repair.
(see Table below)

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DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction. It requires a free 3′-
OH group (located on the sugar) to which it can add the next nucleotide by forming a
phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This
essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how
does it add the first nucleotide? The problem is solved with the help of a primer that provides
the free 3′-OH end. Another enzyme, RNA primase, synthesizes an RNA primer that is about
five to ten nucleotides long and complementary to the DNA. RNA primase does not require a
free 3′-OH group. Because this sequence primes the DNA synthesis, it is appropriately called the
primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that
are complementary to the template strand (Figure 2).

The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only
extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know,
the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and the other
is oriented in the 3′ to 5′ direction. One strand, which is complementary to the 3′ to 5′ parental
DNA strand, is synthesized continuously towards the replication fork because the polymerase
can add nucleotides in this direction. This continuously synthesized strand is known as

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the leading strand. The other strand, complementary to the 5′ to 3′ parental DNA, is extended
away from the replication fork, in small fragments known as Okazaki fragments, each
requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese
scientist who first discovered them. The strand with the Okazaki fragments is known as
the lagging strand.
The leading strand can be extended by one primer alone, whereas the lagging strand needs a
new primer for each of the short Okazaki fragments. The overall direction of the lagging strand
will be 3′ to 5′, and that of the leading strand 5′ to 3′. A protein called the sliding clamp holds
the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-
shaped protein that binds to the DNA and holds the polymerase in
place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the
replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA
helix a-o-nd then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA pol
I, which breaks down the RNA and fills the gaps with DNA nucleotides. The nicks that remain
between the newly synthesized DNA (that replaced the RNA primer) and the previously
synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of
phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of
the other fragment.
(My notes: I think this process is almost impossible to visualize from reading text. I
strongly recommend that you watch a couple of animations / videos like the one
available here.)
Once the chromosome has been completely replicated, the two DNA copies move into two
different cells during cell division. The process of DNA replication can be summarized as
follows:
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended in both
directions.
3. Single-strand binding proteins coat the DNA around the replication fork to
prevent rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent
supercoiling (over-winding).
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase III starts adding nucleotides to the 3′-OH (sugar) end of the
primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed and gaps are filled with DNA by DNA pol I.
9. The gaps between the DNA fragments are sealed by DNA ligase.
DNA replication has been extremely well-studied in prokaryotes, primarily because of the small
size of the genome and large number of variants available. Escherichia coli has 4.6 million base
pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes,
starting from a single origin of replication and proceeding around the chromosome in both
directions. This means that approximately 1000 nucleotides are added per second. The process
is much more rapid than in eukaryotes.

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