DNA REPLICATION

The growth, development, and reproduction of organisms rely on cell division, or the process by which a
single cell divides into two usually identical daughter cells. This requires first making a duplicate copy of
every gene in the genome in a process called DNA replication. The copies are made by specialized
enzymes known as DNA polymerases.

Theories of DNA Replication

DNA replication must have high fidelity to avoid dramatic and rapid random changes in the sequence of
genes which would cause an extreme reduction in viability

During the 1950's, three theories were proposed by Mathew Meselson and Franklin Stahl to explain how
DNA might be replicated. They went by the names Conservative, Dispersive and Semi-conservative DNA
replication.

Conservative Replication

The parental molecule directs synthesis of an entirely new double-stranded molecule, such that after
one round of replication, one molecule is conserved as two old strands. This is repeated in the second
round. Following replication, one daughter molecule contains both of the parental strands. The other
daughter molecule contains two newly synthesized DNA strands.

This theory was proven to be incorrect.

Dispersive Replication
Material in the two parental strands is distributed more or less randomly between two daughter
molecules. In the model shown here, old material is distributed symmetrically between the two
daughter molecules. Other distributions are possible.

This theory was proven to be incorrect

Semi-Conservative Replication

Each parental strand acts as a template for the synthesis of one new daughter strand. Therefore, in
daughter molecules a newly synthesized strand is base paired to one of the original parental strands.
Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one old
and one new strand, the DNA double helix is said to be replicated “semiconservatively” by DNA
polymerase

The semi-conservative model is the intuitively appealing model, because separation of the two strands
provides two templates, each of which carries all the information of the original molecule.

The Semi-conservative theory is correct

DNA replication is semiconservative and proceeds in three major stages:

1.Unwinding

DNA molecules consists of two individual strands of linked nucleotides coiled around each other in a
double helix. Before any form of replication can occur, these two intertwined strands have to be
separated.
Special unwinding proteins attach to the DNA. The weak but numerous forces, called hydrogen bonds,
that hold the base pairs together are further weakened until the base pairs separate and the strands can
be pulled further and further apart. This creates a "Y"-shaped structure called a replication fork, and it is
at this fork that the next stage in the replication process takes place.

Additional replication proteins are needed to help in opening the double helix and thus provide the
appropriate single-stranded DNA template for the DNA polymerase to copy. Two types of proteins
contribute to this process—DNA helicases and single-strand DNA-binding proteins.

Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins

These bind tightly and cooperatively to exposed single-stranded DNA strands without covering the
bases, which therefore remain available for templating. These proteins are unable to open a long DNA
helix directly, but they aid helicases by stabilizing the unwound, single-stranded conformation.

2.Complementary pairing up of the nucleotide bases

Each separate DNA strand now has its sequence of bases exposed and unpaired. Enzymes match up
each one of these exposed bases, in turn, with free nucleotide triphosphates; A with T, and G with C.
DNA polymerase creates the new strand, but it needs some help in finding the correct place to begin, so
primase lays down a short section of RNA primer. A primer is a short single-stranded nucleic acid used
by all living organisms in the initiation of DNA synthesis. Once this short section of primer is laid, DNA
polymerase can bind to the DNA molecule and start connecting nucleotides in the correct order to
match the sequence of nitrogenous bases on the template (original) strand.

It is therefore the sequence of bases on the old, original strand which dictates and specifies the
complementary order of bases on the newly created strand
3 Completing the joins

Other enzymes, called polymerases, link up the free, matched nucleotide triphosphates by removing the
terminal di-phosphate and using energy so released to carry out the very non-spontaneous chemical
reaction of joining the phosphate to the deoxyribose sugar. New DNA chains, therefore, can only "grow"
or elongate in one direction .

4 Continuous and Discontinuous replication

The two strands of DNA run antiparallel to one another. DNA polymerase is an enzyme which can only
work in one direction on the DNA molecule. This means that one strand of DNA can be replicated in one
long string, as DNA polymerase follows helicase as it unzips the DNA molecule. This strand is termed the
“leading strand”. The other strand, however, can only be replicated in small chunks since the DNA
polymerase replicates in the opposite direction that helicase is unzipping. This strand is termed the
“lagging strand”. These small chunks of replicated DNA on the lagging strand are called Okazaki
fragments. Once DNA polymerase has replicated the DNA, a third enzyme called ligase completes the
final stage of DNA replication, which is repairing the sugar-phosphate backbone. This connects the gaps
in the backbone between Okazaki fragments. Once this is complete, the DNA coils back into its classic
double helix structure.

Enzymes Involved in DNA Replication

DNA replication is a highly enzyme-dependent process. There are many enzymes involved in the DNA
replication which includes the enzymes DNA-dependent DNA polymerase, helicase, ligase, etc. Among
them, DNA-dependent DNA polymerase is the main enzyme.

DNA-dependent DNA polymerase

It helps in the polymerization and catalyzes and regularizes the whole process of DNA replication with
the support of other enzymes. Deoxyribonucleoside triphosphates are the substrate as well as the
energy provider for the replication process. DNA polymerase is of three types:

DNA Polymerase I

It is a DNA repair enzyme. It is involved in three activities:

 5′-3′ polymerase activity

 5′-3′ exonuclease activity

 3′-5′ exonuclease activity

DNA Polymerase II

It is responsible for primer extension and proofreading.

DNA Polymerase III

It is responsible for in vivo DNA replication.

Helicase
Helicase is the enzyme which unzips the DNA strands by breaking the hydrogen bonds between them.
Thus, it helps in the formation of the replication fork.

Ligase

Ligase is the enzyme which glues the discontinuous DNA strands.

Primase

This enzyme helps in the synthesis of RNA primer complementary to the DNA template strand.

Endonucleases

These produce a single-stranded or a double-stranded cut in a DNA molecule.

Single-stranded Binding Proteins

It binds to single-stranded DNA and protects it from forming secondary structures.

Proof reading ability of DNA polymerase

As discussed at the beginning of this chapter, the fidelity of copying DNA during replication is such that
only about 1 mistake is made for every 109 nucleotides copied

If the DNA polymerase did nothing special when a mispairing occurred between an incoming
deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be
incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA
replication, however, depends not only on complementary base-pairing but also on several
“proofreading” mechanisms that act sequentially to correct any initial mispairing that might have
occurred.

The first proofreading step is carried out by the DNA polymerase, and it occurs just before a
new nucleotide is added to the growing chain

By contrast, theRNA polymerase enzymes involved in gene transcription do not need efficient
exonucleolytic proofreading: errors in making RNA are not passed on to the next generation, and the
occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are
thus able to start new polynucleotide chains without a primer.

Most cancers arise from cells that have accumulated multiple mutations and cells deficient in mismatch
proofreading therefore have a greatly enhanced chance of becoming cancerous. Fortunately, most of us
inherit two good copies of each gene that encodes a mismatch proofreading protein; this protects us,
because it is highly unlikely that both copies would mutate in the same cell.

The strand-directed mismatch repair system detects the potential for distortion in the DNA helix that
results from the misfit between noncomplementary base pairs. But if the proofreading system simply
recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched
nucleotides, it would mistakenly “correct” the original template strand to match the error exactly half
the time, thereby failing to lower the overall error rate

Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new
strands, nucleotide by nucleotide, at the replication fork as it moves from one end of a DNA molecule to
the other. But because of the antiparallel orientation of the two DNA strands in the DNA double helix
this mechanism would require one daughter strand to polymerize in the 5′-to-3′ direction and the other
in the 3′-to-5′ direction.

Such a replication fork would require two different DNA polymerase enzymes. One would polymerize in
the 5′-to-3′ direction, where each incoming deoxyribonucleoside triphosphate carried the triphosphate
activation needed for its own addition. The other would move in the 3′-to-5′ direction), this does not
occur in DNA synthesis; no 3′-to-5′ DNA polymerase has ever been found.

A replication fork therefore has an asymmetric structure. The DNA daughter strand that is synthesized
continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter
strand that is synthesized discontinuously, known as the lagging strand.

For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of
DNA chain growth. Lagging-strand DNA synthesis is delayed because it must wait for the leading
strand to expose the template strand on which each Okazaki fragment is synthesized. The synthesis of
the lagging strand by a discontinuous “backstitching” mechanism means that only the 5′-to-3′ type
of DNA polymerase is needed for DNA replication.

This enigma was resolved by experiments showing that only one strand of DNA is synthesized in a
continuous manner in the direction of overall DNA replication; the other is formed from small,
discontinuous pieces of DNA that are synthesized backward with respect to the direction of movement
of the replication fork. These small pieces of newly synthesized DNA (called Okazaki fragments after
their discoverer) are joined by the action of DNA ligase, forming an intact new DNA strand. The
continuously synthesized strand is called the leading strand, since its elongation in the direction of
replication fork movement exposes the template used for the synthesis of Okazaki fragments (the
lagging strand).