2. Introduction
DNA is located in chromosomes inside the nucleus of a cell.
DNA replication is the biological process of producing two identical replicas of
DNA from one original DNA molecule.
This process occurs in all living organisms and is the basis for biological
inheritance.
The cell possesses the distinctive property of division, which makes replication of
DNA essential.
3. Structure of DNA
DNA is made up of a double helix of two complementary strands.
The two DNA strands are called polynucleotides since they are composed of
simpler monomer units called nucleotides.
Each nucleotide is composed of one of four nitrogen-
containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]),
a sugar called deoxyribose, and a phosphate group.
The nucleotides are joined to one another in a chain by covalent bonds between
the sugar of one nucleotide and the phosphate of the next, resulting in an
alternating sugar-phosphate backbone.
4. The nitrogenous bases of the two separate polynucleotide strands are bound
together, according to base pairing rules (A with T and C with G), with hydrogen
bonds to make double-stranded DNA.
5. Steps in duplications
Before the replication process starts DNA needs to be prepared for replication.
After that replication process occurs as follows
1. Initiation
2. Replication fork formation
3. Elongation
4. Replication machinery
5. Termination
6. Enzymes
DNA replication would not occur without enzymes that catalyze various steps in the
process. Enzymes that participate in the eukaryotic DNA replication process include:
DNA helicase - unwinds and separates double stranded DNA as it moves along
the DNA. It forms the replication fork by breaking hydrogen bonds between
nucleotide pairs in DNA.
DNA primase - a type of RNA polymerase that generates RNA primers. Primers are
short RNA molecules that act as templates for the starting point of DNA
replication.
DNA polymerases - synthesize new DNA molecules by adding nucleotides to
leading and lagging DNA strands.
Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent
the DNA from becoming tangled or supercoiled.
7. Exonucleases - group of enzymes that remove nucleotide bases from the end of a
DNA chain.
DNA ligase - joins DNA fragments together by forming phosphodiester bonds
between nucleotides.
8. Initation
This process is initiated at particular points in the DNA, known as "origins", which
are targeted by initiator proteins.
Once the origin has been located, these initiators recruit other proteins and form
the pre-replication complex, which unwinds the double-stranded DNA.
9. Replication Fork Formation
It is created by helicases, which break the hydrogen bonds holding the two DNA
strands together.
The resulting structure has two branching "prongs", each one made up of a single
strand of DNA.
These two strands serve as the template for the leading and lagging strands.
10. Leading Strand
The leading strand is the strand of nascent DNA which is being synthesized in the
same direction as the growing replication fork.
A polymerase "reads" the leading strand template and adds
complementary nucleotides to the nascent leading strand on a continuous basis.
11. Lagging Strand
The lagging strand is the strand of nascent DNA whose direction of synthesis is
opposite to the direction of the growing replication fork.
Because of its orientation, replication of the lagging strand is more complicated as
compared to that of the leading strand.
As a consequence, the DNA polymerase on this strand is seen to "lag behind" the
other strand.
12. Replication Machinery
The lagging strand is synthesized in short, separated segments.
On the lagging strand template, a primase "reads" the template DNA and initiates
synthesis of a short complementary RNA primer.
A DNA polymerase extends the primed segments, forming Okazaki fragments.
The RNA primers are then removed and replaced with DNA, and the fragments of
DNA are joined together by DNA ligase.
13. Okazaki Fragments
The lagging strand is synthesized in short, separated segments.
On the lagging strand template, a primase "reads" the template DNA and initiates
synthesis of a short complementary RNA primer.
A DNA polymerase extends the primed segments, forming Okazaki fragments.
The RNA primers are then removed and replaced with DNA, and the fragments of
DNA are joined together by DNA ligase.
14. Termination
Termination requires that the progress of the DNA replication fork must stop or be
blocked.
Termination at a specific locus, when it occurs, involves the interaction between
two components:
(1) a termination site sequence in the DNA, and
(2) a protein which binds to this sequence to physically stop DNA replication.
In various bacterial species, this is named the DNA replication terminus site-
binding protein, or Ter protein.
16. PCR uses a pair of primers to span a target region in template DNA, and then
polymerizes partner strands in each direction from these primers using a
thermostable DNA polymerase.
Repeating this process through multiple cycles amplifies the targeted DNA region.
At the start of each cycle, the mixture of template and primers is heated, separating
the newly synthesized molecule and template.
Then, as the mixture cools, both of these become templates for annealing of new
primers, and the polymerase extends from these.
As a result, the number of copies of the target region doubles each
round, increasing exponentially.
