Replication

1. PRESENTED BY,
K.GOKILA
MSFBI1802

2.  DNA replication.
 Meselson stahl experiment.
 Molecular mechanism of DNA replication.
 Bidirectional and rolling circle replication.

3.  In molecular biology, DNA replication is
the biological process of producing two identical
replicas of DNA from one original DNA molecule.
 DNA replication occurs in all living organisms acting
as the basis for biological inheritance. The cell
possesses the distinctive property of division, which
makes replication of DNA essential.

4. DNA is made up of a double helix of
two complementary strands. During replication, these strands
are separated. Each strand of the original DNA molecule then
serves as a template for the production of its counterpart, a
process referred to as semiconservative replication.
As a result of semi-conservative replication, the new helix will
be composed of an original DNA strand as well as a newly
synthesized strand. Cellular proofreading and error-
checking mechanisms ensure near perfect fidelity for DNA
replication.[

6. Models of Replication:

7. Meselson-Stahl experiment:
Matt Meselson and Franklin Stahl originally met in the summer of
1954, the year after Watson and Crick published their paper on the
structure of DNA.
Although the two researchers had different research interests, they
became intrigued by the question of DNA replication and decided to
team up and take a crack at determining the replication.
Meselson and Stahl conducted their famous experiments on DNA
replication using E. coli bacteria as a model system.

8. They began by growing E. coli in medium, or nutrient broth,
containing a "heavy" isotope of nitrogen N (15). An isotope is just
a version of an element that differs from other versions by the
number of neutrons in its nucleus.)
When grown on medium containing heavy N(15)the bacteria took
up the nitrogen and used it to synthesize new biological molecules,
including DNA.
After many generations growing in the medium, the nitrogenous
bases of the bacteria's DNA were all labeled with heavy N(15) .
Then, the bacteria were switched to medium containing a "light “
N(14) isotope and allowed to grow for several generations.

9. Meselson and Stahl knew how often E. coli cells divided,
so they were able to collect small samples in each
generation and extract and purify the DNA. They then
measured the density of the DNA using density gradient
centrifugation.
This method separates molecules such as DNA into bands
by spinning them at high speeds in the presence of another
molecule, such as cesium chloride, that forms a density
gradient from the top to the bottom of the spinning tube.
Density gradient centrifugation allows very small
differences—like those between N (14) and N(15) , labeled
DNA—to be detected.

12. What is mechanism of DNA replication?
DNA replication is semi conservative. Each strand in the
double helix acts as a template for synthesis of a new, complementary
strand. New DNA is made by enzymes called DNA polymerases,
which require a template and a primer (starter) and synthesize DNA in
the 5' to 3' direction.
The two strands are easily separable because the hydrogen bonds which
hold the two strands are very weak in contrast to other chemical bonds.
When these two strands separate out, each part of one strand constitutes
the complementary part of other strand.

15. Two parental strands do not separate completely, but are opened
up at what is known as replication fork. As a result, opposite A,
T would fit, opposite C; G would come and so on. Due to this
process exact nucleotide sequence would be automatically
formed.
Thus, regeneration of DNA helix occurs, with one strand of
original helix combining with freshly formed complement to
constitute a double stranded DNA molecule. This type of
replication has also been called as zipper duplication.

17. Details of DNA replication can be discussed under the
following headings:
Activation of deoxyribonucleiosides
Recognition of initiation point.
Unwinding of DNA molecule.
Formation of RNA primer.
Formation of new DNA chains.
Removal of RNA primer.
Proof reading &DNA repair.

19. Bidirectional Replication:
Bidirectional replication is a method of DNA replication found in
organism from each of the main kingdoms. Bidirectional
replication involves replicating DNA in two directions at the same
time resulting in a leading strand (were replication occurs more
rapidly) and a lagging strand (with slower replication).
The properties of each of these strands is caused by DNA
polymerase and its ability to only replicate in the 5' to 3' direction.
In the leading strand, a single DNA polymerase can replicate large
portions of the strand (approximately X1000-5000 bases before it
falls off the DNA due to its high processivity) before dissociating.

21. However, in the lagging strand, the DNA is replicate in chunks
which are called Okasaki fragments. Each of these fragments is
later fused together by DNA ligase to produce the full, un
fragmented strand.
At all replication origins, replication takes place in a
bidirectional format which results in the formation of
‘replication bubbles’. These bubbles grow in size as replication
continues. Eventually, two replication forks (at each end of a
bubble) meet, at which point they fuse together producing a
larger bubble.

24. Ultimately, all the replication bubbles along
the chromosome merge into one large bubble joint only at
the telomeres these split to give two identical strands of DNA.
This process continues to produce a many strands of DNA which
are then passed on to daughter cells
Rolling circle replication:
Rolling circle replication describes a process of unidirectional
nucleic acid replication that can rapidly synthesize multiple
copies of circular molecules of DNA or RNA, such as plasmids,
the genomes of bacteriophages, and the circular RNA genome
of viroids Some eukaryotic viruses also replicate their DNA
or RNA via the rolling circle mechanism.

25. Rolling circle replication
produces multiple copies
of a single circular
template.
As a simplified version of natural
rolling circle replication, an
isothermal DNA amplification
technique, rolling circle
amplification (RCA) was
developed.
The RCA mechanism is widely
used in molecular biology &
biomedical nanotechnology
especially in the field
of biosensing (as a method of
signal amplification).