DNA replication occurs semi-conservatively to produce two identical copies of DNA before cell division. It involves unwinding of the DNA double helix by helicase, followed by synthesis of new strands complementary to the original strands. RNA primers are required for DNA polymerase to begin DNA synthesis. The leading strand is synthesized continuously while the lagging strand is synthesized in fragments called Okazaki fragments. DNA polymerase proofreads and repairs any errors with its exonuclease activity to maintain high fidelity of DNA replication.

1. MOLECULAR GENETICS

2. INTRODUCTION
• Cells
• Fundamental working units of every living
system.
• Every organism is composed of one of two
radically different types of cells:
• prokaryotic cells
• eukaryotic cells which have DNA inside a nucleus.
• Prokaryotes and Eukaryotes are descended from
primitive cells and the results of
3.5 billion years of evolution.

3. Cont-------
• All Cells have common Cycles
• Born, eat, replicate, and die

5. Common features of organisms
• Chemical energy is stored in ATP
• Genetic information is encoded by DNA
• Information is transcribed into RNA
• There is a common triplet genetic code
• some variations are known, however
• Translation into proteins involves ribosomes
• Shared metabolic pathways
• Similar proteins among diverse groups of
organisms

6. All Life depends on 3 critical molecules
1.DNAs (Deoxyribonucleic acid)
• Hold information on how cell works
2.RNAs (Ribonucleic acid)
• Act to transfer short pieces of information to different
parts of cell
• Provide templates to synthesize into protein
3.Proteins
• Form enzymes that send signals to other cells and
regulate gene activity
• Form body’s major components

7. DNA, RNA, and the Flow of
Information
The central dogma
• Formulated by Francis Crick
• Defines three major steps in the processing of
genetic information
• 1. replication
• 2. Transcription
• 3. Translation

8. The central dogma

9. DNA Replication
• Replication is the duplication of DNA content
before mitosis, i.e., the two DNA copies are
divided equally between the new daughter
cells. Each old strand will be copied into a
complementary new strand.
• DNA replication is the process by which the
genetic material is copied prior to distribution
into daughter cells

10. Cont----
• The original DNA strands are used as
templates for the synthesis of new strands
• It occurs very quickly, very accurately and at
the appropriate time in the life cycle of the
cell
• DNA replication relies on the
complementarities of DNA strands
• The AT/GC rule.

11. Cont-------
• This process is called semi-conservative
because it conserves only half of the original
(parent) DNA molecule in the two daughter
DNA molecules. One strand in each daughter
molecule is completely new.
• which means that each daughter cell will
receive a DNA strand from the mother cell and
a complementary newly synthesized strand.

13. Cont----
Basic requirements for replication
A. Substrates
• The four deoxyribonucleosides triphosphate
• dATP
• dGTP
• dCTP
• dTTP

14. Cont----
• B. Template
• For DNA replication to occur the two chains
have to unwind and separate.
• The separated strands serve as template for
the synthesis of the new daughter strands.
• A template is required to direct the addition
of the appropriate complimentary nucleotide
to the newly synthesized DNA strand.

15. Cont----
• C. Enzymes
• DNA synthesis is catalyzed by enzymes called
DNA dependent DNA polymerase
• DNA polymerase I, is responsible for gap filling by
nick translation of the RNA primers used during
DNA replication into DNA sequence.
• DNA polymerase II, has proofreading and DNA
repair function.
• DNA polymerase III, is the major enzyme
responsible for DNA replication.

16. Cont----
DNA helicase
• Unwinds the double helix into two single-
stranded DNA.
• It breaks down hydrogen bonds between the
bases at expense of ATP. Two ATP molecules
are consumed for each base pair broken. Once
separated, the single stranded DNA formed is
stabilized by proteins called DNA-binding
proteins.

17. Cont----
The single-stranded DNA binding proteins
(SSB)
• that prevent reannealing of DNA helix by
binding the single stranded DNA created by
helicase without interfering with DNA
polymerase function.

18. Cont----
DNA gyrase
• Removes positive supercoils and introduces
negative supercoils.
Topoisomerases
• Catalyzes the relaxation of supercoiled DNA by
breaking just one strand of DNA.

19. Primase
• DNA-dependent RNA polymerase that
synthesizes short RNA primers (5-200 bases)
with the help of DNA binding protein
(primosome).
• RNA primer complementary and anti-parallel
to the DNA is synthesized by primase.
• The DNA polymerase III requires a primer to
elongate at the beginning of replication. The
primer has a free 3'-OH to accept deoxy-
ribonucleotides polymerized by DNA
polymerase III.

20. Cont----
DNA ligase
• joins the free 3'-OH on C3 of one end to 5'-OH
of phosphate on C5 of the other end of DNA.

21. STAGES OF REPLICATION
The process of replication can be divided in to
three stages.
• Initiation
• Elongation
• Termination

22. Cont----
• Initiation
• DNA replication is initiated by the binding of
DnaA proteins to the DnaA box sequences
• This binding stimulates the cooperative
binding of an additional 20 to 40 DnaA
proteins to form a large complex.
• This causes the DNA to twist and puts torque
on the nearby AT-rich region to denature and
form a replication bubble

23. Cont----
–AT base pairs are held together by only
2 H bonds
–CG base pairs are held together by 3 H
bonds
–Therefore, AT-rich regions of DNA
denature more easily than CG-rich
regions of DNA

24. Cont----
• In the next step, DnaB (also called helicase)
binds to each strand of the separated double
helix. It’s job is to move along the DNA,
progressively expand the replication fork in
both directions.
• DNA helicase separates the two DNA strands
by breaking the hydrogen bonds between
them

25. Cont----
• This generates positive supercoiling ahead
of each replication fork so another enzyme,
topoisomerase, travels ahead of the
helicase and alleviates these supercoils
• Single-strand binding proteins (SSBPs) are
also needed to bind to the separated DNA
strands and keep them apart
– Otherwise, the strands would simply reanneal

26. Cont----
• After the helicase, gyrase, and SSBPs are in
place, short (10 to 12 nucleotides) RNA
primers are synthesized by DNA primase
• These short RNA strands start, or prime, DNA
synthesis because DNA polymerase, the
enzyme that copies DNA, cannot start a new
strand on its own
• The RNA primers are later removed and
replaced with DNA

27. Cont----
• Elongation
• DNA polymerase III begins the process of
elongation by adding free deoxy-ribonucleotides
to the free 3'-OH end of the RNA primer in the
5'3' direction in a continuous manner (the
leading strand) to the end of the DNA molecule
or till reaching the flanking replication fork.
• This is done according to the base pairing rule: A-
T and G-C using the 3'5' DNA strand as a
template.

28. Cont----
• The other 5'3' the lagging strand of DNA, lags
behind for a while until at least 1000 to 5000
nucleotides in prokaryotes (but only 150 - 250
nucleotides in eukaryotes) are exposed, where
primase put a primer at the upper end of it at the
replication fork, i.e., in 5'3' direction.
• DNA polymerase III covers the downstream(away
from the replication fork) sequence by
synthesizing at least 1000 DNA nucleotides.

29. Cont----
• This process is repeated when a new enough
stretch of DNA is again exposed due to the
continuous progress of the leading strand,
where, primase puts a new primer that is
elongated by DNA polymerase III until it
reaches the previous RNA primer, and so on.
Each area enough for the DNA polymerase III
to work on the lagging strand is called Okazaki
fragment.

30. Cont----
• Thus, one strand is built towards the
replication fork 5’3’, i.e., the Leading strand,
while the other strand is built in the opposite
direction but also in 5'3', i.e., Lagging
strands.
• Leading strand
• One RNA primer is made at the origin
• DNA pol III attaches nucleotides in a 5’ to 3’ direction as
it slides toward the replication fork

31. Cont----
Lagging strand
• Synthesis is also in the 5’ to 3’ direction
–However it occurs away from the
replication fork
• Many RNA primers are required
• DNA pol III uses the RNA primers to
synthesize small DNA fragments (1000 to
2000 nucleotides each)
–These are termed Okazaki fragments
after their discoverers

32. Cont----
• Finally, the RNA primers are removed by the
5'3' nick translation by DNA polymerase I
activity with a replacement into DNA
nucleotides.
• The 5'-end of DNA fragments, built by DNA
polymerase III, is linked to the 3'-end of the
DNA fragment, built by the DNA polymerase I,
by ATP-dependent ligase

33. Cont----
• All DNA polymerases, whether bacterial or
eukaryotic, share 2 very important limitations:
• 1. They cannot initiate DNA synthesis on their
own. They require an RNA primer be laid
down on the DNA first by DNA primase.
• 2. They can only “grow” a new DNA chain in
the 5’ to 3’ direction.

34. Cont----
• Proofreading
• DNA replication exhibits a high degree of fidelity.
• Mistakes during the process are extremely rare
• There are several reasons why fidelity is high:
• 1. Instability of mismatched pairs
– Complementary base pairs have much higher stability
than mismatched pairs
– This feature only accounts for part of the fidelity
– It has an error rate of 1 per 1,000 nucleotides

35. Cont----
• 2. Configuration of the DNA polymerase active
site
– DNA polymerase is unlikely to catalyze bond
formation between mismatched pairs
• 3. Proofreading function of DNA polymerase
– DNA polymerases can identify a mismatched
nucleotide and remove it from the daughter
strand
– The enzyme uses its 3’ to 5’ exonuclease activity
to remove the incorrect nucleotide

36. Cont----
• C. Termination
• A specific protein, ter binding protein, binds
termination sequences and prevents helicase
from further unwinding of DNA and facilitates
the termination of replication.

39. 5' 3'
Primase
RNA Primer
DNA polymerase III
Single-strand
binding protein
dATP
dGTP
dCTP
dTTP
ATP, GTP, CTP, TTP
PPI
Leading strand
DNA polymerase I
DNA ligase
Helicase
Lagging strand
PPi
5'
5'
5'
3'
3'
5'
Topoisomerases (Gyrase),
Remove supercoiling