DNA REPLICATION PROCESS

1. DNA replication

2. How does DNA Replicate?
• The data collected by Meselson and Stahl were consistent with
the SEMICONSERVATIVE model of replication.
– Remember, this was the model that was suggested by the
complementary structure of DNA molecules.
– The real beauty of the Meselson-Stahl experiment was the fact that
they could distinguish among all possible models.
Conservative Semiconservative Dispersive

3. Features of DNA Replication
• DNA replication is semi-conservative
– Each strand of template DNA is being copied.
• DNA replication is bidirectional
– Bidirectional replication involves two replication forks,
which move in opposite directions
• DNA replication is semi-discontinuous
– The leading strand copies continuously
– The lagging strand copies in segments (Okazaki
fragments) which must be joined

4. Steps in DNA Replication
• Replication involves 3 stages:
- initiation
- elongation
- termination
• Replication begins at site called origin of
replication
- 1 in prokaryotes
- numerous in eukaryotes

5. • Opening DNA double helix
– initiating replication
– unwinding duplex
– stabilizing single strands
– relieve torque
• Building a primer
• Assembling complementary strands
• Removing the primer
• Joining Okazaki fragments
Replication Process

6. Overview
1. An initiator protein binds to and separates the strands at the
replication origin
2. Helicase separates the strands at the replication fork
3. Gyrase relieves supercoiling as it develops
4. Primase creates a short RNA primer
5. DNA polymerase III extends the strand
6. DNA polymerase I replaces the RNA primer with DNA
7. Ligase repairs missing phosphodiester bonds between fragments

7. Replication as a process
• Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two strands are made.
Each has one new and one old
DNA strand.

8. The process of DNA Replication
• Unwinding of DNA with helicase: Unwinding
a portion of the DNA double helix using energy
derived from ATP
• Binding Single-stranded proteins :Keeps the
unwound DNA strands from re-annealing
• Gyrases: Eliminates super coiling that
accompanies unwinding removes torsion strain
by opening double helix.

10. The process of DNA Replication
• RNA Primase: Attaches RNA primers (5-15 bp) on
to DNA template to initiate DNA synthesis. Initiation
of okazaki fragment on lagging strand by primosome-
a protein complex contain DNA primase and DNA
helicase.
• DNA Polymerase delta (ð): Binds to the 5' - 3'
strand in order to bring nucleotides and create the
daughter leading strand.

11. The process of DNA Replication
• DNA Polymerase epsilon (å): Binds to the 3' - 5'
strand in order to create discontinuous segments starting
from different RNA primers.
Exonuclease (DNA Polymerase I): Finds and
removes the RNA Primers
DNA Ligase: Adds phosphate in the remaining gaps of
the phosphate - sugar backbone
Nucleases: Remove wrong nucleotides from the
daughter strand

12. Initiation
• Initiator protein (Dna A) is responsible for
intial step in unwinding the helix, binds to the
origin and separates strands

13. A eukaryotic chromosome have hundreds or even
thousands of replication origins
Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
The bubbles expand laterally, as
DNA replication proceeds in both
directions.
Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete.
1
2
3
Origin of replication
Bubble
Parental (template) strand
Daughter (new) strand
Replication fork
Two daughter DNA molecules
In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome. In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
(b)
(a)
0.25 µm

14. Replication fork
1. As the strands of DNA
unwind, an area of
replication called the
replication fork is
created
2. As nucleotides are
added, the replication
fork moves down the
parental strand
3. Replication is completed
when the replication
fork reaches the end of
the parent strand

16. DNA Replication Fork

17. DNA polymerases
• Add nucleotides against a DNA template to the 3' end of
the growing strand (5’3’ growth)
Requirements
1. A template
2. Deoxyribonucleoside
triphosphates which
serve both as the
source for the
nucleotide and as
the energy source
3. A primer

18. Primase
• DNA polymerase needs a primer
• Short bits of RNA serve as the primer
• The primer is added by a DNA-dependent RNA
polymerase called primase
• RNA polymerases are different from DNA
polymerases as they can initiate a new strand
based on a template strand

19. Primer
• Only one primer is needed for synthesis of
the leading strand
– But for synthesis of the lagging strand, each
Okazaki fragment must be primed separately

20. Type I Topoisomerases
• One strand is
“nicked,” allowing
the remaining intact
phosphate backbone
to twist under
torsion
• The nicked strand is
then rejoined
• No net energy
expenditure

21. DNA polymerase I
(α in eukaryotes)
• Does all the stuff DNA poly III does
– 5‘3' polymerase
– 3‘5' exonuclease
• Also a 5‘3' exonuclease
– removes damaged bases
– removes primers
• Slower than DNA poly III. abundant

22. DNA polymerase III
(δ in eukaryotes)
The main polymerase in bacteria consists of 10 peptide sub
units
5‘3' polymerase
3‘5' exonuclease for proof reading

23. Elongation of DNA Replication
• DNA polymerase binds to 3'-end of RNA primer
- makes DNA in 5' --> 3' direction
- slides along template strand in 3' --> 5'
direction
• Elongation can occur continuously on one of the
template strands as helix opens in front of it
- strand with continuous replication is called
leading strand

24. Termination of DNA Replication
• RNA primers removed from newly
synthesized DNA molecules and replaced
by DNA
• DNA ligase seals gaps between these
fragments

25. Replication Process
• Single stranded breaks are sealed by DNA
ligase

26. Ligation and Primer removal

27. Figure 16.16
Overall direction of replication
Leading
strand
Lagging
strand
Lagging
strand
Leading
strand
OVERVIEW
Leading
strand
Replication fork
DNA pol III
Primase
Primer
DNA pol III Lagging
strand
DNA pol I
Parental DNA
5
3
4
3
2
Origin of replication
DNA ligase
1
5
3
Helicase unwinds the
parental double helix.
1
Molecules of single-
strand binding protein
stabilize the unwound
template strands.
2 The leading strand is
synthesized continuously in the
5 3 direction by DNA pol III.
3
Primase begins synthesis
of RNA primer for fifth
Okazaki fragment.
4
DNA pol III is completing synthesis of
the fourth fragment, when it reaches the
RNA primer on the third fragment, it will
dissociate, move to the replication fork,
and add DNA nucleotides to the 3 end
of the fifth fragment primer.
5
DNA pol I removes the primer from the 5 end
of the second fragment, replacing it with DNA
nucleotides that it adds one by one to the 3 end
of the third fragment. The replacement of the
last RNA nucleotide with DNA leaves the sugar-
phosphate backbone with a free 3 end.
6
DNA ligase bonds
the 3 end of the
second fragment to
the 5 end of the first
fragment.
7
A summary of DNA replication

28. Replication forks