DNA Replication, Replication Fork, Proofreading www.facebook.com/careeratfoodscience

1. REPLICATION
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2. The Molecular Basis of Inheritance
C
T
A
A
T
CG
GC
A
C G
AT
AT
A T
TA
C
TA
0.34 nm
3.4 nm
(a) Key features of DNA structure
G
1 nm
G
(c) Space-filling model
T

4. • Structure was discovered in 1953 by James
Watson and Francis Crick

5. The Structure of DNA
• DNA is composed of four nucleotides,
each containing: adenine, cytosine,
thymine, or guanine.
• The amounts of A = T, G = C, and
purines = pyrimidines [Chargaff’s Rule].
• DNA is a double-stranded helix with
antiparallel strands [Watson and Crick].
• Nucleotides in each strand are linked by
5’-3’ phosphodiester bonds
• Bases on opposite strands are linked by
hydrogen bonding: A with T, and G with
C.

7. The Basic Principle: Base Pairing to
a Template Strand
• The relationship between structure and
function is manifest in the double helix
• Since the two strands of DNA are
complementary each strand acts as a
template for building a new strand in
replication

8. DNA replication
• The parent molecule unwinds, and two
new daughter strands are built based on
base-pairing rules
(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
A
C
T
A
G
A
C
T
A
G
A
C
T
A
G
A
C
T
A
G
T
G
A
T
C
T
G
A
T
C
A
C
T
A
G
A
C
T
A
G
T
G
A
T
C
T
G
A
T
C
T
G
A
T
C
T
G
A
T
C

9. DNA Replication is “Semi-conservative”
• Each 2-stranded
daughter molecule
is only half new
• One original strand
was used as a
template to make
the new strand

10. DNA Replication
• The copying of DNA is remarkable in its speed and
accuracy
• Involves unwinding the double helix and synthesizing
two new strands
• More than a dozen enzymes and other proteins
participate in DNA replication
• The replication of a DNA molecule begins at special
sites called origins of replication, where the two
strands are separated

11. Origins of Replication
• A eukaryotic chromosome may 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

12. Mechanism of DNA Replication
• DNA replication is catalyzed by DNA polymerase which
needs an RNA primer
• RNA primase synthesizes primer on DNA strand
• DNA polymerase adds nucleotides to the 3’ end of the
growing strand

13. Mechanism of DNA Replication
• Nucleotides are added by complementary base pairing
with the template strand
• The substrates, deoxyribonucleoside triphosphates, are
hydrolyzed as added, releasing energy for DNA synthesis.

14. The Mechanism of DNA Replication
• DNA synthesis on the leading strand is continuous
• The lagging strand grows the same general direction
as the leading strand (in the same direction as the
Replication Fork). However, DNA is made in the 5’-
to-3’ direction
• Therefore, DNA synthesis on the lagging strand is
discontinuous
• DNA is added as short fragments (Okasaki
fragments) that are subsequently ligated together

17. DNA polymerase I degrades the
RNA primer and replaces it with
DNA

18. The Mechanism of DNA Replication
• Many proteins assist in DNA replication
• DNA helicases unwind the double helix, the
template strands are stabilized by other
proteins
• Single-stranded DNA binding proteins make
the template available
• RNA primase catalyzes the synthesis of
short RNA primers, to which nucleotides are
added.
• DNA polymerase III extends the strand in
the 5’-to-3’ direction
• DNA polymerase I degrades the RNA
primer and replaces it with DNA
• DNA ligase joins the DNA fragments into a
continuous daughter strand

19. Enzymes in DNA replication
Helicase unwinds
parental double helix
Binding proteins
stabilize separate
strands
DNA polymerase III
binds nucleotides
to form new strands
Ligase joins Okazaki
fragments and seals
other nicks in sugar-
phosphate backbone
Primase adds
short primer
to template strand
DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases

20. Binding proteins prevent single strands from rewinding.
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
5’
3’
5’
3’
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
3’5’
5’3’
Replication

21. Overall direction
of replication
5’3’
5’
3’
5’
3’
3’5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
Replication

22. DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
5’
5’
Overall direction
of replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication

23. 5’
5’3’
5’
3’
3’
5’
3’
Overall direction
of replication
Leading strand synthesis continues in a
5’ to 3’ direction.
Replication

24. 3’5’ 5’
5’3’
5’
3’
3’
5’
3’
Overall direction
of replication
Okazaki fragment
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

25. 5’ 5’
5’3’
5’
3’
3’
5’
3’
Overall direction
of replication
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Okazaki fragment
Replication

26. 5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

27. 3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

28. 5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease activity of DNA polymerase I
removes RNA primers.
Replication

29. Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’
Replication

30. Replication Fork Overview

31. Proofreading
• DNA must be faithfully replicated…but mistakes
occur
– DNA polymerase (DNA pol) inserts the wrong
nucleotide base in 1/10,000 bases
• DNA pol has a proofreading capability and can correct errors
– Mismatch repair: ‘wrong’ inserted base can be removed
– Excision repair: DNA may be damaged by chemicals,
radiation, etc. Mechanism to cut out and replace with
correct bases

32. Mutations
• A mismatching of base pairs, can occur at a rate of
1 per 10,000 bases.
• DNA polymerase proofreads and repairs
accidental mismatched pairs.
• Chances of a mutation occurring at any one gene
is over 1 in 100,000
• Because the human genome is so large, even at
this rate, mutations add up. Each of us probably
inherited 3-4 mutations!

33. Proofreading and Repairing DNA
• DNA polymerases proofread
newly made DNA, replacing
any incorrect nucleotides
• In mismatch repair of DNA,
repair enzymes correct errors
in base pairing
• In nucleotide excision DNA
repair nucleases cut out and
replace damaged stretches of
DNA
Nuclease
DNA
polymerase
DNA
ligase
A thymine dimer
distorts the DNA molecule.
1
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
2
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
3
DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
4

34. •Thank you