“This structure has novel features which are of considerable biological interest.” This may be the science most famous statement, which appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

1. DNA REPLICATION AND REPAIR
Nirajan Shrestha
Biomedical Research Institute
Chonbuk National University Medical School

2. DNA: Introduction
• DNA (Deoxyribonucleic acid) is the hereditary
material in humans and almost all other
organisms.
• Most DNA is located in the nucleus (Nuclear
DNA), but a small amount of DNA can also be
found in the mitochondria.
• The information in DNA is stored as a code
made up of four chemical bases: adenine (A),
guanine (G), cytosine (C), and thymine (T).
Human DNA consists of about 3 billion bases.

3. Structure of DNA
• DNA exists as a double stranded
molecule, in which the strands
wind around each other, forming a
double helix.
• Double helix structure of DNA was
proposed by James Watson and
Francis Crick in April 1953, on the
basis of X-ray diffraction model
proposed by Rosalind Franklin and
Maurice Wilkins.
• Nine years later, in 1962, Watson
and Crick shared the Nobel Prize in
Physiology and Medicine with
Maurice Wilkins.

4. • “This structure has novel
features which are of
considerable biological
interest.”
• This may be the science most
famous statement, which
appeared in April 1953 in the
scientific paper where James
Watson and Francis Crick
presented the structure of the
DNA-helix.
• “It has not escaped our notice
that the specific pairing we
have postulated immediately
suggests a possible copying
mechanism for the genetic
material."

6. Do not forget her….

7. • DNA is poly-deoxyribonucleotide
that contain many mono-deoxy
ribonucleotide covalently linked by
3’-5’ phosphodiester bond.
• The chains are paired in anti-parallel
manner.
• In the DNA helix, the hydrophilic
deoxyribose-phosphate backbone is
on outside of the molecule, where as
the hydrophobic bases are stacked
inside.
• Base pairing: A–T /G-C (H Bond)

8. DNA REPLICATION
• DNA replication is the process of synthesis of
two daughter DNA from single parental DNA
molecule.
• When the two strands of DNA double helix
separated, each strand can contribute as a
template for the daughter DNA.
• In a single daughter DNA, one strand comes
from parent and next is newly synthesized.
• Hence, DNA replication is semi-conservative in
nature.

9. Semi-conservative nature of DNA Replication

10. DNA Replication in Prokaryotes
• The replication process described in this section were
first known from studies of the bacterium E. coli. DNA
synthesis in higher organisms is less well understood,
but involves the same types of mechanisms with few
exception.
A. Separation of two complementary Strands
B. Formation of replication fork
C. Direction of DNA replication
D. Synthesis of RNA primer
E. Chain elongation
F. Excision of RNA primer and their replacement by DNA
G. DNA ligase action
H. Termination

11. A. Separation of two complementary Strands
• In order to replicate the parent DNA, they must first separate.
• Replication begins at the point called “Origin of Replication”.
• At the origin of replication, DnaA protein bind to specific
nucleotide sequence. This energy requiring process cause the
dsDNA to separate. As the dsDNA is unwound, a replication
bubble forms.

12. B. Formation of Replication Fork
• As the two strands unwind and separate, they form a “Y
shaped” where active synthesis occurs. This region is
called the replication fork.
• DNA helicase unwinds the double helix.
• The replication fork moves at the rate of 1000 nucleotides
per second.
• SSB protein helps to keep the strand separated.
• As the two strands of the double helix are separated, a
problem is encountered, namely, super-coiling in the
region of DNA ahead of the replication fork.

13. • The accumulating positive supercoils interfere with further
unwinding of the double helix
• To solve the problem of super-coiling, there is a group of
enzymes called DNA topoisomerases, which are responsible for
removing supercoils in the helix.
• These enzymes reversibly cut one strand of the double helix.
They have both nuclease (strand-cutting) and ligase (strand-
resealing) activities.

14. C. Direction of Replication
• The DNA polymerases responsible for replication are only able to
“read” the parental nucleotide sequences in the 3′→5′ direction,
and they synthesize the new DNA strands only in the 5′→3′ (anti-
parallel) direction.
1. Leading Strand: This strand is extended towards the replication
fork and synthesized continuously.
2. Lagging strand: This strand is extended away from the
replication fork and synthesized discontinuously in small
fragments known as Okazaki fragments, each requiring a
primer to start the synthesis. Okazaki fragments are named
after the scientist who first discovered them.

15. Picture showing Replication Fork

16. D. RNA Primer
• DNA polymerases cannot initiate synthesis of a complementary
strand of DNA on a totally single-stranded template. Rather, they
require an RNA primer, with a free hydroxyl group on the 3′-end
of the RNA strand.
• A specific RNA polymerase, called Primase (DnaG), synthesizes the
short stretches of RNA (approximately ten nucleotides long) that
are complementary and anti-parallel to the DNA template.
• These short RNA Primer are constantly being synthesized at the
replication fork on the lagging strand, but only one RNA sequence
at the origin of replication is required on the leading strand.

17. E. Chain Elongation
• DNA polymerases elongate a new DNA strand by adding deoxy-
ribonucleotides, one at a time, to the 3′-end of the growing chain.
• DNA chain elongation is catalyzed by DNA polymerase III.
• The new strand grows in the 5′→3′ direction, anti-parallel to the
parental strand .
• Pyrophosphate (PPi) is released when each new deoxynucleoside
monophosphate is added to the growing chain.

18. F. Excision of RNA primers and their
replacement by DNA
• DNA POL I removes the RNA
primer and fills the gap
between Okazaki fragments.

19. G. DNA Ligase Action
• The final phosphodiester linkage between the 5′-phosphate
group and the 3′-hydroxyl group on the chain is catalyzed
by DNA ligase.
• DNA ligase is an enzyme that catalyzes the sealing of nicks
remaining in the DNA.
• The joining of these two stretches of DNA requires energy,
which in most organisms is provided by the cleavage of ATP
to AMP + PPi.

20. H. Termination
• Termination of DNA replication in E. coli is mediated
by binding of the protein, TUS (Terminus Utilization
Substance) to replication termination sites (Ter sites)
on the DNA, stopping the movement of DNA
polymerase.

21. Proof-reading Function of DNA POL III
• The addition of an incorrect base can take place by a
process called tautomerization.
• If the wrong base is inserted then the bond is unstable.
• DNA polymerase (I and III) have the ability to
proofread, using 3' → 5' exonuclease activity.
• When an incorrect base pair is recognized, DNA
polymerase reverses its direction by one base pair of
DNA and excises the mismatched base. Following base
excision, the polymerase can re-insert the correct base
and replication can continue.

22. Proofreading…………….
• For example, if the template base is Thymine and the
enzyme mistakenly inserts an cytosine instead of a
Adenine into the new chain, the 3′→5′
exonuclease activity hydrolytically removes the
misplaced nucleotide. The 5′→3′ polymerase activity
then replaces it with the correct nucleotide.
• The proofreading exonuclease activity requires
movement in the 3′→5′ direction, not 5′→3′ like
the polymerase activity. This is because the excision
must be done in the reverse direction from that of
synthesis.

23. Proofreading in figure

24. Eukaryotic DNA Replication
• The process of eukaryotic DNA replication closely
follow that of Prokaryotic DNA Synthesis.
Prokaryotic DNA Replication Eukaryotic DNA Replication
Single Origin of Replication Multiple Origin of replication
Three types of DNA Polymerase Five types of DNA POL
DNA POL I,II, III DNA POL α, β, Υ, δ and ε
DNA POL III carries out both
initiation and elongation
Initiation is carried out by DNA
polymerase α while elongation by
DNA polymerase δ and ε
DNA repair and gap filling are
done by DNA polymerase I
DNA polymerase β and ε performs
this function
RNA primer is removed by DNA
polymerase I
Removed by DNA polymerase β
DNA POL Υ replicates
mitochondrial DNA.

25. DNA Repair
• Any manufacturing company tests its product in several
ways to see whether its has been assembled correctly.
Production mistakes are rectified before the item goes on
market. The same is true for DNA synthesis.
• DNA replication is incredibly accurate- only about 1 in
100,000 bases is added incorrectly. In addition to the proof-
reading capabilities of the DNA polymerase, repair enzymes
further assure the accuracy of DNA replication. This
mechanism is called DNA repair.
• A failure to repair DNA produces a mutation. Luckily, Cells
are interestingly efficient at repairing the damage done to
their DNA.

26. Agents that Damage DNA

27. Types DNA Repair
DNA repair can be grouped into two major functional
categories:
A. Direct Damage reversal
B. Excision of DNA damage

28. A. Direct Damage Reversal
• It is the simplest repair
mechanism.
• Process in a single-reaction step
• It involves enzymatic properties
which binds to the damage and
restores the DNA to its normal
state.
i) DNA photolyases
ii) DNA- alkyltransferases

29. B. Excision of DNA damage
I ) Base excision repair (BER)
II) Nucleotide excision repair (NER)
III) Mismatch repair (MMR)

30. I ) Base Excision Repair (BER)
Base excision-repair of DNA
• The enzyme uracil DNA
glycosylase removes the
uracil created by
spontaneous deamination of
cytosine in the DNA.
• An endonuclease cuts the
backbone near the defect
• An endonuclease removes a
few bases
• The defect is filled in by the
action of a DNA polymerase.
• Finally, the strand is rejoined
by a ligase.

31. • In Escherichia coli, there
are three specific proteins,
called UvrA, B and C,
involved in lesion
recognition.
• This fragment is released
by UvrD helicase action,
generating a gap that is
finally submitted to repair
synthesis.
II) Nucleotide excision repair (NER)

32. III) Mismatch Repair (MMR)
• When a mismatch occurs, the
proteins responsible for
removal of mispaired
nucleotides must be able to
discriminate between the
template strand and newly
synthesized strand .
• Newly synthesized strand is
distinguished because it has not
been methylated.
• When the new strand
containing mismatch is
identified, an exonuclease
removes mismatched bases.

33. • The gap left by removal of the mismatched nucleotides
is filled by using DNA polymerase I.
• A defect in mismatch repair in human has been
identified to cause Hereditary Nonpolyposis Colon
Cancer (HNPCC).
• HNPCC is one of the most common inherited diseases;
it affects one in 200 people and is responsible for
about 15% of all colorectal cancers in the United
States.
• The relationship between HNPCC and defects in
mismatch repair was discovered in 1993.

35. Thank You!!