The document provides information on DNA, including its identification as the genetic material, its structure and replication. Some key points: - Experiments in the 1920s-1950s identified DNA as the genetic material, including Griffith's work on bacterial transformation and the experiments of Avery, MacLeod and McCarty and Hershey and Chase. - DNA is made up of nucleotides containing phosphate groups, sugars and nitrogenous bases. Watson and Crick discovered its double helix structure in 1953, with two anti-parallel strands held together by hydrogen bonds between complementary bases. - DNA replication is semi-conservative and involves unwinding of the DNA strands, synthesis of new complementary strands, and production of identical double

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Anilkumar C
PALM 3001

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DNA: A genetic material, its
replication, damage and repair

3. In this session...
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Identification of genetic material
Components of DNA
Structure of DNA
Replication
Damage and repair
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4. Introduction
 The progeny of organism develops characters similar to that
organism
 The resemblance of offspring to their parents depends on the
precise transmission of principle component from one generation
to the next
 That component is-
The Genetic Material
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5. What is genetic material?
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6. Four requirements for a genetic material
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• Must carry information
– Cracking the genetic code
• Must self replicate
– DNA replication
• Must allow for information to
change
– Mutation
• Must govern the expression of
the phenotype
– Gene function

7. 7
Identification of genetic material:
RNA
DNA
PROTEINDNA

8.  The process of identification of genetic material began in 1928
with experiments of Griffith and concluded in 1952 with the
studies of Hershey and Chase.
 Between these two experiments other three scientists, Avery,
Macloed and McCarty were did an experiment to identify the
genetic material.
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9. Discovery of Transformation in Bacteria:
In 1928, Frederick Griffith discovered bacterial transformation.
He worked on Streptococcus pneumonieae (Pneumococcus)
Pneumococci have various strains which can be classified by-
1. The presence or absence of a polysaccharide capsule
2. The molecular composition of the capsule
When grown on blood agar medium, pneumococci with capsules
are virulent and form large, smooth colonies and designated as
typeIII S
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10. S pneumococci mutate to an avirulent form that has no
capsules.
When grown on blood agar medium, these noncapsulated
pnuemococci form small, rough-surfaced colonies and
designated as typeII R
Based on the molecular composition of the capsule, these
pneumococci cells are type I, II, III, and so forth.
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11. (Griffith,1928)
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12.  Based on these observations he concluded that some of the
cells of typeIIR had changed into typeIIIS due to influence of
dead typeIIIS cells
 He called this phenomenon as transformation
 Principle Component of typeIIIS cells which induced the
conversion of type IIR cells into type IIIS was named
transforming principle
Griffith’s Conclusions:
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13.  Griffith’s transforming principle was the genetic
material
 Transformation assay to identify actual biomolecule
 Major constituents - DNA, RNA, proteins,
carbohydrates & lipids
 Made cell extracts from type IIIS cells containing
each of these macromolecules
1944 - Avery, MacLeod & McCarty Identify the
Genetic Material
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14. Avery, MacLeod, McCarty Experiment:
The transforming principle is DNA
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(Avery, et al., 1944)

15. (Avery, et al., 1944)
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16.  The evidence presented supports the belief that a nucleic acid
of the deoxyribose type is the fundamental unit of the
transforming principle of Pneumococcus TypeIIIS.
(Avery, et al., 1944)
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17. Genetic information is transmitted by DNA only
 The final evidence that DNA transmits genetic
information was provided by Hershey and Chase in
1952.
 They experimented with T2 bacteriophages, viruses
that attack bacteria.
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18. (Hershey and Chase, 1952)
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19. (Hershey and Chase, 1952)
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20. • The sulphur containing protein of resting phage particles is
confined to a protective coat that is responsible for the adsorption
to bacteria, and functions as an instrument for injection of the
phage DNA into the cell. This protein probably has no role in
growth of intracellular phage. The DNA has some function.
Their conclusion:
(Hershey and Chase, 1952)
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21. What is DNA?
• nitrogen base and sugar make a nucleoside.
• Phosphate group and a nucleoside make a
nucleotide.
•DNA is deoxyribo nucleic acid. A German
chemist,Friedrich Miescher, discovered
DNA in 1869.
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•DNA contains three main components
(1) Phosphate (PO4) groups;
(2) Five-carbon sugars; and
(3) Nitrogen-containing bases called
purines and pyrimidines.

22. Components of DNA:
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23. Assembly into nucleotides
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24. Nucleotides linked in a chain
The phosphate group of one
nucleotide is attached to the
sugar of the next nucleotide in
line.
• The result is a “backbone” of
alternating phosphates and
sugars, from which the bases
project
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25. 5’ PO4
PO4 5’
3’ OH
3’ OH
Structure of DNA:
• Two polynucleotide
chains are held
together by
hydrogen bonding
between bases in
opposing strands.
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26. Watson and Crick’s structure :
 They proposed that DNA as
a right handed double helix
with two poly nucleotide
chains are coiled about one
another in a spiral.
(Watson and Crick,1953)26

27. The strands of DNA are antiparallel
The strands are complimentary
There are Hydrogen bond forces
There are base stacking interactions
There are 10 base pairs per turn
Properties of a DNA double helix
(Watson and Crick,1953)
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28. 28 Watson and Crick with their model of DNA structure

29. Basis for double helix:
 Rosalind Franklin’s DNA X-
ray diffraction photograph.
 Central cross mark indicates –
helical structure of DNA.
 Top and bottom dark bands
indicates bases perpendicular
to axis of molecule.
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30. Chargaff’s base pairing rule:
Percent of adenine = percent of thymine (A=T)
Percent of cytosine = percent of guanine (C=G)
A+G = T+C (or purines = pyrimidines)
(Chargaff et al.,1950)
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31. DNA Replication:
 Replication is one of the most
important requirement for a genetic
material.
 The parent molecule unwinds, and two
new daughter strands are built based on
base-pairing rules.
It has not escaped our notice that the specific pairing we have
postulated immediately suggests a possible copying mechanism for
the genetic material’.
(Watson and Crick,1953)
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32.  extreme accuracy of DNA replication is necessary in order
to preserve the integrity of the genome in successive
generations.
 DNA has to be copied before a cell divides
 DNA is copied during the S or synthesis phase of interphase
 New cells will need identical DNA strands
Biological significance:
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33. Models of DNA replication:
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34. Semiconservative model of DNA replication
(Meselson and Stahl,1958)34

35. Steps in DNA replication:
Initiation
Proteins bind to DNA and open up double helix
Prepare DNA for complementary base pairing
Elongation
Proteins connect the correct sequences of nucleotides
into a continuous new strand of DNA
Termination
Proteins release the replication complex
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36. 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’
Proteins in replication:
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37. Overall direction
of replication
5’3’
5’
3’
5’
3’
3’5’
 DNA polymerase III enzyme adds DNA nucleotides
to the RNA primer.
 DNA polymerase proofreads bases added and replaces
incorrect nucleotides.
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38. 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.
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39. 5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
 Exonuclease activity of DNA polymerase I
removes RNA primers.
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40.  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’
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41. Origin of replication:
Initiator proteins identify specific base sequences on
DNA called sites of origin.
Prokaryotes – single origin site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator) E.g.
yeast(ARS)
Prokaryotes Eukaryotes
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42.  Most eukaryotes except for budding yeast have ill-defined
origins of replication that rely on epigenetic mechanisms for
molecular recognition by initiator proteins.
 Replication is initiated at multiple origins along the DNA
using a conserved mechanism that consists of four steps:
origin recognition, assembly of a prereplicative initiation
complex, followed by activation of the helicase and loading of
the replisome.
(Sclafani and Holzen,2007)
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43. Uni or bidirectional
Replication forks move in one or opposite directions
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44. Replication Fork
 View of bidirectional movement of the DNA replication machinery
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45. Semi-discontinuous replication
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’
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46. DNA Replication Fork
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47. DNA synthesis only in 5’ 3’:
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48. Eukaryotic enzymes:
Five common DNA polymerases from mammals.
1. Polymerase  (alpha): nuclear, DNA replication, no proofreading
2. Polymerase  (beta): nuclear, DNA repair, no proofreading
3. Polymerase  (gamma): mitochondria, DNA replication,
proofreading
4. Polymerase  (delta): nuclear, DNA replication, proofreading
5. Polymerase  (epsilon): nuclear, DNA repair, proofreading
 Polymerases vary by species.
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49. Model of DNA Replication:
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50. Replication of circular DNA in E. coli:
1. Two replication forks
result in a theta-like
() structure.
2. As strands separate,
positive supercoils
form elsewhere in the
molecule.
3. Topoisomerases
relieve tensions in the
supercoils, allowing
the DNA to continue
to separate.50

51. 1. Common in several bacteriophages
including .
2. Begins with a nick at the origin of
replication.
3. 5’ end of the molecule is displaced and
acts as primer for DNA synthesis.
4. Can result in a DNA molecule many
multiples of the genome length
5. During viral assembly the DNA is cut
into individual viral chromosomes.51
Rolling circle model of DNA Replication:

52. End-replication problem:
 Every time a linear chromosome replicates, the laggaing strand at each end
gets shorter by about 150 nucleotides. Because there is a minimum length
of DNA needed for initiation of an Okazaki fragment.
 DNA polymerase/ligase cannot fill gap at end of chromosome after RNA
primer is removed. If this gap is not filled, chromosomes would become
shorter each round of replication.
Eukaryotes have tandemly repeated sequences at the ends of their
chromosomes.
Telomerase binds to the terminal telomere repeat and catalyzes the
addition of of new repeats.
Compensates by lengthening the chromosome.
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53. DNA Damage and Repair:
 DNA polymerase do great job during DNA replication by
proof reading the new DNA strand.
 But its not enough to maintain the 100% fidelity in
replication.
 Several kinds of damage occurs by endogenous and
exogenous agents.
 DNA has its own mechanisms to repair this damages and
maintain the accuracy of copying mechanism.
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Natural polymerase error
Endogenous DNA damage
oxidative damage
depurination
Exogenous DNA damage
radiation
chemical adducts
“Error-prone” DNA repair
Sources of
damage

55. DNA Damage Response(DDR):
 To respond to these threats, eukaryotes have evolved the
DNA Damage Response (DDR).
 The DDR is a complex signal transduction pathway that has
the ability to sense DNA damage and transduce this
information to the cell to influence cellular responses to
DNA damage.
(Ciccia and Elledge, 2010)
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56. “Mutation is rare because of repair”
Over 200 human genes known to be involved in DNA repair
Major DNA repair pathways:
1. Base excision repair (BER)
2. DNA Mismatch repair (MMR)
3. Nucleotide excision repair (NER)
4. DNA strand break repair pathways:
Single strand break repair (SSBR)
Double-strand break repair pathways (DSBR)
Homologous Recombination (HR)
Nonhomologous end joining (NHEJ)
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57.  Direct reversal of damage - Photoreactivation (bacteria, yeast,
some vertebrates - not humans) Two thymines connected together
by UV light.
 Excision Repair - removal of defective DNA. There are three
distinct types
 1) base-excision
 2) nucleotide-excision
 3) mismatch repair
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58. Base-excision repair(BER):
 Presence of the Uracil in DNA is a great example of this type
 Special enzymes replace just the defective base
 snip out the defective base
 cut the DNA strand
 Add fresh nucleotide
 Ligate gap
N
N
NH2
O
O
H2
C
O
O
N
H
N
O
O
O
H2
C
O
O
deoxycytosine deoxyuracil
1’
2’3’
4’
5’
12
3
4
5
6
CH3
thymine
glycosidic bond
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59. Nucleotide-excision repair(NER):
 Same as previous except that-
 It removes entire dmaged nucleotide
 Remove larger segments of DNA
Example:Xeroderma pigmentosum
• Extreme sensitivity to sunlight
• Predisposition to skin cancer
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60. Mismatch repair (MMR):
 Despite extraordinary fidelity of DNA synthesis, errors do
persist
 Such errors can be detected and repaired by the post-
replication mismatch repair system
 Special enzymes scan the DNA for bulky alterations in the
DNA double helix
 These are normally caused by mismatched bases
A G
A C
C T
 These are excised and the DNA repaired
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61.  MMR also processes mispairs that result from heteroduplex DNA
formed during genetic recombination: act to exclude
“homeologous” recombination.
 Repair involving two or more close sites in same heteroduplex
occur much more often on the same strand than the opposite
strands.
 Analysis of the pattern of repair suggest that repair tracks initiates
at mismatches and propagate preferentially in 5’ to 3’ direction.
(Wagner and Meselson, 1976)
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62. The problem of strand discrimination:
 MMR can only aid replication fidelity if repair is targeted to
newly synthesized strand
 The cell has a mechanism of identifying new strand synthesis by
leaving nicks that DNA. There are enzymes which scan these
new regions looking for errors.
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63. Other forms of DNA damage:
 Depurination - the base is simply ripped out of the DNA molecule
leaving a gap.
 Deamination - An amino group of Cytosine is removed and the
base becomes Uracil.
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64. Basic mechanism is the
same for all three types
1) Remove damaged
region
2) Resynthesis DNA
3) Ligate
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