The document discusses the structure and replication of DNA. It describes how DNA is made up of nucleotides containing phosphate, sugar and a nitrogenous base. The nucleotides bond together via hydrogen bonds between complementary bases to form the DNA double helix structure. DNA replication involves unwinding the double helix, synthesizing new strands complementary to each original strand, and resulting in two identical copies of the original DNA molecule. Key enzymes involved in replication include helicase, DNA polymerase, primase, ligase and others.

1. GUMEDE ESTHER NTOMBIFUTHI 3RD YEAR
UNIVERSITY OF JOHANNESBURG 2014

2. NUCLEIC ACIDS :

3. DNA Structure
DNA consists of two molecules that are
arranged into a ladder-like structure called a
Double Helix.
A molecule of DNA is made up of millions of
tiny subunits called Nucleotides.
Each nucleotide consists of:
1. Phosphate group
2. Pentose sugar-Deoxyribose
3. Nitrogenous base

4. Nucleotides
Phosphate
Nitrogenous
Base
Pentose
Sugar

5. DNA Structure Helps
Explain How It Duplicates
DNA is made up of two nucleotide
strands held together by hydrogen bonds
Hydrogen bonds between two strands
are easily broken
Each single strand then serves as
template for new strand

6. DEOXYRIBONUCLEIC ACID
DNA usually exists as a double-stranded
structure, with both strands coiled
together to form the characteristic
double-helix.
Each single strand of DNA is a chain of
four types of nucleotides having the
bases:
Adenine
Cytosine
Guanine
Thymine
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7. Nucleotides
The phosphate and sugar form the
backbone of the DNA molecule, whereas the
bases form the “rungs”.
There are four types of nitrogenous bases.

8. Orientation of
DNA on the sugar ring are
The carbon atoms
numbered for reference. The 5’ and 3’
hydroxyl groups (highlighted on the left) are
used to attach phosphate groups.
 The directionality of a DNA strand is due to the orientation of
the phosphate-sugar backbone.

9. Nucleotides
A
Adenine
C
Cytosine
T
Thymine
G
Guanine

10. Nucleotides
Each base will only bond with one other
specific base.
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
Form a base pair.
Form a base pair.

11. DNA Structure
A gene is a section of DNA that codes for a
protein.
Each unique gene has a unique sequence of
bases.
This unique sequence of bases will code for the
production of a unique protein.
It is these proteins and combination of proteins
that give us a unique phenotype.

12. DNA
Gene
Protein
Trait

13. A Nucleoside is a combination of
Pentose sugar & Nitrogen Base
A Nucleotide is a combination
of nucleoside & Phosphoric Acid
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14. P
T
A
5’
C
G
P
3’
P
DNA has directionality.
P
P
P
C
Two nucleotide chains
together wind into a helix.
G
A
P
P
T
P
P
G
Hydrogen bonds between
paired bases hold the two
DNA strands together.
C
P
P
G
3’
C
DNA strands are antiparallel.
P
P
A sugar and phosphate
“backbone” connects
nucleotides in a chain.
5’

15. DNA BACKBONE
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16. Nucleotides are matched between strands
through hydrogen bonds to form base pairs.
Adenine pairs with thymine and cytosine
pairs with guanine
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17. These terms refer to the
carbon atom in
deoxyribose to which the
next phosphate in the
chain attaches.
Directionality has
consequences in DNA
synthesis, because DNA
polymerase can synthesize
DNA in only one direction
by adding nucleotides to
the 3' end of a DNA strand.
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18. The pairing of bases in DNA
through hydrogen bonding
means that the information
contained within each strand is
redundant. The nucleotides on a
single strand can be used to
reconstruct nucleotides on a
newly synthesized partner
strand.
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19. Functions
DNA is used to store genetic information
It is replicated before cell division
DNA is very important so it is stored in
the nucleus.
It never leaves the nucleus
Your DNA stores the code for your
proteins, which exhibit your “traits”
The DNA gets converted to RNA in order
to move out into the cytoplasm

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21. DNA replication is a biological
process that occurs in all living
organisms and copies their exact
DNA. It is the basis for biological
inheritance.
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22.  Each old strand
stays intact
 Each new DNA
molecule is half
“old” and half
“new”
Fig. 1-7, p.212

23. The first major step for the DNA
Replication to take place is the
breaking of hydrogen bonds between
bases of the two antiparallel
strands.
The unwounding of the two strands is
the starting point. The splitting
happens in places of the chains
which are rich in A-T. That is
because there are only two bonds
between Adenine and Thymine.
There are three hydrogen bonds
between Cytosine and Guanine.
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25. Helicase is the enzyme that
splits the two strands. The
structure that is created is
known as "Replication
Fork".
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26. 03/07/14
In order for DNA replication to begin,
the double stranded DNA helix must first
be opened. The sites where this process
first occurs are called replication origins.
Helicase unwinds the two single strands 26
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27. Replication
3’
3’
5’
5’
3’
5’
3’
5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.

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

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

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

31. rk
o
F
o n The replication fork is a structure that
ti
forms within the nucleus during DNA
ca
i
pl
replication. It is created by helicases,
e
R
which break the hydrogen bonds
holding the two DNA strands together.
The resulting structure has two
branching "prongs", each one made up
of a single strand of DNA.
These two strands serve as the template
for the leading and lagging strands,
which will be created as DNA
polymerase matches complementary
nucleotides to the templates; The
templates may be properly referred to
as the leading strand template and the
lagging strand template
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32. 3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.

33. 3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

34. 3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’
5’

35. 3’
5’
3’
5’
3’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

36. 5’
3’
3’
5’
3’
3’5’
5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.

37. 3’
3’
5’
3’
5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate
backbone.

38. One of the most important steps of DNA
Replication is
the
binding
of RNA
Primase in the initiation point of the 3'-5'
parent chain.
RNA Primase can attract RNA nucleotides
which bind to the DNA nucleotides of the 3'-5'
strand due to the hydrogen bonds between
the bases. RNA nucleotides are the primers
(starters) for the binding of DNA nucleotides.
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39. In the lagging strand the DNA Pol IExonuclease- reads the fragments and
removes the RNA Primers. The gaps are
closed with the action of DNA
Polymerase which adds complementary
nucleotides to the gaps and DNA
Ligase which acts as a glue to attach
the phosphate to the sugar by forming
phosphodiester bond.
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40. Enzymes in Replication
Enzymes (Helicases) unwind the two
strands
DNA polymerase needed for the synthesis of
complementary strand
DNA ligase joins pieces of the lagging strand
together

41. Helicase unwinds
parental double helix
DNA polymerase
binds nucleotides
to form new strands
Binding proteins
stabilize separate
strands
Exonuclease removes
RNA primer and inserts
the correct bases
Primase adds
short primer
to template strand
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone

42. DNA Replication models
There are three possible models that
describe the accurate creation of the
daughter chains:
Semiconservative Replication
Conservative Replication
Dispersive Replication

43. Figure 11.2
11-6

44. Replicatoin can’t just start:
1. All DNA polymerases need a primer
2. The primer can be a piece of RNA or DNA
3. It must be “base-paired” with the “template” and with
primer
3’OH
Thus:
5’
3’
3’
Synthesis direction
5’
Template strand

45. “Proof-reading” is essential at DNA replication
•What is proof-reading?
1. If there is a wrong base built in, then there is
no base paring possible.
2. The DNA polymerase can’t continue on
building in the next base.
3. The DNA polymerase removes the “wrong” base
and starts over

46. Klenow Fragment (of pol I)
(The proof-reading activity)

47.  Bacterial DNA polymerases may vary in their
subunit composition

However, they have the same type of catalytic subunit
Structure resembles a human
right hand
Template DNA thread
through the palm;
Thumb and fingers
wrapped around the DNA

48. Proteins involved in E. coli replication
Terms to be known

49. DNA replication mistakes
The most errors in DNA sequence occur during
replication.
Reparation takes place after replication is
finished
DNA polymerases can get the right sequence
from the complementary strand and repair,
along with DNA ligase, the wrong bases.

50. Is a method in which multiple repetitions of DNA
replication are performed in a test tube.
Mix in test tube:
DNA template
DNA to be amplified
Primers
one complementary to each strand
Nucleotides
dATP,d GTP, dCTP, and dTTP
DNA polymerase
heat stable form
from thermophilic bacteria

51. DNA template is denatured with heat to separate strands.
5’
3’
G
C
5’
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’

52. DNA template is denatured with heat to separate strands.
5’
3’
G
A
C
T
A
G
C
G
C
5’
A
T
T
G
A
T
C
G
C
3’

53. Each DNA primer anneals, binding to its
complementary sequence on the template DNA
5’
3’
G
C
A
T
A
T
5’
C
T
A
G
C
G
3’
5’
3’
C
5’
T
T
G
A
T
G
C
C
G
G
C
3’

54. DNA polymerase creates a new strand of DNA
complementary to the template DNA starting from the
primer.
3’
G
C
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’
5’
5’
3’
G
C
5’
5’
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’

55. Denaturation
DNA template is denatured with
high heat to separate strands.
Annealing
Each DNA primer anneals, binding
to its complementary sequence
on the template DNA
Extension
DNA polymerase creates a
new strand of DNA complementary
to the template DNA
starting from the primer.
Multiple rounds of denaturation-annealing-extension are
performed to create many copies of the template DNA
between the two primer sequences.

56. Visualization of PCR products

57. agarose gel-electrophoresis of DNA
Top
Negative pole
DNA is
Negatively charged
Small molecules
dissolve faster
separation based
on size
Bottom
Positive pole
Slab of agarose gel (ethidium bromide staining)