DNA Replication
Biology 12
•Cells can contain 6-9 feet of DNA. If all the DNA in your body
was put end to end, it would reach to the sun and back over 600
times.
•DNA in all humans is 99.9 percent identical. It is about one tenth
of one percent that makes us all unique, or about 3 million
nucleotides difference.
•DNA can store 25 gigabytes of information per inch and is the
most efficient storage system known to human. So, humans are
better than computers!!
•In an average meal, you eat approximately 55,000,000 cells or
between 63,000 to 93,000 miles of DNA.
•It would take a person typing 60 words per minute, eight hours a
day, around 50 years to type the human genome.
DNA is composed of units called NUCLEOTIDES, which are composed of three
sub-molecules:
1. Pentose Sugar (deoxyribose)
2. Phosphate
3. Nitrogen Base (purine or pyrimidine)
Two Kinds of Bases in
DNA
N
 Pyrimidines
are
single ring bases.
are
double ring
bases.
N
O
C
C
C
N
 Purines
C
N
N
C
C
C
N
C
N
N C
4
DNA is composed of two complimentary strands of
nucleotides joined by hydrogen bonds:
Adenine with Thymine (A-T or T-A)
They join with 2 hydrogen bonds
Cytosine with Guanine (C-G or G-C)
They join with 3 hydrogen bonds
DNA twists into a double helix
Chargraff’s Rule:
Adenine and Thymine
always join together
•
A
T
Cytosine and Guanine
always join together
•
C
6
G
Bonding 1
10
The bases always pair up in the same way to form base pairs.
Adenine forms a bond with Thymine
Adenine
Thymine
and Cytosine bonds with Guanine
Cytosine
Guanine
11
Bonding 2
PO4
PO4
adenine
thymine
PO4
PO4
cytosine
guanine
PO4
PO4
PO4
PO4
Replication
Copying the genetic
material is
REPLICATION.
Replication occurs prior to
cell division, because the
new, daughter cell will
also need a complete
copy of cellular DNA.
Replication
DNA Replication Problem Solving

On the surface….
The replication of
DNA is pretty
simple. Just unzip,
plug in the the spare
parts by
complementary
base pairing, and
stitch up the new
backbone.

There are a lot of
irritating details and
problems with this
process.

The solutions to
these problems
involve a list of vital
enzymes.
DNA Replication Complexities:
DNA is a very stable molecule but its
stability depends on its double stranded
nature. Single stranded DNA is vulnerable to
a number of kinds of damage. So…..to take
a huge DNA molecule and seperate its two
strands for its entire length....is a very bad
idea.
 So how can long DNA molecules be
replicated without making them single
stranded for long periods of time?


DNA is very long. VERY long. How can a
very long DNA molecule get itself replicated
in a relatively short period of time?

The primary DNA replication enzyme, DNA
Polymerase, is highly specific in a number of
ways. One of those specificities is that it can
only add new nucleotides to an already
existing growing strand of nucleotides. This
is a problem because it means that DNA
polymerase is not capable of actually
starting the process itself. How does
replication get started?

DNA polymerase is also highly specific in
that it can only build new polynucleotide
strands in the 5' to 3' direction--new strands
must always run from 5' to 3'. But the two
strands of DNA are antiparallel to each
other--one runs 3' to 5', the other from 5' to
3'. Both sides have to be replicated. How is
this puzzle solved in DNA replication?
What's the name of the scientist credited
with this discovery?

DNA is double stranded, and the two strands
twist around each other. These two strands
need to be pulled apart, thus tightening the
twist between Origin points. This would lead
to accidental breakage of the polynucleotide
strands as the twists got compressed into
smaller and smaller lengths. How is this
problem avoided in DNA replication? What is
the name of the enzyme needed to solve this
problem (it has several; any of them will do)?

Finally, the solutions to the previous
problems leave us with an embarrassing
problem: single stranded breaks in the
polynucleotides of our new DNA molecules.
How are these breaks repaired? Again,
you'll need to identify the enzyme involved.
DNA Replication Overview
1.
2.
3.

Enzyme breaks weak hydrogen bonds
DNA strands open up
Free nucleotides (from our food) fill in the open side
(free nucleotides are a significant component of the
nucleoplasm in any cell.

Using complimentary
base pairing
End Result:

2 identical DNA strands
How Does DNA Replicate?

Three Hypotheses:
Conservative
2 strands of
the parent
stay together,
daughter gets
new two
strands
SemiConservative
2 strands of parent
separate, daughter
gets 1 strand of
DNA from parent
Dispersive
New DNA is
made of a
random
Mixture of parent
and daughter
DNA
Meselson-Stahl
Parental (old)
DNA molecule



Discovered the
‘semi-conservative’ model
Experimented with
bacteria
Replication fork
(site of replication)
Daughter
(new) strand
Daughter
DNA molecule
(double helices)
Figure 10.6
Semi-Conservative
Replication
Origin of name:

Original parent
strands conserved
BUT are not still
attached together
End Result:

Each new daughter
DNA strand is ½
“old” and ½ “new”
Three Steps In DNA Replication

1. Initiation – Replication begins at a location
on the double helix known as “oriC” to which a
certain initiator proteins bind and trigger
unwinding. Enzymes known as helicases
unwind the double helix by breaking the
hydrogen bonds between complimentary base
pairs, while other proteins keep the single
strands from rejoining. The topoisomerase
proteins surround the unzipping strand and
relax the twisting that migh damage the
unwinding DNA.
2. Elongation


With the primer as the starting point for the leading
strand, a new DNA strand grows one base at a time.
The old (existing) strand is the template for the new
strand. The enzyme DNA polymerase controls
elongation, which can only occur in the leading
direction.
The lagging strand unwinds in small sections that DNA
polymerase replicates in the leading direction. The
resulting “Okazaki fragments” can contain between 100
to 200 bases. The fragments terminate in an RNA
primer that is later removed so that enzymes stitch the
back together into one long strand.
3. Termination
After the elongation is completed, two new
double helices have replaced the original
one.
 During termination the last primer must be
removed from the end of the lagging strand.
 Enzymes proofread the new double helix
and remove mispaired bases.

Step #1 Separating DNA Strands
Gyrase
Relieves tension by unwinding
Helicase
Breaks hydrogen bonds
Unzips the DNA
Terminates at fork
- Enzyme breaks the weak
hydrogen bonds
- Splitting the parent DNA strand
- Leaving two separated strands
Separating DNA Strands
SSBs (Single-stranded binding proteins)
Bind to the exposed DNA
 Keep from “annealing”

• Reattaching with complimentary base pair
The two sides of the molecule are
separated for a short distance.
Since DNA is most stable (and
least vulnerable to damage) in its
double stranded configuration,
as little of it as possible will be
single stranded at once.
DNA Replication

Priming:
1. RNA primers: before new DNA strands can
form, there must be small pre-existing
primers (RNA) present to start the addition of
new nucleotides (DNA Polymerase).
2. Primase: enzyme that polymerizes
(synthesizes) the RNA Primer.
DNA polymerase performs only one job, following the complementary
base pairing rule. It adds the new free nucleotides in the new strand
of a replicating DNA molecule.
This also means that DNA polymerase cannot actually start the process
of replication.
An enzyme called primase (an RNA polymerase) actually begins the
replication process. It builds a short piece of RNA called a primer. This
primer is later removed by RNAse H and replaced by DNA
nucleotides.
Step #2 Building Complimentary
Strands
Replication proceeds along
Both sides of the replication
fork.
- Free nucleotides with the assistance of a protein called
DNA polymerase, attach to original parent DNA strand
which serves as a template.
- Nucleotides in the new strand are selected using
complimentary base pairing
- A with T
and
C with G
DNA polymerase is only capable of
building a new strand from the 5’ end
to the 3’ end. This is a problem
because the two sides of the DNA are
antiparallel.
 One side of the new strand (called the
leading strand) can be directly and
continuously constructed from its 5’
end to its 3’ end.

DNA Replication

Synthesis of the new DNA Strands:
1. DNA Polymerase: with a RNA primer in
place, DNA Polymerase (enzyme) catalyze
the synthesis of a new DNA strand in the
5’ to 3’ direction.
5’
3’
Nucleotide
DNA Polymerase
RNA
Primer
5’
DNA Replication
2. Leading Strand: synthesized as a
single polymer in the 5’ to 3’
direction.
5’
3’
5’
Nucleotides
DNA Polymerase
RNA
Primer
Figure 5
Single-stranded binding proteins
Parent strand
Replication fork
III
The other strand (called the lagging
strand) must be constructed in short
segments, built backwards.
 These short strands of new DNA are
called Okasaki fragments after the
man who discovered them.
 The Okasaki fragments will eventually
be connected by an enzyme named
DNA ligase. DNA ligase specializes
in healing single stranded nicks in
DNA. It simply seals the bond
between one nucleotide and the
neighboring nucleotide.

DNA Replication
3. Lagging Strand: also synthesized in
the 5’ to 3’ direction, but
discontinuously
against overall direction of
replication.
Leading Strand
5’
3’
DNA Polymerase
3’
5’
RNA Primer
5’
3’
3’
5’
Lagging Strand
DNA Replication
4. Okazaki Fragments: series of short
segments on the lagging strand.
DNA
Polymerase
Okazaki Fragment
RNA
Primer
5’
3’
5’
3’
Lagging Strand
DNA Replication
5. DNA ligase: a linking enzyme that
catalyzes the formation of a covalent bond
from the 3’ to 5’ end of joining stands.
Example: joining two Okazaki fragments together.
DNA ligase
5’
Okazaki Fragment 1
3’
Okazaki Fragment 2
3’
5’
Lagging Strand
DNA ligase
 Joins Okazaki fragments together
Completes backbone
 Lagging strand only!

Building Complimentary
Strands
Leading strand
 Built continuously
 From parent 3’ end toward replication
fork
Building Complimentary
Strands
Lagging strand
 Built in short Okazaki fragments
between RNA primers
Discontinuous
 From parent 3’ end away from
replication fork

Single-stranded binding proteins
Okazaki fragments
Parent strand
Replication fork
III
Step 1
Step 2
Step 3
DNA is extremely long. It would take a
very long time to replicate the whole
molecule from end to end using only a
single replication fork.
 Each of these long molecules have
many sites called Origins. This forms
a configuration called a replication
bubble. Each replication bubble
actually has two forks, one on each
end of the bubble and travelling in
opposite directions.

Fork vs. Bubble
Origin of
replication
Replication Bubble:
 Begins at multiple
sites
 Produces multiple
forks
 Proceeds in both
directions
 Result:
 Faster replication
Origin of
replication
Origin of
replication
Parental strand
Daughter strand
Bubble
Two daughter DNA molecules
Figure 10.8
DNA cannot be double stranded and not twist. Unless
there is a way to relax the twists between replication
bubbles, the helical twists would compress putting
increasing stress on the molecule causing random breaking
and damage. This problem is solved by the creation of single
stranded breaks (swivels) between origin sites.
These nicks are made by an enzyme called
DNA topoisomerase (previously called “unwindase”,
then “swivelase”.
After the replication bubbles meet, ‘the single strands are
healed by DNA Ligase.
Proofreading
DNA polymerase I and III
 Check for proper base
pairing
 If mistake found:
Removes
 Replace with proper
nucleotide

End Result
- Results in two identical daughter DNA strands
- ½ new and ½ old
Leading
vs.
Lagging
Leading Strand
Lagging Strand
Toward
Direction
Away from
replication fork complimentary replication fork
strand is built
Yes
Built
No
continuously? (Okazaki fragments)
Only once
Number of
Multiple times
times RNA
(as replication
primers are
fork moves)
needed
Leading vs. Lagging

Lagging ONLY


DNA ligase completes backbone
bonds
Both:

Built from parent 3’ to 5’ end
• From daughter 5’ to 3’ end

Use DNA polymerase III & I
Leading vs. Lagging
Why do we need leading and lagging
strands?



DNA polymerase III can only build from
parent 3’ end
DNA runs antiparallel
When “unzipping”:


Leading: 3’ end IS available
Lagging: 3’ end IS NOT available
Human Genome

3 BILLION base pairs


In each cell = 46 DNA strands
Mistakes can occur with mismatched pairs

Called mutations
DNA Repair

If no repair
In sex cells  inherited diseases
 Example Tay-Sachs disease - the body
lacks hexosaminidase A, a protein that
helps break down a chemical found in
nerve tissue
 caused by a defective gene on
chromosome 15
 In somatic cells  cancer

Replication Mistakes

Change in the nucleotide base
sequence of a genome; rare.

Almost always deleterious
(bad). A deleterious mutation
has a negative effect on the
phenotype, and thus
decreases the fitness of the
organism. (A harmful mutation)
Rarely lead to a protein having
a novel property that improves
ability of organism and its
descendents to survive and
reproduce.

Effect of Mutation
Sickle cell anemia
is a disease passed
down through families
in which red blood
cells form an abnormal
sickle or crescent
shape. Red blood cells
carry oxygen to the
body and are normally
shaped like a disc.
Causes bone pain.