Cell / Organism of the Day

Thermus aquaticus
• Lives in hot springs
• Grows at temperatures 65–70 °C (149–
158 °F)
• Can live at temperatures of 50–80 °C
(122–176 °F)
• In order to live at high temps, cells need
to make heat stable proteins.
• DNA polymerase from T. aquaticus is
used to replicate DNA in the lab via PCR
• PCR requires repeated cycles of heating
and cooling (heating to 95C)
• Heating to 95C would irreversibly
denature typical DNA polymerases – but
not Taq!
 Learning Objectives

DNA replication in vivo – inside cells

Targeted:
•Compare and contrast DNA replication in vitro (PCR) and in vivo (in cells).
•Place DNA replication within the cell cycle of a bacterial or eukaryotic cell.
•Explain the logistics of the DNA replication process including how the cell solves
the problems of:
• Separating the DNA strand.
• Replicating both strands simultaneously in the 5’ to 3’ direction.
• Synthesizing primers and providing primers for the leading and the
lagging strands during replication (DNA synthesis).
• Replicating the ends of the DNA.
•Distinguish between and label the leading and the lagging strands of DNA in a
replication fork.
•Explain what an Okazaki fragment is and its role in replication.
•Predict the types of non-covalent interactions that enable interactions between
DNA and DNA-binding proteins.

©2022_UBC_ K.Smith all rights reserved

 Function of DNA
Replication

DNA encodes all of the proteins

and RNA (rRNA, tRNA) in the cell to
allow for proper cell function.

New cells will need a copy of the

genome i.e. all DNA

“DNA replication in vivo” Shown here:

bacterial cells
means taking place in a living organism
©2022_UBC_ K.Smith all rights reserved
dividing by binary
fission
 DNA is copied by DNA Polymerase
Key Properties of DNA Polymerase:
• DNA Pol “reads” the sequence on a
template and links nucleotides together.
i.e., reads the template 3’ to 5’ and
synthesizes new DNA in the direction 5’ to
3’.
• DNA polymerase cannot “start on its
own”.
It must always start from an existing 3’OH
end of an existing template (e.g., a primer!)
In the cell, the primer is RNA.
©2022_UBC_ K.Smith all rights reserved
https://en.wikipedia.org/wiki/DNA_polymerase
 Polymerization
Monomers added to the Polymer

DNA polymerase
reads the template
strand 3’ – 5’.

The newly
synthesized strand
is elongated
(synthesized/grows)
5’ – 3’.

Activated monomers (triphosphates) are used to

provide energy to drive the reaction.
©2022_UBC_ K.Smith all rights reserved
Polymerization of DNA / RNA
1. Requires an enzyme (DNA or RNA Polymerase)
2. Requires a form of energy.
This energy form is a monomer with 2 or more phosphates
Most common forms = triphosphates
Can be ATP, CTP, GTP, TTP
5’ carbon of monomer
Shown here is
Adenine
triphosphate.
If Cytosine needs
to be added
then it would be
CTP etc

New monomer 3’ carbon of polymer

Polymerization of DNA or RNA:

The 5’ carbon of a new monomer is added to the 3’ carbon of the existing strand (or 1st
monomer). ©2022_UBC_ K.Smith all rights reserved
Why are monomers shown as a
triphosphate (3 phosphates:
ATP, CTP, GTP, TTP)?
This is called an
“activated
monomer”

-binding of three phosphates creates an unfavorable

state
(3 negative ions in close proximity).
-removing the outermost phosphates reduces free
energy of the system.
-the reaction is energetically favorable
©2022_UBC_ K.Smith all rights reserved
 DNA Replication
is semi-conservative

= “newly
synthesized
strand
Each cell will receive an
original template strand that
is base-paired with a newly
synthesized strand
(daughter). ©2022_UBC_ K.Smith all rights reserved
 Overview: The Process of
Replication

Semiconservative DNA replication: each new DNA duplex

consists of one strand that was originally part of the
parental duplex and one newly synthesized strand.
©2022_UBC_ K.Smith all rights reserved
 Rate of replication is ∼1000 base
pairs/second
Circular DNA Linear DNA

E. coli bacteria cell Human cell - eukaryote

Genome = 4.6 million Genome = 3.9 billion

base pairs base pairs
Time to replicate entire
genome with a single origin
of replication site

∼1 hour ∼45 days!

But! Takes a few hours due to multiple
origin of replication sites (called OriR)
©2022_UBC_ K.Smith all rights reserved
Know the function of
these enzymes
c DNA Pol. I DNA Ligase
D
b Primase

a Helicase

e
g f

Topoisomerase
h i

SSBPs DNA Pol. III ©2022_UBC_ K.Smith all rights reserved

 DNA replication

The replication bubble is shown by

# ______, and the replication fork is
A. 4, 2
shown by # _______. B. 3, 2
C. 4, 1
4 D. 3, 1
1

©2022_UBC_ K.Smith all rights reserved

For Practice:
Shown here: part of
the genome from a
cell

OriR (origin of replication)

5’ 3’

3’ 5’

On your paper/tablet:

1. Draw the replication bubble.

2. Label the ends of the DNA.
3. Draw the Leading strand (label the ends) on both sides.
4. Draw the Lagging strand showing Okazaki fragments
(label the ends) on both sides.
5. Draw an arrow showing the direction of replication of
each of the strands.
6. Draw an arrow that points to each replication fork.
©2022_UBC_ K.Smith all rights reserved
 OriR (origin of replication)

5’ 3’
l
3’ 5’

©2022_UBC_ K.Smith all rights reserved

Figures you should know
Replication Bubble – consists of 2 replication forks

Replication Fork Replication Fork

For each bubble,
there is one origin of
replication sequence
for both strands.

©2022_UBC_ K.Smith all rights reserved

 Replicating
Linear vs. Circular DNA

Easy-peasy! Bacterial cells

can replicate in a circle –both
directions! The ends will meet
up! Usually has 1 origin of Oh-oh. Eukaryotic DNA
replication (called OriC) can be big & linear so
need more than one
origin
©2022_UBC_ K.Smith all rights reserved of replication (OriR)
 To replicate DNA
Cell has three “problems” to solve:
1. Separate the DNA strands only a little at a time.
• Can’t separate an entire genome – very messy!
2. Make primers for DNA Polymerase.
• Cell makes a RNA primer - can start without a 3’OH.
3. Allow synthesis to happen simultaneously from the two
template strands
• DNA strands are anti-parallel yet DNA
polymerization has to occur in the 5’ – 3’ direction.
• Synthesis has to happen in opposite directions!

©2022_UBC_ K.Smith all rights reserved

 To replicate DNA
Cell has three “problems” to solve:

1.Separate the DNA strands only a little at a

time.
• Can’t separate an entire genome –
very messy!

Helicase
Unwinds the DNA at the replication fork.

©2022_UBC_ K.Smith all rights reserved

 To replicate DNA
Cell has three “problems” to solve:

2. Make primers for DNA Polymerase.

DNA Polymerase needs a primer (short sequence of RNA
or DNA) to synthesize DNA
Why? Needs 3’OH group to add on a new incoming
monomer (dNTP- nucleotide tri-phosphate TTP, CTP, GTP
or ATP)
Cannot start synthesis on its own.

Solution - RNA Primase

RNA polymerase – or RNA Primase makes RNA
primers. DNA Polymerase can add dNTPs to the 3’OH
end of this short RNA sequence.
©2022_UBC_ K.Smith all rights reserved
 Primers direct the start of
polymerization
No DNA synthesis shown here as there is no 3”OH to
add to!
DNA polymerase cannot
“start on its own.
It must always start from an
existing 3’OH end of an
existing template (e.g. a
primer!) In the cell, the DNA synthesis can start with DNA polymerase
III as there is a 3’ OH to add to
primer is RNA. Primase

Primase is an enzyme that

creates the primer!

©2022_UBC_ K.Smith all rights reserved

 To replicate DNA
Cell has three “problems” to solve:
3. Allow synthesis to happen simultaneously from the two
template strands
• DNA strands are anti- parallel, yet DNA
polymerization has to occur in the 5’ – 3’ direction.

• From the replication fork – synthesis occurs in the

opposite directions
i.e., in one direction on one strand and in the other
direction on the other strand
• But overall, replication must progress on both
strands in the direction of the growing replication
fork.
©2022_UBC_ K.Smith all rights reserved
 As the replication fork opens…

• At first, all is good – both strands i.e. leading and

lagging strands are synthesized at the replication
fork (although in opposite directions).
©2022_UBC_ K.Smith all rights reserved
 As the replication fork opens…
the lagging strand has a problem i.e. gap

The replication fork has “gap” 1st Okazaki fragment

opened further to the “left”
so need to start a new
Or lagging strand
Okazaki fragment

Leading strand

• Here is the problem – as the replication fork opens further, the “top”
strand has DNA Polymerase moving (synthesizing) to the right &
leaving a gap at the fork. See the red arrow
• SOLUTION – Add another Okazaki fragment to join the first fragment
©2022_UBC_ K.Smith all rights reserved
Okazaki Fragments to the
Rescue!

©2022_UBC_ K.Smith all rights reserved

 As the replication fork opens…
a 2nd Okazaki fragment is
added
i.e. RNA primer , then 1st Okazaki fragment
elongated by DNA Pol III.

©2022_UBC_ K.Smith all rights reserved

You should know how to label both sides of the
bubble i.e. each replication fork

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Both sides of the replication fork

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 Discontinuous vs
Continuous Synthesis
Leading strands are referred to as continuous synthesis
Lagging strands are referred to as discontinuous synthesis

©2022_UBC_ K.Smith all rights reserved

 DNA Pol I & DNA Ligase
As DNA replication proceeds, the cell will have to:
• Remove RNA primers and replace with nucleotides that are DNA
• Link all the fragments together to form one continuous strand.

• DNA polymerase I
• (a different polymerase
than DNA Pol III) removes
the RNA primer (red) and
replaces it with DNA.

• DNA ligase – joins all the

fragments together
-joins all Okazaki fragments
-joins leading and lagging strands
at the origin of replication (see
examples in pink circles) ©2022_UBC_ K.Smith all rights reserved
 Other “players” on the DNA
Replication “Team”
• Topoisomerase – relieves stress on the
winding helix.
• Single-stranded binding proteins –
keep the single stranded DNA apart.
• DNA polymerase I (a different
polymerase) removes the RNA primer
and replaces it with DNA.
• DNA ligase – joins the fragments
together.

©2022_UBC_ K.Smith all rights reserved

 DNA replication

The directionality of the top strand in this

example is… lagging leading

A. 3’ to 5’
B. 5’ to 3’
C. Can’t tell
lagging
leading DNA daughter strands

OriR

One replication bubble

©2022_UBC_ K.Smith all rights reserved

How is the lagging strand different from the
leading strand? Choose all that apply

1. Lagging strands are single-stranded.

2. Lagging strands use Primase, DNA Pol III,
SS-binding proteins, DNA Pol I, Ligase.
3. Lagging strands are synthesized in the
direction away from the direction of the
replication fork.
4. Lagging strands are synthesized in the 5’
to 3’ direction.
5. Lagging strands need many RNA primers.

©2022_UBC_ K.Smith all rights reserved