Today start on the material in Chapter 4.

1. DNA replication
2. Mutations causes and effects.
3. How genes move between cells in Bacteria
and Archaea: transposons, bacteriophage
(viruses), plasmids and “transformation”.

DNA replication.
1. Explain the logic of the process of DNA replication.
2. Explain the function of the following enzymes:
DNA polymerase III
helicase/primase
single strand binding proteins
DNA polymerase I
DNA ligase
3. Explain what an Okazaki fragment is and it’s role in replication
4. Explain how the Meselson Stahl experiment was set up
and how it determined the DNA replication is semi-
conservative.

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 The cell needs to make a duplicate of
this every generation: DNA replication

2 µm

DNA replication

Two parts.

1. Control of when to start the process of DNA

replication. Need to replicate exactly once
per cell cycle.

2. Enzymatic process of DNA synthesis. The

principles are simple: and we have already
covered them.

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Quick review of DNA structure:

Points:
1. DNA is double stranded.

2. Strands are anti-parallel (5’ to 3’ on one strand,

3’ to 5’ on the other).

3. Bases on the two strands are complementary.

5’ 3’ 5’
3’

3’
5’
5’ 3’

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 ating
An odd –and useful plic
com property of DNA polymerase.

DNA polymerase cannot “start on it’s own”.

It must always start from an existing 3’OH end.

(and therefore it only synthesizes in one direction)

DNA polymerase elongates this: 5’ OH 3’

3’ 5’

DNA polymerase will NOT use this: 3’ 5’

DNA polymerase will NOT use this: 5’ 3’

3’ 5’

Quick review of what we learned about DNA synthesis.

What happens in DNA amplification by PCR?

Starting point: template, DNA polymerase,

primers (dNTPs –not shown).

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 What happens in DNA amplification by PCR

95 C: template denatured

What happens in DNA amplification by PCR

60 C: primers
bind

Note: The two DNA strands are copied separately. A primer for each
strand is needed

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 What happens in DNA amplification by PCR.

72 C: DNA polymerase extends primers

g
l i c atin
An odd –and useful
p property of DNA polymerase.
com
Therefore:
To replicate DNA, we have three “problems” to solve

1. Separate the DNA strands

2. Allow synthesis to happen in one direction on one strand
and in the other direction on the other strand. The
strands need to be replicated separately.
3. Provide primers

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Here’s a diagram from the book that shows most of the
proteins needed for DNA replication in bacteria. Lets look at
what the proteins are needed for.

Problem 1. Need to separate the DNA strands

helicase

There is a specific enzyme called helicase that unwinds the

DNA strands.
Note that the DNA is copied right after the strands are unwound. This is
unlike the PCR experiment!
The enzyme DNA gyrase is needed to remove kinks from the DNA
as the DNA replication process continues.

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 Problem 1. Need to separate the DNA strands

SSB

There are specific proteins that are needed to keep the strands apart long
enough for the rest of the process.
These are called “SSB” which stands for single strand binding proteins.
Many of these proteins are used, they come off the DNA as the new
strands are synthesized.

Problem 2. Need to replicate the strands separately.

The enzyme complex that replicates DNA has lots of

subunits (two are shown). DNA polymerase III and
the β subunit the “sliding clamp”. Note that there are
two complexes: one for each of the strands being
replicated.
DNA polymerase III

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 Problem 2. The two strands need to be copied separately.

We need to consider the two DNA strands separately.

One of the two strands is called “the leading strand”. The
other is called “the lagging strand”.
We’ll look at the leading strand first.
Leading strand
3’ end

5’ end

5’ end
3’ end

Problem 3. Need to provide primers. The leading strand.

DNA synthesis on the leading strand is proceeding 5’ to 3’

continuously as we go left to right on the picture. The
template is 3’ to 5’.
We need a primer at the very beginning of the replication.
But once replication has started no additional primers are
needed.
3’ end of template

5’ end

5’ end of new strand

3’ end

We will need to return to “the very beginning of replication”.

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 Problem 2. The two strands need to be copied separately.
The lagging strand.

The DNA polymerase cannot bind to the 5’ end of the

lagging strand and copy it moving left to right in the diagram.
This would be “backwards”.

5’ end

3’ end of new strand 3’ end

Lagging strand
5’ end of template

Problem 2. The two strands need to be copied separately.

So the DNA polymerase has to move right to left in the

diagram. Remember in PCR this problem was solved by
completely separating the DNA strands. In a cell, the DNA
is too big to do this. So DNA synthesis using the lagging
strand is done in small pieces (called Okazaki fragments).

5’ end

3’ end of new strand 3’ end

Okazaki fragment
5’ end of template Note direction of synthesis

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Problem 3. Need to provide primers. The lagging strand.

Each time the DNA polymerase starts a new Okazaki

fragment it needs a new primer. These primers are made by
a specific enzyme “primase”. This primer is a short piece of
RNA (not DNA). The primase product stays on the DNA in a
helix (like the primer in PCR) and DNA polymerase can use
the 3’OH to start synthesis.

5’ end

3’ end

primase

Problem 2. The two strands need to be copied separately.

DNA polymerase synthesizes the new Okazaki fragment that

will end when it bumps into the previous one that was made.

5’ end

3’ end

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Problem 2. The two strands need to be copied separately.

As DNA synthesis proceeds, the RNA attached to the

Okazaki fragments is removed, leaving a gap that is filled by
a second DNA polymerase (DNA polymerase I). The
enzyme DNA ligase (we used this in our cloning experiment)
joins up the ends to make the new molecule.

5’ end

3’ end

Problem 2. The two strands need to be copied separately.

Here’s an alternate picture of the events with the Okazaki

fragments.
1. New RNA primer made by primase

2. DNA polymerase elongates a new Okazaki fragment

3. The previous Okazaki fragment primer is removed.

4. DNA polymerase I fills in the gap.

5. DNA ligase seals the single nucleotide gap to link fragment to the DNA

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•www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf

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