Student Case Study

Case Study - How does DNA replicate?

Introduction

The following is a case study that is designed to support you in answering the
question How does DNA replicate? It is assumed that you already know the
structure of DNA and you now want to find out how DNA makes copies of
itself during cell division.

The case study is designed to provide you with an opportunity to find out the
answer to the question yourself, using the real experimental data from the
scientists who first successfully answered it in 1958.

It is intended that by completing this case study you should not only learn the
mechanism by which DNA replicates, but should also have had the
opportunity to develop the scientific enquiry skills.

Learning outcomes

Higher Biology
1. b. (i) DNA can direct its own replication.

Useful weblinks

http://www.sumanasinc.com/webcontent/animations/content/meselson.htm
l
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?
it=swf::535::535::/sites/dl/free/0072437316/120076/bio22.swf::Meselson
%20and%20Stahl%20Experiment
http://www.learnerstv.com/animation/animation.php?ani=20&cat=biology
http://www.dnalc.org/view/15331-Proposed-models-of-DNA-replication-
Matthew-Meselson-.html
http://www.dnalc.org/view/15880-Models-of-DNA-replication.html
http://www.dnalc.org/view/15879-Semi-conservative-replication.html
HOW DOES DNA REPLICATE?

Revision: What is the structure of DNA?

Activity 1

In April 1953 Francis Crick and James D.

Watson had a paper published in the Journal
Nature entitled A Structure for Deoxyribose
Nucleic Acids.

In this paper they wrote:

We wish to put forward a radically different

structure for the salt of deoxyribose nucleic
acid. This structure has two helical chains
each coiled around the same axis. Watson & Crick
Image from Preoccupation
Watson and Crick (1953)

Use this information and your prior learning to draw an annotated

diagram of Watson and Cricks structure of DNA.

Activity 2

So, we know the structure of DNA but why does it replicate?

Why does DNA need to be able to copy itself?

Activity 3

DNA replication occurs as part of a process that occurs in nearly every cell in
our bodies.

Name this process and summarise it as a diagram in your jotter.

 Student Case Study

Hypothesising: How might DNA replicate?

Activity 4

In the same 1953 paper, Watson and Crick went on to say:

If an adenine forms one member of a pair, on either chain, thenthe other

member must be thymine; similarly for guanine and cytosineIt 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)

DNA can direct its own replication, but how does this happen? Watson and
Crick hinted at a possible mechanism, but they were not able to supply any
experimental evidence to support their theory. What do you think? How does
one DNA molecule turn into two?

Perhaps an entire new DNA molecule is made from an old DNA molecule,
like this:

In this hypothesis, the

two new DNA strands
DNA occur together in one
REPLICATION daughter cell, the two
old in the other.
Conservative model
old DNA old DNA new DNA

There are at least two other possible hypotheses to explain how new DNA
strands might be made. Draw these in your jotter. Once you have drawn
the other models, describe them and try to find out their names.
HOW DOES DNA REPLICATE?

Experimental procedure: How can we find out how DNA

replicates?

Activity 5

Matthew Meselson and Franklin W. Stahl set

out to answer this very question. In 1958
they published a paper that outlined their
experimental procedure for determining the
mechanism of DNA replication. They
introduce the paper by saying:

Hypotheses for the mechanism of DNA Meselson & Stahl

replication differ in the predictions they Image from achievement.org

make concerning the distribution among

progeny molecules of atoms derived from parental molecules. Radioisotopic
labels have been employed in experiments bearing on the distribution of
parental atoms among progeny molecules in several organisms. We
anticipated that a label which imparts to the DNA molecule an increased
density might permit an analysis of this distribution by sedimentation
techniques.
Meselson and Stahl (1958)

What does this mean? Look up any words you do not understand and
explain in your own words what Meselson and Stahl are saying in the
above quote.

Activity 6

Meselson and Stahls experiment relied on the different properties of the two
isotopes of nitrogen, 14 N and 15 N. Isotopes can be distinguished on the basis
of their mass: 14 N is lighter than 15 N; neither of these isotopes is radioactive.

Why is it important that these isotopes are not radioactive?

Activity 7

Meselson and Stahl grew bacteria in a medium where their nitrogen source,
used to synthesise nucleotides and eventually DNA, was the ammonium ion
(NH 4 + ) containing heavy 15 N. After several generations, all the DNA in these
bacteria would be heavy.

Where are the nitrogen atoms in DNA?

 Student Case Study

The parental bacteria were then placed in a fresh medium containing light
( 14 N) ammonium ions and allowed to divide and copy their DNA just once to
produce generation 1. A sample removed now from this beaker would contain
generation 1 DNA.

Some of the generation 1 bacteria were then allowed to divide once more in
14
N medium to produce generation 2 bacteria.

Bacteria Bacteria DNA sample taken

cultured in transferred after 20 minutes.
medium to medium Generation 1
containing containing
heavy light
nitrogen nitrogen DNA sample taken
(15N) (14N) after 40 minutes.
Generation 2

To find out which form of replication is occurring, Meselson and Stahl

isolated and separated the DNA from generation 1 and generation 2. This was
done by density gradient centrifugation. This technique allows the separation
of molecules on the basis of their density.

After centrifugation, the tubes were irradiated with ultraviolet light. DNA
absorbs this light so the position of the DNA in the centrifuge tube shows up
as a black band.

Light 14 N DNA is found at the top of the centrifuge tube and heavy 15 N DNA
is located at the bottom of the tube. Any mixture of 14 N and 15 N DNA is found
in the centre of the tube.

light 14N DNA

14
N and 15N hybrid DNA
heavy 15N DNA
HOW DOES DNA REPLICATE?

Prediction: What would you expect to happen?

Before you find out which of the models described above is the correct one,
you must first work out what actual results Meselson and Stahl would have
obtained for each of the predicted models.

Activity 8

In the table below, draw the expected centrifuge tube for each of the
predictions for generation 1 and generation 2. The centrifuge tubes for
the conservative model have been done for you.

Prediction
Semi-
Model Conservative Dispersive
conservative

Generation 1

Generation 2

Activity 9

On the following page you will find the actual results from Meselson and
Stahls experiments. On their diagram the heavier DNA is found on the
right.

They conducted a range of experiments. You will need to identify which of

two results show generations 1 and 2. Compare Meselson and Stahls
results with your own predictions above to determine which of the models
of DNA replication their experiment supports.
Student Case Study
HOW DOES DNA REPLICATE?

Analysis: Which model of DNA replication is supported?

Activity 10

Which is the correct model of DNA replication?

Explain how you arrived at this conclusion.

Homework

How does DNA replicate?

You now need to demonstrate your understanding of how DNA replicates and
take your understanding further.

Your task is to prepare a resource that could be used by students learning

about DNA replication using the following as your title:

How does DNA replicate?

It is up to you to choose how to present your understanding but through your

resource you must:

demonstrate a clear understanding of semi-conservative DNA

replication
explain why the data from Meselson and Stahls experiment supports
semi-conservative DNA replication over other hypotheses
outline the steps involved in semi-conservative DNA replication.

You should consider the following questions when preparing your resource:

Why and when does DNA replicate?

What is the model of DNA replication?
What evidence supports this model of DNA replication?
How does the structure of DNA relate to the mechanism of replication?
Which enzymes catalyse DNA replication?
What is the sequence of steps involved in DNA replication?

Use your jotter to plan your resource.

 Student Case Study

Extension: How was DNA polymerase discovered?

Now that you know how the model of DNA replication was tested, what about
the enzyme which catalyses DNA replication DNA polymerase? What do
we know about this enzyme? Who discovered it? When did they discover it?
What methods did they use?

Find out what you can about the discovery of DNA polymerase and
summarise your findings.

Useful weblinks

http://www.jbc.org/content/278/37/34733.full?
maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRS
TINDEX=30&resourcetype=HWCIT
http://www.dnalc.org/resources/nobel/kornberg_a.html