1. Using a Viral Protein to Study DNA Replication
Mrs. Foster
University of Delaware
Department of Biological Sciences

2. Eukaryotic DNA Replication: A well understood process?
Molecular Cell Biology. 4th ed. (2000) Lodish.
PCNA
RPA
helicase
origin
RFC
All cells have DNA
During normal cell growth and division, our cells need to make more DNA so that new cells will have the same amount.
DNA replication is a very complex and regulated process within the cell. It initiates at certain sequences of DNA called origins of replication and many proteins are required to carry out the process.
So far we know that there are many different origins of replication within our DNA and we know of most of the proteins involved. The question lies in exactly how this process is carried out on the DNA and how the proteins specifically interact to do their jobs.
The Cell: A Molecular Approach. (2000) Cooper.

3. How can we overcome the problem of studying complex systems?
Modeling: Use a simplified system to study a complicated process
Examples of models:
Animal models to study human body systems
Bacterial and viral models to study eukaryotic cell processes
With this great problem of complex processes such as DNA replication, how then can we study and learn more about these processes?
Use a technique called modeling
Essentially we can study the simple model system and make inferences into how more complex systems will work.
In my lab, I am using a virus as a model system to study DNA replication, specifically the monkey virus SV40.

4. Why use viruses to study DNA replication?
Viruses can’t replicate their DNA alone, they rely on infecting other cells
Viruses use cell proteins for replication
Ori-unlike euk. With many uncharacterized oris
T-ag- unlike euk. Rep. SV40 initiation requires few proteins including the only viral protein needed T-ag all others recruited from the host cell
Structure- supercoiled ds DNA assoc. w/ histones H2A, H2B, H3 H4 forming a minichromosome structure

5. What’s special about Simian Virus 40?
Structure of SV40 chromosome comparable to eukaryotic DNA
Single, well-defined origin of replication
T-ag is the only viral protein required
Large T-ag
Small t-ag
Agno
VP1
VP2
VP3
Ori-unlike euk. With many uncharacterized oris
T-ag- unlike euk. Rep. SV40 initiation requires few proteins including the only viral protein needed T-ag all others recruited from the host cell
Structure- supercoiled ds DNA assoc. w/ histones H2A, H2B, H3 H4 forming a minichromosome structure

6. How do we use T-ag? ACTGTTACG T TGACAATGC A
Making mutations in the DNA
sequence that encodes T-ag
ACTGTTACG T
TGACAATGC A
Results in
Mutated T-ag protein

7. What about my research?
I am making mutations to investigate the ability of T-ag to interact with DNA
By mutating these amino acids, I hope to disrupt DNA binding thus proving the function of these amino acids.

8. In vitro DNA Replication
No T-ag WT
E177K
K178E
K178R
H201F
H201N
Y211F
K214Q
RIs
Replication Intermediates
CCC
Covalently Closed Circular DNA
Form I
Reactions include origin containing plasmid DNA, 1.5 µg FL T-ag, 293 cell
extract supplemented with 100 ng Topo I and 32P labelled dCTP.
After 1 hr incubation at 37C reactions were stopped and DNA was purified
and run on a 1.5% agarose gel. Replicated DNA was visualized by
autoradiography.

9. Thank you!

10. How I make mutant T-ag Plasmid DNA containing gene for SV40 T-ag
Target for mutation
Primer with mutated base
Denature/separate DNA
Allow primers DNA to attach
Copy the rest of the plasmid
Enzymes