using a viral protein to study dna replication
DESCRIPTION
Using a Viral Protein to Study DNA Replication. Mrs. Foster University of Delaware Department of Biological Sciences. Eukaryotic DNA Replication: A well understood process?. RPA. origin. helicase. PCNA. Molecular Cell Biology. 4th ed. (2000) Lodish. RFC. - PowerPoint PPT PresentationTRANSCRIPT
Using a Viral Protein to Study DNA Replication
Mrs. FosterUniversity of Delaware
Department of Biological Sciences
Eukaryotic DNA Replication:A well understood process?
The Cell: A Molecular Approach. (2000) Cooper.
Molecular Cell Biology. 4th ed. (2000) Lodish.
PCNA
RPA
helicase
origin
RFC
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
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
http://www.influenzareport.com/ir/pathogen.htm
Large T-agSmall t-ag
Agno
VP1
VP2
VP3
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
www.protein.osaka-u.ac.jp
How do we use T-ag?
Re
sults in
Making mutations in the DNAsequence that encodes T-ag
Mutated T-ag protein
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.
In vitro DNA Replication
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 purifiedand run on a 1.5% agarose gel. Replicated DNA was visualized by autoradiography.
No
T-a
g
WT
E17
7K
K17
8E
K17
8R
H20
1F
H20
1N
Y21
1F
K21
4Q
Replication Intermediates
Covalently Closed Circular DNA
Form I
RIs
CCC
How I make mutant T-agPlasmid DNA containinggene for SV40 T-ag
Primer with mutated base
Denature/separate DNA
Allow primers DNA to attach
Copy the rest of the plasmid
Target for mutation
Enzymes