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Using a Viral Protein to Study DNA Replication

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.

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Using a Viral Protein to Study DNA Replication

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  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? RPA origin helicase PCNA Molecular Cell Biology. 4th ed. (2000) Lodish. RFC 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

  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 http://www.influenzareport.com/ir/pathogen.htm

  5. What’s special about Simian Virus 40? VP1 VP3 VP2 Agno Large T-ag Small t-ag • 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

  6. How do we use T-ag? Making mutations in the DNA sequence that encodes T-ag ACTGTTACG Results in T TGACAATGC A 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 E177K K178E K214Q H201N K178R Y211F H201F WT 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

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