1. Isolating and Purifying DNA Polymerase ζ Yesenia Correa Biochemistry & Biophysics Mentor: Dr. John Hays Environmental and Molecular Toxicology http://users.rcn.com/jkimball.ma.ultranet/BiologyPages /A/Arabidopsis.html

2. DNA Polymerases  DNA polymerases are able to make accurate copies of DNA strands but in certain situations damaged areas can stop replication.  Translesion polymerases are specialized DNA polymerases that are able to synthesize DNA past a damaged template.

3. DNA Damage  Endogenous damage:  Oxygen radicals produced from metabolic byproducts. http://content.answers.com/main/content/wp/ en/2/2f/DNA_UV_mutation.gif  Exogenous damage:  Ultraviolet radiation  Human made mutagenic chemicals  Certain plant toxins

4. DNA Damage  Damage in cells causes:  Cell-cycle arrest  Apoptosis  Mutation  Unregulated cell division can lead to formation of a cancerous tumor http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/apoptosis.jpg

5. Polymerase ζ and Arabidopsis thaliana  Polymerase ζ is a translesion polymerase and is essential for life in mammals.  There is not very much known about the activity of polymerase ζ in higher eukaryotic organisms, because knockouts are lethal in mammals.  Isolating polymerase ζ in Arabidopsis thaliana would serve as a good model for working with polymerase ζ in human cells.

6. Background  Yeast polymerase ζ has been purified and studied biochemically, but human and Arabidopsis thaliana polymerase ζ have not been purified.  Polymerase ζ is a two subunit DNA polymerase containing Rev7 and Rev3.

7. Rev7 and Rev3 Rev3 Rev7  cDNA available  648 base pairs  215 amino acids  cDNA not available  5673 base pairs  1890 amino acids

8. Objective  To express the genes that together encode the DNA polymerase ζ.  To analyze the ability of polymerase ζ to extend primer sequences on normal and damaged DNA templates.

9. Hypothesis  Polymerase ζ in Arabidopsis thaliana is more effective at bypassing DNA damage than yeast polymerase ζ.

10. Overview Isolate DNA subunitsClone DNA subunitsPurify the proteinAnalyze polymerase ζ Express DNA

11. Isolating cDNA for Rev3  Attempted using PCR to amplify Rev3 using a cDNA library. 7/06/07 Appears to be correct size of the Rev3 gene.

12. Isolating Rev3  Attempted cloning the PCR product of the correct size but were unsuccessful.  The Rev3 gene is underrepresented in the cDNA libraries available.

13. Plaque Hybridization  A method used to screen a cDNA library to isolate a specific gene.  The cDNA library is combined with E.coli and plated on LB agar plates.  A nitrocellulose membrane is then placed on the LB agar and marked asymmetrically.  The nitrocellulose membrane serves as an identical copy for the plate.

14. Plaque Hybridization  A probe of a significant size is necessary to isolate the gene.  Using PCR and genomic DNA a probe for Rev3 was isolated.  This probe is then radioactively labeled.

15. http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2030.jpg

16. Isolating Rev7  Streaked for isolation  Cloned into pET21a expression vector

17. Expressing Rev7 Rev7 subunit expressed as a protein. +) expression induced —) expression not induced

18. What is next  Continue with plaque hybridization to isolate Rev3.  Proceed to clone Rev3 and assure the purity of the product.  Express Rev3 and Rev7 as polymerase ζ.

19. Acknowledgements  Howard Hughes Medical Institute  Dr. John Hays  Pete Hoffman  Buck Wilcox  Dr. Kevin Ahern