1 / 19

Isolating and Purifying DNA Polymerase ζ

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. DNA Polymerases.

issac
Download Presentation

Isolating and Purifying DNA Polymerase ζ

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 Rev7 • cDNA available • 648 base pairs • 215 amino acids Rev3 • 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. Isolate DNA subunits Clone DNA subunits Purify the protein Analyze polymerase ζ Overview Express DNA

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

  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.jpghttp://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

More Related