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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.

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isolating and purifying dna polymerase

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
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.
dna damage
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
dna damage4
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

polymerase and arabidopsis thaliana
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.
background
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.
rev7 and rev3
Rev7 and Rev3

Rev7

  • cDNA available
  • 648 base pairs
  • 215 amino acids

Rev3

  • cDNA not available
  • 5673 base pairs
  • 1890 amino acids
objective
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.
hypothesis
Hypothesis
  • Polymerase ζ in Arabidopsis thaliana is more effective at bypassing DNA damage than yeast polymerase ζ.
overview

Isolate DNA subunits

Clone DNA subunits

Purify the protein

Analyze polymerase ζ

Overview

Express DNA

isolating cdna for rev3
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

isolating rev3
Isolating Rev3
  • Attempted cloning the PCR product of the correct size but were unsuccessful.
  • The Rev3 gene is underrepresented in the cDNA libraries available.
plaque hybridization
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.
plaque hybridization14
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.
slide15

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

isolating rev7
Isolating Rev7
  • Streaked for isolation
  • Cloned into pET21a expression vector
expressing rev7
Expressing Rev7
  • Rev7 subunit expressed as a protein.

+) expression induced

—) expression not induced

what is next
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.
acknowledgements
Acknowledgements
  • Howard Hughes Medical Institute
  • Dr. John Hays
  • Pete Hoffman
  • Buck Wilcox
  • Dr. Kevin Ahern