Isolating and Purifying DNA Polymerase ζ

Yesenia CorreaBiochemistry & BiophysicsMentor: Dr. John HaysEnvironmental and Molecular Toxicology

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Arabidopsis.html

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 Endogenous

damage: Oxygen radicals

produced from metabolic byproducts.

http://content.answers.com/main/content/wp/en/2/2f/DNA_UV_mutation.gifExogenous damage:

Ultraviolet radiationHuman made mutagenic chemicalsCertain plant toxins

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 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 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

Rev3

Rev7

cDNA available 648 base pairs 215 amino acids

cDNA not available 5673 base pairs 1890 amino acids

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 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

Express DNA

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.

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 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 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.

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

Isolating Rev7 Streaked for

isolation Cloned into

pET21a expression vector

Expressing Rev7

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Rev7 subunit expressed as a protein.

+) expression induced—) expression not induced

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