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 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.
Exogenous
damage:
Ultraviolet
http://content.answers.com/main/content/wp/
en/2/2f/DNA_UV_mutation.gif
radiation
Human made mutagenic chemicals
Certain 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
Rev7



cDNA available
648 base pairs
215 amino acids
Rev3



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
Express DNA
Purify the protein
Analyze polymerase ζ
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


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

Rev7 subunit expressed as a protein.
QuickTi me™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
+) 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