isolating and purifying dna polymerase ζ yesenia correa biochemistry & biophysics mentor: dr....
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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.
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.