Polymerase η
Translesion Synthesis in
Arabidopsis thaliana
Eric Brooks
Mentor: Dr. John Hays
Environmental and Molecular Toxicology
Relevance
In humans there is a metabolic disease
associated with nonfunctional Pol η known
as xeroderma pigmentosum variant (XPV).
 XPV patients are hypersensitive to UV
induced DNA damage leading to skin
cancer.

Goals of the Study

To localize expression of DNA Polymerase
η (Pol η) in Arabidopsis

To quantify Pol η expression levels in
various plant tissues
Background
Since 1999 ten novel polymerases have
been discovered.
 Now 19 eukaryotic DNA polymerases
known to exist
 Once recently discovered family is the
translesion synthesis polymerases. Pol η is
a member of this family.

Background Cont.
Translesion polymerases are capable of
inserting nucleotides into the growing strand
opposite template DNA lesions.
 Pol η especially proficient at bypassing
cyclobutadiene pyrimidine dimers (CPDs).

Hypothesis

Polymerase  in plants is more efficient at
bypassing DNA photoproducts than its
Human or Yeast homologues.
– Based on this hypothesis we would predict
significant differences in the crystal structures
– There may also be differences in expression
patterns
Experimental Design

Develop a method or several methods to
reliably locate and quantify Pol η
expression in Arabidopsis tissue extracts
or in planta.
Methods

Western Blot
– Generated antibody in rabbits against 16 aa
peptide sequence found in the polymerase
core

RT-PCR
– Quantify mRNA transcript levels using reverse
transcriptase and PCR amplification
techniques.
Results
STD BSA Talon Eta
Results (Cont)
1
2
3
4
5
6
7
8
1: Standards
2: BSA
3: Root Extract
4: Root Extract
+eta spike
5: Meristem
Extract
6: Meristem
Extract + eta
spike
7: Leave Extract
8: Leave Extract +
eta spike
Conclusions
The antibody binds to the Arabidopsis eta
protein with some specificity (e.g. it does
not seem to have a high affinity for BSA)
 The antibody does not seem to be etaspecific in whole protein extract.

Work in Progress
Use RT-PCR to specifically determine Pol η
 Perform crystallography on Arabidopsis Pol
η and compare structure with humans and
yeast.
 Compare tissue-specific and overall
expression of Pol η in humans, yeast and
Arabidopsis.

Acknowledgments
Dr. John Hays
Dr. Marc Curtis
Peter Hoffman
Dr. Kevin Ahern
Frances Cripps and The Cripps Foundation
The Howard Hughes Medical Institute