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). 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. 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 localize expression of DNA Polymerase η (Pol η ) in Arabidopsis To quantify Pol η expression levels in various plant tissues To quantify Pol η expression levels in various plant tissues

Background Since 1999 ten novel polymerases have been discovered. Since 1999 ten novel polymerases have been discovered. Now 19 eukaryotic DNA polymerases known to exist 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. 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. 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). 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. 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. Develop a method or several methods to reliably locate and quantify Pol η expression in Arabidopsis tissue extracts or in planta.

Methods Western Blot Western Blot –Generated antibody in rabbits against 16 aa peptide sequence found in the polymerase core RT-PCR RT-PCR –Quantify mRNA transcript levels using reverse transcriptase and PCR amplification techniques.

Results STDBSATalonEta

Results (Cont) : 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 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 eta- specific in whole protein extract. The antibody does not seem to be eta- specific in whole protein extract.

Work in Progress Use RT-PCR to specifically determine Pol η Use RT-PCR to specifically determine Pol η Perform crystallography on Arabidopsis Pol η and compare structure with humans and yeast. 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. 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