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Polymerase η Translesion Synthesis in Arabidopsis thaliana

Study on the role of DNA Polymerase η in Arabidopsis, comparing its efficiency in bypassing DNA lesions with humans and yeast. Methods include Western Blot and RT-PCR for quantifying expression levels.

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Polymerase η Translesion Synthesis in Arabidopsis thaliana

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  1. Polymerase η Translesion Synthesis in Arabidopsis thaliana Eric Brooks Mentor: Dr. John Hays Environmental and Molecular Toxicology

  2. 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.

  3. 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

  4. 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.

  5. 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).

  6. 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

  7. Experimental Design • Develop a method or several methods to reliably locate and quantify Pol η expression in Arabidopsis tissue extracts or in planta.

  8. 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.

  9. Results STD BSA Talon Eta

  10. 1 2 3 4 5 6 7 8 Results (Cont) 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

  11. 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 eta-specific in whole protein extract.

  12. 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 ηin humans, yeast and Arabidopsis.

  13. Acknowledgments Dr. John Hays Dr. Marc Curtis Peter Hoffman Dr. Kevin Ahern Frances Cripps and The Cripps Foundation The Howard Hughes Medical Institute

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