The Effect of Overexpressed AtOgg1 on Spontaneous Guanine-to-Thymine Mutation

1. The Effect of Overexpressed AtOgg1 on Spontaneous Guanine-to-Thymine Mutation Colin Tominey Dr. John Hays Oregon State University June 27, 2008

2. Background • All organisms experience a certain number of mutations due to chemical and environmental interactions with their DNA. • Point mutations have two general causes: • Chemical modification of a DNA base • DNA replication error

3. Arabidopsis as a Model Organism • Arabidopsis thaliana is often used as a model differentiating organism. • Advantages: • Short generation times (~6 weeks) • Small, well-documented genome • Transgenic varieties are easily created • Survives multiple gene knockouts

4. Arabidopsis and Mutation Research • Insertion of modified GUS genes into Arabidopsis created mutation-reporting plants. • Each line is designed to report one class of point mutation (e.g., G→T) • Reversion of the designated base reactivates the GUS gene and produces blue spots on that plant.

5. -C G T-G A A-A T C- -G C A-C T T-T A G- -C G T-G C A-A T C- -G C A-C G T-T A G- GUS Mutation Reporters wt GUS Codon 11: Active Protein GUS G→T Reporter Codon 11: Inactive Protein • When a mutation causes the reporter codon to revert to the wt codon, active protein is produced and plant cells are stained blue.

6. Unexpected Observation • Spontaneous reversion in the GUS G  T lines was much higher than that in other GUS plants:

7. Spontaneous Reversions

8. Potential Sources of G → T Mutation • 8-oxoguanine has been implicated in G → T mutation: • Pairs strongly with adenine, causing mutation during replication • Generated by reactive oxygen species attacking guanine in the DNA strand

9. GUS G→T Mutation Reporter Reversion -GCA- -CGT- -GCA- -CGT- O X O 8 O- -GAA- -CGT- O X O 8 Top strand retains the pre-existing codon. REPLICATION* Bottom strand forms a pre-mutagenic lesion. -GAA- -CTT- REPLICATION* Top strand results in fixation of mutation (reversion to wt) and production of active GUS protein. * Assumes replication without repair of the lesion.

10. Defending DNA • AtOgg1, AtFpg, and AtMyh all code for proteins that protect Arabidopsis. • Many bacteria and eukaryotes (including humans) have these same kinds of proteins. • Are these defenses sufficient?

11. Hypothesis • High levels of 8-oxoguanine have overwhelmed the plant’s natural defenses (OGG1, FPG, and MYH), thus causing the unexpectedly-high rate of spontaneous G → T mutation.

12. Prediction • Plants with enhanced AtOgg1 expression will show significantly less spontaneous G → T mutation than their siblings with normal AtOgg1 expression.

13. The Experiment • Create a line of Arabidopsis plants that overexpress the AtOgg1 gene. • Cross these mutants with existing GUS G → T reporter lines. • Compare spontaneous GUS reversion in new mutants with non-overexpressing siblings.

14. Acknowledgments • Howard Hughes Medical Institute • Oregon State University URISC • Dr. John Hays • Dr. Marc Curtis • Dr. Stephanie Bollmann • Peter Hoffman • Dr. Kevin Ahern