Testing the Role of Error-Prone DNA Polymerases in Genetic Instability of Gene Duplications

Project Summary

Organisms like Arabidopsis thaliana activate error-prone DNA polymerases when under extreme stress, such as conditions causing high damage to DNA. We used DNA alkylating agent, ethylmethane sulfonate (EMS), to mutagenize a gene duplication mutant of Arabidopsis, called bal. This duplication causes a dwarfed phenotype involving shrunken rosettes. Mutants are stable under natural conditions, but after treating mutant seeds with EMS, a high frequency of progeny show bal phenotype suppression. This project was designed to determine whether error-prone DNA polymerases are involved in the high number of suppressed progeny. My experiments involved planting mutagenized and non-mutagenized seeds of various Arabidopsis genotypes: bal control and bal/error-prone DNA polymerase double mutants. Adult plants were characterized based on number of individuals containing suppressed phenotypes (e.g., expanded, flat leaves; tall flowering stems). Effectiveness of EMS mutagenesis was evaluated by opening Arabidopsis siliques to score plants giving rise to aborted seeds.  This data confirmed mutagenesis induced lethal mutations in treated populations unlike mock-treated lines. As expected, EMS treatment produced a higher return in plants suppressing the balphenotype compared to seeds without mutagenesis. However, the difference in the return of the suppressed phenotype, between mutagenized seeds of bal control and bal/error-prone DNA polymerase double mutants, was not significant. These findings suggest these error-prone DNA polymerases are not involved in the high rate of recovery of suppressed bal phenotype plants after EMS mutagenesis. These error-prone polymerases may act redundantly, and it will be important to compare sector frequencies in further error-prone polymerase mutants.

My Experience

Being at Boyce Thompson Institute has set the bar high for future internships I will be involved in. This was my first summer research experience, and I have learned so much by being immersed in this community. It has conceptually been a great help to handle protocols and lab procedures that were once just words read in a textbook, words mentioned by my professors, or material that was not even a part of my curriculum. With the tremendous support of the members of the Richards lab, I have been making a transition in my way of thinking. Rather than just taking in information, they have helped me to apply it, and I have enjoyed the process. This experience has also given me a better understanding of paths my life can take with opportunities I now know are available. I am excited more than ever about what my future holds.