Travis Hunt and Dr. Brent Nielsen, Microbiology and Molecular Biology
The overall goal of the project was to study the role of DNA recombination in maintenance of plant mitochondrial DNA (mtDNA). Recombination is the process by which a molecule of DNA is nicked or broken and then the end is joined to a different but homologous DNA molecule.
This project is important because the mitochondria are the principle site of energy production in the cell. Mitochondria convert nutrients into energy as well as doing many other specialized tasks. Mitochondria contain DNA, which encodes only some of the proteins needed for mitochondrial function. By studying how and when mtDNA recombination occurs we can learn more about how the DNA is maintained, replicated and repaired when damage occurs. This research may help us understand mitochondrial genetic diseases associated with mutations in humans.
We narrowed our research to studying a particular protein found in the mitochondria of plants; this protein is called Twinkle. Twinkle primase/helicase is thought to be the single functional mtDNA primase in eukaryotes. Eukaryotes are organisms ranging from yeast to humans, which have nucleated cells. Twinkle is essential for mtDNA maintenance and may be involved in regulation of mtDNA copy number in animal cells. This protein functions both as a DNA primase to initiate DNA replication and as a DNA helicase to unwind the DNA for replication. Replication is the identical duplication of DNA.
Dr. Nielsen’s lab at BYU identified a putative mitochondrial-targeted Twinkle homologue in Arabidopsis thaliana. Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard family. To study the twinkle protein we did Western blot analysis using an antibody specific for a unique region of this protein. Western blot analysis confirmed that it is localized only to mitochondria. We did experiments on bacterial expressed recombinant protein to see if the twinkle protein had helicase and primase activity. We found that bacterial expressed recombinant protein has DNA helicase activity and DNA primase activity. Homozygous T-DNA insertion mutants in the promoter of this gene show significantly reduced growth of plants, which abort before flowering. An insertion in an exon near the middle of the gene results in less effect on plant growth.
We followed common molecular biology procedures to discover more about the Twinkle protein. T-DNA insertion mutant plants for the Arabidopsis putative primase/helicase homologue were analyzed for growth and development. Insertions in either the promoter or an early exon of the gene showed significant defects in plant growth. In biology, a promoter is a regulatory region of DNA located upstream of a gene. Western blot analysis showed localization of the protein in mitochondria but not chloroplasts. Western blot is a method of quantifying the amount of a protein of interest in a cell extract. The gene was overexpressed in E. coli and the protein was recovered and shown to have DNA helicase and DNA primase activities.
From our results, we have determined that T-DNA insertions in the primase/helicase gene cause growth defects. Our Western blot analysis shows the protein is targeted to mitochondria and is expressed in different ages of plants. To mention again the protein has been expressed in E. coli, which has allowed us to perform experiments in which we have found the protein has DNA helicase activity and DNA primase activity.
We are excited with the results that we have found so far and current work includes quantitative PCR to examine differences in mtDNA levels in mutant versus wild-type plants, western blot and reverse transcriptase PCR analysis of expression levels at various stages of growth, and transmission electron microscopy to examine differences in mitochondrial morphology in the mutants.
This is the first time that a single mitochondrial targeted protein has been identified and shown to have both primase and helicase activities. Primase/helicase proteins have been found previously in bacteriophages, viruses that attack and kill bacterial cells. It has been suggested that primase/helicase proteins are present in mitochondria but this is the first evidence for a mitochondrial protein with both activities. These findings have laid the ground work for further studies.
Doing research in a molecular biology lab was a valuable experience for me. I have graduated and I am beginning my first year of pharmacy school. The skills and knowledge that I gained from doing this research will help me in pharmacy school, and help prepare me to serve my community as a pharmacist.
References
- Manchekar Medha., et al. “DNA Recombination Activity in Soybean Mitochondria.” J. Mol. Biol. (2006) 356: 288-299.
- Shutt Timothy, Gray Michael. “Twinkle, the Mitochondrial Replicative DNA Helicase, Is Widespread in the Eukaryotic Radiation and May Also Be the Mitochondrial DNA Primase in Most Eukaryotes.” Journal of Molecular Evolution. (2005) 62: 588-599.
- Tyynismaa, Henna, et al. “Twinkle Helicase is Essential for mtDNA Maintenance and Regulates mtDNA Copy Number.” Human Molecular Genetics. (2004) 13: 3219-3227.