Functional Analysis of an Arabidopsis mitochondrial‐targeted DNA primase/helicase

Abstract

Twinkle primase/helicase is thought to be the single functional mitochondrial DNA (mtDNA) primase in eukaryotes. This protein is be 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. We have identified a putative mitochondrial‐targeted Twinkle homologue in Arabidopsis thaliana. Western blot analysis using an antibody specific for a unique region of this protein confirms that it is localized only to mitochondria. Bacterial expressed recombinant protein has DNA helicase activity and is being tested for 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. 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 research is funded by a grant from the NIH and by a BYU Mentoring Environments Grant.