A bifunctional mitochondrial DNA primase/helicase (AMH) identified in Arabidopsis thaliana: Preliminary biochemical characterization of its functions

Defects in mitochondrial DNA maintenance lead to dysfunction of mitochondria, which is implicated in a number of human diseases and even in the aging process. Replication of mammalian mitochondrial DNA is essential for maintaining normal genome level and proper organelle function, but the mechanism of replication remains elusive so far. Primary sequence analysis showed the DNA replication apparatus of mitochondria in mammals bears a striking resemblance to that of bacteriophage T7. The strongest similarity is between the mitochondrial DNA helicase and the T7 gene 4 protein (T7 gp4). T7 gp4 is a bi-functional protein in which the primase and helicase activities reside at two ends of the same polypeptide. Despite the high amino acid sequence identity to T7 gp4, studies have shown that the mitochondrial DNA (mtDNA) helicase in higher eukaryotes has only a vestigial primase region with deficient primase activity linked to a functional helicase domain. For example, TWINKLE -- which is the human mtDNA helicase -- has helicase activity, but its primase activities have not been detected; the situation is the same for primase activities of mtDNA helicase homologues from many other metazoan sources. The lost primase activities of mtDNA helicases is likely to be linked to the current mitochondrial genome replication model adopted in mammals, which is predicted to partly account for the high mutation rates in mitochondrial genome which are associated with aging and many severe diseases in humans.;In this study, we have detected primase activity by the mtDNA helicase found within Arabidopsis. It is the first report of detecting bi-functional primase/helicase activities in a mitochondrial protein from a higher eukaryote. Biochemical analyses were performed to characterize both the helicase activities and primase activities of this mitochondrial DNA helicase.;The bi-functional primase/helicase we have identified here from the mitochondria of Arabidopsis thaliana raises the possibility of determining the effect of an active primase on the human mitochondrial replisome. When added into the mammalian mitochondrial DNA replisome, it could potentially resume mtDNA synthesis in a mode similar to that in prokaryotes and might lead to a reduction of the high genome mutation rates. This provides an alternative way of thinking about the replication mechanism of mitochondrial DNA in mammals and also the pathogenesis of related human diseases and even the aging process.