Research Project
7779788
DESCRIPTION (provided by applicant): The maintenance of genome integrity in mitochondria is essential for cell survival as well as a major factor in the development of disease. We are interested in the effect that an active primase has on mitochondrial DNA (mtDNA) replication. Mitochondria replicate their genomes using a unique asynchronous process. A primase enzyme is required to replicate DNA in a manner similar to genomic DNA where leading- and lagging-strand synthesis is performed in a highly synchronized manner. The asynchronous mechanism of mtDNA replication has been postulated to be partially responsible for the high mutation rate of mtDNA due to the increased susceptibility of long-lived, single-stranded DNA intermediates to mutation. In comparison, the synchronous mode exposes a very small amount of single-stranded DNA during the replication cycle. Thus, we hypothesize that a replisome with a functional primase will result in a lower rate of mutation in the mitochondrial genome. We are interested in answering two questions: 1) Are there any naturally occurring mitochondrial primase-helicases that facilitate synchronous DNA replication?,and 2) Can the human mitochondrial replisome support synchronous DNA replication with the addition of an active primase? We have isolated and purified the mitochondrial DNA helicase from Arabidopsis thaliana and we have detected primase activity by this enzyme which is the first time ever for a mitochondrial helicase from a higher eukaryote. We are presently characterizing this primase domain structurally and biochemically. The modular nature of primase-helicases allows for the re-allying of primase and helicase activities by constructing chimeric proteins with domains from different organisms. For example, a chimeric protein will be fashioned from the active primase domain from the T7 gene 4 protein and the helicase domain from the human mitochondrial helicase. We are able to detect primase activity by reconstituted replisomes in two different ways: first, by using a mini-circle DNA construct where leading- and lagging-strand synthesis can be monitored independently, and, in a collaborative effort, using single-molecule microscopy to directly observe primase activity of individual replisomes. PUBLIC HEALTH RELEVANCE: Mutations in mitochondrial genomic DNA are associated with a wide range of human diseases as well as the ageing process. We propose to study mitochondrial DNA replication in order to resolve mechanisms of mutagenesis and, potentially, to find ways to decrease the rate of mutation in mitochondrial DNA.
