Primase Activity of the Mitochondrial Replisome

描述（由申请人提供）：线粒体基因组完整性的维持对细胞存活至关重要，也是疾病发展的主要因素。我们对活性引物酶对线粒体DNA （mtDNA）复制的影响很感兴趣。线粒体通过一种独特的异步过程复制它们的基因组。需要引物酶以类似基因组DNA的方式复制DNA，其中前导链和滞后链的合成以高度同步的方式进行。由于长寿命的单链DNA中间体对突变的易感性增加，mtDNA复制的异步机制被认为是mtDNA高突变率的部分原因。相比之下，同步模式在复制周期中暴露了非常少量的单链DNA。因此，我们假设具有功能性引物酶的复制体将导致线粒体基因组的较低突变率。我们有兴趣回答两个问题：1)是否有任何自然发生的线粒体引物解旋酶促进同步DNA复制？2)人类线粒体复制体能否在添加活性引物酶的情况下支持同步DNA复制？我们从拟南芥中分离纯化了线粒体DNA解旋酶，并通过该酶检测了引物酶活性，这是首次从高等真核生物中检测到线粒体解旋酶。我们目前正在对这个引物酶结构域进行结构和生物化学表征。引物解旋酶的模块化特性允许通过构建来自不同生物体结构域的嵌合蛋白来重新整合引物酶和解旋酶的活性。例如，嵌合蛋白将由T7基因4蛋白的活性引物酶结构域和人类线粒体解旋酶的解旋酶结构域形成。我们能够通过两种不同的方式检测重组复制体的引物酶活性：首先，通过使用微型环状DNA结构，可以独立监测前导链和滞后链的合成，并且，在合作的努力下，使用单分子显微镜直接观察单个复制体的引物酶活性。

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.