Primase Activity of the Mitochondrial Replisome

描述(由申请人提供)：保持线粒体基因组的完整性是细胞生存所必需的，也是疾病发展的主要因素。我们感兴趣的是有活性的引物酶对线粒体DNA(MtDNA)复制的影响。线粒体使用一种独特的异步过程复制它们的基因组。复制DNA需要一种底物酶，这种方式类似于基因组DNA，在基因组DNA中，领先和滞后链的合成是以高度同步的方式进行的。线粒体DNA复制的异步机制被认为是线粒体DNA高突变率的部分原因，因为长寿命的单链DNA中间体对突变的敏感性增加。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，我们假设具有功能启动子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)有没有天然存在的线粒体启动酶-解旋酶可以促进同步DNA复制？2)人类线粒体复制体能在增加活性启动子的情况下支持同步DNA复制吗？我们从拟南芥中分离纯化了线粒体DNA解旋酶，并用该酶检测到了底物酶的活性，这是首次从高等真核生物中检测到线粒体解旋酶。我们目前正在从结构和生物化学的角度描述这个初级酶结构域的特征。底物酶-解旋酶的模块化性质允许通过构建具有不同生物体结构域的嵌合蛋白来重新结合底物酶和解旋酶的活性。例如，T7基因4蛋白的活性启动域和人类线粒体解旋酶的解旋酶结构域将形成嵌合蛋白。我们能够通过两种不同的方式通过重组的复制体来检测启动酶活性：第一，通过使用迷你环DNA结构，其中领先和滞后链的合成可以被独立地监测，并且在合作的努力下，使用单分子显微镜来直接观察单个复制体的启动酶活性。 公共卫生相关性：线粒体基因组DNA突变与多种人类疾病以及衰老过程有关。我们建议研究线粒体DNA复制，以解决突变的机制，并潜在地寻找降低线粒体DNA突变率的方法。

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.