Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

细胞代谢依赖于线粒体DNA（mtDNA）的调节和维持。每个细胞中存在数百至数千个 mtDNA 拷贝，但由于线粒体缺乏组蛋白或其他对核基因组压缩很重要的机制，因此 mtDNA 如何包装成单个核仁仍然悬而未决。在这项研究中，我们使用长读长单分子可及性作图来测量单个全长 mtDNA 分子在接近单核苷酸分辨率下的压缩。我们发现，与核基因组不同，人类 mtDNA 在很大程度上经历了全有或全无的全局压缩，大多数核素都处于难以接近的非活性状态。高度可及的线粒体核由转录和复制成分共同占据，并选择性地形成三链置换环结构。此外，我们还发现，初级类核相关蛋白 TFAM 在体内和体外直接调节不可接近的类核的比例，与成核和扩散机制一致地作用，以包裹和压缩线粒体核。这些发现共同揭示了人类细胞中 mtDNA 包装和调节的主要结构。