Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom.
PLoS Genet. 2019 Jan 3;15(1):e1007781. doi: 10.1371/journal.pgen.1007781. eCollection 2019 Jan.
Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3'-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.
人线粒体 DNA（mtDNA）复制首先在 H 链复制的起始点启动。该起始依赖于从上游启动子（LSP）转录生成的 RNA 引物。在这里，我们使用纯化的转录和复制因子在体外重建这一过程。大多数源自 LSP 的转录事件在大约 120 个核苷酸后过早终止，形成稳定的 R 环。这些新生的 R 环不能直接启动 mtDNA 合成，但必须首先被 RNase H1 处理，以产生可被 DNA 聚合酶 γ 用于起始 DNA 合成的 3' 末端。我们的发现与最近对敲除小鼠模型的研究一致，该研究表明 RNase H1 是体内 R 环处理和 mtDNA 维持所必需的。R 环形成和 DNA 复制起始都受到线粒体单链 DNA 结合蛋白的刺激。在 RNase H1 缺陷的患者细胞系中，mtDNA 复制的精确起始丢失，DNA 合成从线粒体控制区的多个位点起始。结合之前发表的体内数据，这里提出的发现表明了一种模型，即 RNase H1 通过 R 环处理指导人线粒体中 DNA 复制的特定起始。
