The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [TruSeq]

Single-stranded DNA (ssDNA) binding proteins protect regions of ssDNA formed during processes such as DNA replication and repair. We here devise a genetic screen and identify the mitochondrial ssDNA-binding protein (mtSSB) as a key regulator of mtDNA levels. In mitochondria, RNA synthesis from the light-strand promoter (LSP) is required for transcription as well as for generating the primers for initiation of mtDNA synthesis. We find that mtSSB is essential for mtDNA replication initiation, as transcription is strongly upregulated from the LSP in an mtSSB knockout mouse model, but cannot support the switch to replication. Using deep sequencing as well as biochemical reconstitution experiments, we find that mtSSB is also necessary to restrict transcription initiation and primer formation to specific promoters and origins of replication both in vitro and in vivo. Pathological mutations in human mtSSB cannot efficiently support primer maturation and origin specific initiation of mtDNA replication in vitro. Total RNA differential gene expression and mitochondrial coverage profiles for Ssbp-/- mouse heart tissue, relative to Ssbp+/+ mice, in triplicate Please note that the Ssbp_585H* and Ssbp_586H* processed data files were generated from both technical replicates and is linked to the corresponding *trep1 sample records.单链DNA（single-stranded DNA，ssDNA）结合蛋白可保护DNA复制、修复等过程中形成的ssDNA区域。本研究设计了一套遗传筛选方案，鉴定出线粒体ssDNA结合蛋白（mitochondrial ssDNA-binding protein，mtSSB）为线粒体DNA（mitochondrial DNA，mtDNA）水平的关键调控因子。在线粒体中，从轻链启动子（light-strand promoter，LSP）启动的RNA合成，既是转录过程的必需环节，也是生成mtDNA合成起始引物的必要条件。我们发现，mtSSB对mtDNA复制起始至关重要：在mtSSB敲除的小鼠模型中，LSP位点的转录水平显著上调，但无法支持向复制模式的转换。通过深度测序与生化重构实验，我们证实mtSSB在体外与体内均可限制转录起始与引物形成，使其仅发生于特定的启动子及复制起点。人类mtSSB的病理性突变无法在体外有效支持引物成熟及mtDNA复制的起点特异性起始。本数据集包含Ssbp-/-小鼠心脏组织相较于Ssbp+/+小鼠的总RNA差异基因表达数据及线粒体覆盖度图谱，所有实验均设置三次重复。请注意，Ssbp_585H*与Ssbp_586H*的处理后数据文件由技术重复样本生成，并与对应的*trep1样本记录相关联。