The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [TruSeq]. 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. Overall design: 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、修复等生理过程中形成的单链DNA区域。本研究通过构建遗传筛选体系，鉴定出线粒体ssDNA结合蛋白（mitochondrial ssDNA-binding protein, mtSSB）为线粒体DNA（mitochondrial DNA, mtDNA）稳态的关键调控因子。在线粒体中，轻链启动子（light-strand promoter, LSP）介导的RNA合成不仅参与转录过程，还可生成启动mtDNA合成所需的引物。本研究发现，mtSSB是mtDNA复制起始的必需因子：在mtSSB敲除小鼠模型中，LSP介导的转录水平显著上调，但无法支持向复制进程的转换。通过深度测序与生化重构实验，本研究进一步证实，mtSSB可在体内外将转录起始与引物形成过程限定于特定启动子及复制起始位点，是该过程不可或缺的调控蛋白。人类mtSSB的病理性突变无法在体外有效支持引物成熟及mtDNA复制的位点特异性起始过程。实验总体设计：以Ssbp基因敲除纯合子（Ssbp-/-）小鼠心脏组织为实验组，以野生型（Ssbp+/+）小鼠为对照组，开展三组生物学重复的总RNA差异基因表达分析及线粒体覆盖度图谱检测。请注意：Ssbp_585H*与Ssbp_586H*的已处理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。