The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication.

Min Jiang,Sara Albarran-Gutierrez,Shan Jiang,Xinping Li,Dusanka Milenkovic,Jelena Misic,Bertil Macao,Ilian Atanassov,Stefan J Siira,Claes M Gustafsson,Xuefeng Zhu,Maria Falkenberg,Aleksandra Filipovska,Jack D Griffith,Yonghong Shi,Louise Jenninger,Nirwan Tandukar,Thomas J Nicholls,Xie Xie,Valerio Carelli,Nils Göran Larsson ,Eike Hoberg ,Zsolt Szilágyi

doi:10.1126/sciadv.abf8631

Abstract

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.

Highlights

The mammalian mitochondrial genome [mitochondrial DNA] is essential for cellular energy conversion, encoding 13 of the key subunits of the oxidative phosphorylation system (OXPHOS) as well as tRNA and ribosomal RNA molecules required for mitochondrial translation
When we monitored global changes in mitochondrial transcription in knockout heart tissue using real-time quantitative polymerase chain reaction (qPCR), we found a profound progressive reduction in steady-state levels of transcripts produced from the heavy-strand promoter (HSP) (Fig. 3, A and B) consistent with the observed mitochondrial DNA (mtDNA) depletion
We demonstrate that mtSSB is crucial for maintaining adequate levels of mtDNA

Summary

Introduction

The mammalian mitochondrial genome [mitochondrial DNA (mtDNA)] is essential for cellular energy conversion, encoding 13 of the key subunits of the oxidative phosphorylation system (OXPHOS) as well as tRNA and ribosomal RNA (rRNA) molecules required for mitochondrial translation. All other mitochondrial proteins are nuclear-encoded, including most of the OXPHOS subunits and all factors required for mtDNA maintenance and expression. The mtDNA copy number varies depending on tissue energy requirements, ranging from hundreds to many thousands per cell [1]. Defects in mtDNA maintenance cause a heterogeneous group of mitochondrial disorders, characterized at the molecular level by mtDNA depletion or mtDNA deletions, which lead to OXPHOS deficiency in affected tissues [3]. The underlying causes for many of these disorders are mutations in the core subunits of the mtDNA replication machinery, including DNA polymerase (POL ), the replicative DNA helicase TWINKLE, and mitochondrial single-stranded DNA (ssDNA) binding protein (mtSSB) [3,4,5,6,7,8,9,10]

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Conclusion