Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL. (23rd August 2021)

• Record Type: Journal Article
• Authors: Sarfallah, Azadeh; Zamudio‐Ochoa, Angelica; Anikin, Michael; Temiakov, Dmitry
• Is Part Of: EMBO journal. Volume 40:Number 19(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-08-23
• Subjects: mitochondrial replication -- mitochondrial transcription -- PolG -- POLRMT -- primosome; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.2021107988
• Languages: English
• ISSNs: 0261-4189

Abstract

Abstract: The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem‐loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre‐binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL —a complex of mtRNAP and PolG—can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases. SYNOPSIS: Mitochondrial DNA replication requires coordinated action of RNA and DNA polymerases loaded onto a transiently formed hairpin structure at the origin of replication OriL. Here, efficient RNA primer synthesis and handover is shown to involve transcript slippage and primosome assembly. Mitochondrial RNA polymerase initiates synthesis of replication primer at OriL at position 5751 in human mtDNA. Transcription initiation at the OriL hairpin involves transcript slippage, which is required for efficient primer synthesis. Mitochondrial RNA polymerase and DNA polymerase PolG form a functional primosome complex at OriL. The primosome ensures efficient primer hand‐off and protects the nascent transcript from nucleases. Abstract : Replication of human mitochondrial DNA involves transcript slippage and RNA primer handover/protection within a primosome formed by mtRNAP and DNA polymerase PolG.