Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element.

Elisabeth Jemt,Örjan Persson,Jay P Uhler,Christoph Freyer,Claes M Gustafsson,Marcela Dávila López,Maria Falkenberg,Majda Mehmedovic,Yonghong Shi,Tore Samuelsson

doi:10.1093/nar/gkv804

Elisabeth Jemt, Örjan Persson + Show 8 more

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https://doi.org/10.1093/nar/gkv804

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Abstract

The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5′-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3′-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.

Highlights

We demonstrate that replication initiated at the origin of heavy-strand replication and transcription coming from the opposite direction are both terminated at an evolutionary conserved sequence, which we term coreTAS
To investigate if mitochondrial transcription could be affected in the same manner, we examined transcription in vitro, using a reconstituted system comprising TFAM, POLRMT, TFB2M, and a double-stranded DNA (dsDNA) template containing the mitochondrial DNA (mtDNA) heavy-strand promoter (HSP)/light-strand promoter (LSP) promoter cassette
Since the discovery of mtDNA more than 50 years ago, significant progress has been made towards understanding the mechanisms of mtDNA replication and expression

Summary

Introduction

The mitochondrion is an organelle existing in most eukaryotic cells It is present in numbers ranging from hundreds to thousands of mitochondria per cell, depending of the energetic needs of the tissue. The eukaryotic nuclear transcription mechanism involves many factors and three different RNAPs, while Escherichia coli (E. coli) has only one RNAP (Berg 2019) Because of their evolutionary similarity to eukaryotic RNAP’s and simplicity of the organisms, prokaryotic systems are used as models for research, and much of our knowledge about transcription processes stems from studying E. coli and bacteriophages. Unlike E. coli and the eukaryotic gene expression systems, T7 bacteriophage has only one factor involved, the T7 RNAP This single subunit enzyme can recognize a promoter sequence, initiate, elongate and terminate transcription on its own. Since DNA polymerases cannot move in the 3' to 5' direction, Okazaki fragments are synthesized in the opposite direction to the moving fork (Alberts 2008, Berg 2019)

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