RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Viktor Posse,Maria Falkenberg,Jay P Uhler,Aurelio Reyes,Massimo Zeviani,Anders R Clausen,Ali Al-Behadili,Claes M Gustafsson,Aleksandra Filipovska

doi:10.1371/journal.pgen.1007781

Abstract

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Highlights

The mitochondrial DNA is replicated by a replication machinery distinct from that operating in the nucleus and mutations affecting individual replication factors have been associated with an array of rare, human diseases
We demonstrate that RNase H1 directs originspecific initiation of DNA replication in human mitochondria and that disease-causing mutations may impair this process
A unique feature of mitochondrial DNA (mtDNA) replication is that primers required for initiation of leading-strand DNA replication are produced by the mitochondrial transcription machinery

Summary

Introduction

Mitochondrial DNA (mtDNA) is a 16.6 kb circular, double-stranded DNA (dsDNA) molecule that contains genes for 13 components of the oxidative phosphorylation (OXPHOS) system as well as the 22 tRNAs and 2 rRNAs required for their translation. Polycistronic transcription of the two strands is initiated from the Lstrand and H-strand promoters (LSP and HSP, respectively) and carried out by a transcription machinery consisting of a single subunit RNA polymerase (POLRMT), and transcription factors A (TFAM) and B2 (TFB2M) [1, 2]. OriL adopts a stem-loop structure and POLRMT initiates synthesis of short RNA primers from a poly-dT stretch in the loop region. These primers are used by POLγ to initiate L-strand synthesis with the parental H-strand as template [14, 15]. H-strand and Lstrand DNA replication subsequently proceed until two complete daughter molecules have been formed and separated in a Topoisomerase 3α-dependent process [16]

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