Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

Anna-Karin Berglund,Claes M Gustafsson,Martin K M Engqvist,Clara Navarrete,Robert W Taylor,Zsolt Szilagyi,Emily Hoberg,Maria Falkenberg,Anders R Clausen,Nils-Göran Larsson

doi:10.1371/journal.pgen.1006628

Anna-Karin Berglund, Claes M Gustafsson + Show 8 more

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https://doi.org/10.1371/journal.pgen.1006628

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Abstract

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

Highlights

Human mitochondrial DNA is a double-stranded circular molecule of 16.6 kb that encodes for key components of the oxidative phosphorylation system
For almost half a century it has been known that mitochondrial DNA (mtDNA) contains ribonucleotides, but their identity and precise location are not known
Our findings demonstrate that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of ribonucleotide excision repair pathways in human mitochondria

Summary

Introduction

Human mitochondrial DNA (mtDNA) is a double-stranded circular molecule of 16.6 kb that encodes for key components of the oxidative phosphorylation system. Previous studies have demonstrated the presence of about 10 to 30 ribonucleotides in each mtDNA molecule [1, 2]. These ribonucleotides can either be incorporated by mtDNA polymerase γ (POLγ) during DNA synthesis or, alternatively, be remnants of partially processed RNA primers used for initiation of DNA synthesis. Ribonucleotide incorporation into DNA may be a consequence of inefficient removal of RNA primers used to initiate DNA synthesis. RNA primers are exchanged to DNA during RNA maturation This process involves several nucleases, including RNase H1 and H2, Flap endonuclease 1 (FEN1), and DNA2 [6,7,8,9]. RNase H1 has a mitochondrial isoform and deleterious mutations in the RNASEH1 gene can disturb mtDNA replication and lead to mitochondrial disease [12]

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