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

Artikel i vetenskaplig tidskrift
Författare Viktor Posse
Ali Al-Behadili
Jay Uhler
Anders R Clausen
A. Reyes
M. Zeviani
Maria Falkenberg
Claes M Gustafsson
Publicerad i Plos Genetics
Volym 15
Nummer/häfte 1
ISSN 1553-7404
Publiceringsår 2019
Publicerad vid Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
Språk en
Länkar dx.doi.org/10.1371/journal.pgen.100...
Ämnesord leading-strand origin, r-loop, polymerase-gamma, accessory subunit, in-vivo, transcription, hybrid, primer, mechanism, cells, Genetics & Heredity, ates of america, v107, p16072, ates of america, v112, p9334, ates of america, v105, p11122, ang dd, 1985, proceedings of the national academy of sciences of the united states of, ang dd, 1985, embo journal, v4, p1559, ang dd, 1987, embo journal, v6, p409
Ämneskategorier Genetik

Sammanfattning

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 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. Author summary Human mitochondria contain a double-stranded DNA genome that codes for key components of the oxidative phosphorylation system. The mitochondrial DNA (mtDNA) 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. In the present work, we demonstrate that RNase H1 directs origin-specific initiation of DNA replication in human mitochondria and that disease-causing mutations may impair this process. A unique feature of mtDNA replication is that primers required for initiation of leading-strand DNA replication are produced by the mitochondrial transcription machinery. A substantial fraction of all transcription events is prematurely terminated about 120 nucleotides downstream of the promoter and the RNA remains firmly associated with the genome, forming R-loops. Interestingly, the free 3-end of these R-loops cannot directly prime initiation of DNA synthesis, but must first be processed by RNase H1. The process is stimulated by the mitochondrial single-stranded DNA binding protein and faithfully reconstitutes replication events mapped in vivo. In combination with mapping of replication events in fibroblasts derived from patients with mutations in RNASEH1, our findings point to a possible model for replication initiation in human mitochondria similar to that previously described in the E. coli plasmid, ColE1.

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Denna text är utskriven från följande webbsida:
http://www.gu.se/forskning/publikation/?publicationId=278534
Utskriftsdatum: 2019-08-22