The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo

Latvijas lauksaimniecibas politika: procesi, problemas, risinajumi

Summary in English, RussianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

POLRMT can initiate primer synthesis from a single dT and mtSSB is abundant <i>in vivo</i>.

<p>(A). POLRMT can initiate primer synthesis from a linear template containing one or more dT (lanes 2 to 7). Deletion of the poly-dT stretch abolishes primer synthesis (lane 1). (B) Representative quantitative Western blot measurement of endogenous mtSSB protein in human Hela cells. Protein extracts (5, 10 or 20 µl) were loaded from a determined number of cells. Purified recombinant mtSSB was used to create standard curve with known protein concentrations.</p

mtSSB governs OriL specificity.

<p>(<b>A</b>) <i>In vitro</i> rolling circle DNA replication reaction with increasing concentrations of mtSSB (0, 10, 100, 500 fmol, 1, 5, 10, 20 and 40 pmol) on the SK+OriL dsDNA template. DNA replication was performed in the presence of [α- 32P]-dCTP in order to label newly synthesized DNA as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004832#pgen.1004832-Fuste1" target="_blank">[6]</a>. The weak labeling of input template in lanes 1 and 2 is most likely due to POLγ idling on the free 3′-end, in the absence of active rolling circle DNA replication. (<b>B</b>) Schematic illustration explaining the replication products formed on lagging-strand DNA. At higher mtSSB levels, primers synthesis is restricted to OriL, but at lower levels primer synthesis can take place also at other sites. During the first round of DNA synthesis, the OriL-depending lagging-strand products have a length of about 2100 nts. In later rounds, the fragments will span the entire distance between two OriL sequences (about 3900 nts) and migrate with the same size as the input template. (<b>C</b>) Reactions were performed as in panel A, but replication products were analyzed by Southern blotting using strand-specific probes to detect leading- or lagging-strand DNA synthesis. For comparison, we used a mutant template (OriL-del) in which the OriL sequence had been deleted.</p

mtSSB <i>in vivo</i> occupancy reflects strand-displacement mtDNA replication.

<p>(<b>A</b>) Occupancy of mtSSB and TFAM analyzed by strand-specific qPCR amplification of ChIP samples. (<b>B</b>) Strand-specific ChIP-seq profile of mtSSB binding to mtDNA. The origins of replication are indicated. The short black bars indicate the location of the primers used for strand-specific qPCR. (<b>C</b>) Schematic illustration of expected occupancy of mtSSB accordingly to the different mtDNA replication models. SDM (Strand displacement mode), SC (Strand coupled mode), and RITOLS.</p

Quantification of mtSSB to mtDNA ratio in HeLa cells.

<p>Quantification of mtSSB to mtDNA ratio in HeLa cells.</p

MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation

SummaryMitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no in vivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease of de novo transcription manifested as reduced 7S RNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter