Molecular insights into mitochondrial DNA replication

Mitochondria are organelles found in eukaryotic cells. These organelles produce most of the adenosine triphosphate that cells use as a source of energy. Mitochondria contain their own genomic material, a circular DNA genome (mtDNA) that encodes subunits of the respiratory chain complexes and RNA components needed for mitochondrial translation. Many aspects of mtDNA replication are still not understood and in this thesis we address some of the molecular mechanisms of this process in mammalian cells. DNA synthesis cannot be initiated de novo, but requires a short RNA primer as a starting point. We here demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the origin of light strand DNA replication (OriL) in human mtDNA. Using purified POLRMT and the core factors of the mitochondrial replisome, we faithfully reconstitute OriLdependent initiation of replication in vitro. During origin activation, OriL is exposed in its single-stranded conformation and adopts a stem-loop structure. POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region and after about 25 nt, POLRMT is replaced by the mitochondrial DNA polymerase ! (POL!) and DNA synthesis is initiated. Our findings also suggest that the mitochondrial single-stranded DNA binding protein directs origin-specific initiation by efficiently blocking unspecific initiation events in other regions of the mtDNA genome. To analyze the requirements of OriL in vivo, we have used saturation mutagenesis in the mouse combined with in vitro biochemistry and demonstrated that OriL is essential for mtDNA maintenance. OriL requires a stable stem-loop structure and a pyrimidine-rich sequence in the template strand for proper origin function. The OriL mechanism appears to be conserved, since bioinformatics analyses demonstrated the presence of OriL in the mtDNA of most vertebrates including birds. Our findings suggest that mtDNA replication may be performed by a common mechanism in all vertebrates and lend support to the strand-displacement model for mtDNA replication. A molecular understanding of the mitochondrial DNA replication machinery is also of medical importance. Today, more than 160 mutations in the gene encoding the catalytic subunit of POL! (POL!A) have been associated with human disease. One example is the Y955C mutation, which causes autosomal dominant progressive external ophthalmoplegia, a disorder characterized by the accumulation of multiple mtDNA deletions. The Y955C mutation decreases POL! processivity due to a decreased binding affinity for the incoming deoxyribonucleoside triphosphate. However, it is not clear why this biochemical defect leads to a dominant disease. We have used the reconstituted mammalian mtDNA replisome and studied functional consequences of the dominant Y955C mutation. Our study revealed that the POL!A:Y955C enzyme is prone to stalling at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The mutant POL!A:Y955C competes with wild-type POL!A for access to the primer template. However, once assembled in the replisome, the wild-type enzyme is no longer affected. Our data therefore provide a mechanism for the mtDNA replication phenotypes seen in patients harboring the Y955C mutation

Latvijas lauksaimniecibas politika: procesi, problemas, risinajumi

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

TZAP overexpression induces telomere dysfunction and ALT-like activity in ATRX/DAXX-deficient cells

Summary: The appropriate regulation of telomere length homeostasis is crucial for the maintenance of genome integrity. The telomere-binding protein TZAP has been suggested to regulate telomere length by promoting t-circle and c-circle excisions through telomere trimming, yet the molecular mechanisms by which TZAP functions at telomeres are not understood. Using a system based on TZAP overexpression, we show that efficient TZAP recruitment to telomeres occurs in the context of open telomeric chromatin caused by loss of ATRX/DAXX independently of H3.3 deposition. Moreover, our data indicate that TZAP binding to telomeres induces telomere dysfunction and ALT-like activity, resulting in the generation of t-circles and c-circles in a Bloom-Topoisomerase IIIα-RMI1-RMI2 (BTR)-dependent manner

Quantification of mtSSB to mtDNA ratio in HeLa cells.

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

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 <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