Purification of the sea urchin mtRNAP from baculovirus-infected insect cells and functional assay

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system"</p><p></p><p>Nucleic Acids Research 2007;35(7):2413-2427.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874651.</p><p>© 2007 The Author(s)</p> () Purification of mtRNAP by metal chelate affinity chromatography. The soluble portion of the insect cell lysate expressing the sea urchin mtRNAP was purified by Ni-NTA chromatography; cleared lysate, C.lys, flow-through, FT, wash, W, 3–5, fractions eluted at 250 mM imidazole, were separated on a 10% SDS–PAGE and revealed by immunoblotting as described in ‘Materials and Methods’ section. () Purification profile of mtRNAP as obtained by Heparin–Sepharose chromatography. Peak fractions from Ni-NTA column were pooled and subjected to Heparin–Sepharose chromatography. Input to the column (I) and fractions eluting between 0.75 and 0.9 M NaCl were analyzed by 7.5% SDS–PAGE and Coomassie Brilliant Blue stained. The molecular weight marker Precision Plus Protein Standards (Bio-Rad) is shown (M). The arrow inside the picture indicates the mtRNAP-containing band, as assessed by MALDI-TOF analysis. () Immunoblotting assay of input to the column (I) and Heparin–Sepharose eluted fractions. () Transcriptional activity of purified mtRNAP. The indicated Heparin–Sepharose fractions were assayed in the presence of [α-]PUTP, as described in ‘Materials and Methods’ section. On the top it is shown the diagram of the 71-bp tailed template, named 71bpDNA, with the open bar referring to the duplex DNA portion and the thin line to the 3′-tail. Run-off transcripts are indicated by arrowed line. Radiolabeled transcripts were separated on a 12% polyacrylamide/7M urea mini-gel followed by phosphorimaging analysis. 15 + R, fraction 15 treated with RNase A. RNA markers corresponding to the 10 nt ladder are indicated on the left

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

The rapid loss of mtDNA is not correlated to a loss of mitochondria, autophagy or apoptosis.

<p>Levels of <b>A</b>. mitochondrial proteins and <b>C</b>. an autophagy marker before induction and during the recovery period after induction of PstI for 2h with doxycycline. Total cell lysates (20 μg) were analyzed by western blot with antibodies against markers of the mitochondrial matrix (PDH), mitochondrial outer membrane (Tomm20), autophagy (LC3) and mitophagy (PINK1). CCCP is used as a positive control and Tubulin is used as loading control. Analysis of <b>B</b>. cell mitochondrial content and <b>D</b>. apoptosis by flow cytometry before induction and during the recovery period. * P ≤ 0.05 versus non-induced PstI cells for each cell population (Student’s t-test).</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

The loss of mtDNA is followed by a slow repopulation with intact mtDNA molecules.

<p><b>A</b>. Southern blot analysis of the control HEK293 cells (WT) and stably transfected cells (PstI cells) after digestion by BamHI or BamHI + PstI for the control. PstI expression was induced for 2h with doxycycline and cells were followed for 20 days. At day 20, a second induction of 2h with doxycycline was performed and the cells were followed during the second recovery period (marked with an apostrophe). <b>B</b>. Quantification of the mtDNA/nDNA ratios in the PstI stably transfected cells, from 0h to 41 days after a 2h induction with doxycycline and from 0h to 20days after a second 2h doxycycline induction. (mean ± s.e., <i>N</i> = 3). <b>C</b>. Quantification of the level of PstI transcripts in the control HEK293 cells (WT) and stably transfected cells (PstI cells), during 2 sequential inductions of PstI expression with doxycycline (mean ± s.e., <i>N</i> = 3). TBP is used as a reference RNA.</p

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

<div><p>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.</p></div

Identification of free 5´- ends at OriH and ribonucleotides at OriL.

<p>(A) Reads per million on H-strand (upper panel) and L-strand (lower panel) at OriH using 5´-End-seq, (B) Reads per million on L-strand at OriL using HydEn-seq (upper panel) and 5´-End-seq (lower panel).</p

Ribonucleotide incorporation in mtDNA from fibroblasts from patients with genetic defects in <i>TK2</i>, <i>DGUOK</i> or <i>MPV17</i>.

<p>(A) Hierarchical clustering of HydEn-seq libraries (undigested or digested with HincII). (B) Ribonucleotide content for H-strand (H) and L-strand (L) for patient-derived mutant cell lines. (C) Ribonucleotide incorporation frequency per 1,000 bases of its complementary nucleotides calculated on H-strand and L-strand for fibroblasts and patient-derived mutant lines. (D) Quantification of ribonucleotides in H-strand and L-strand per mtDNA molecule in fibroblasts and patient-derived mutant lines.</p