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• W2086372007 abstract "•TFAM can compact DNA and reconstitute nucleoid-like structures in vitro•TFAM packaging determines the number of active mtDNA molecules•Nucleoid formation blocks mtDNA transcription and replication The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. A mammalian cell contains multiple copies of mtDNA, a circular molecule of 16,569 bp that encodes for 13 essential subunits of the respiratory chain. The genome is essential for normal cellular function; mtDNA mutations can cause mitochondrial disease and have also been implicated in human aging (Park and Larsson, 2011Park C.B. Larsson N.G. Mitochondrial DNA mutations in disease and aging.J. Cell Biol. 2011; 193: 809-818Crossref PubMed Scopus (218) Google Scholar). In vivo, mtDNA exists in a compact nucleoprotein complex, denoted the nucleoid (Bogenhagen, 2012Bogenhagen D.F. Mitochondrial DNA nucleoid structure.Biochim. Biophys. Acta. 2012; 1819: 914-920Crossref PubMed Scopus (196) Google Scholar). Most nucleoids contain a single mtDNA molecule, which is fully coated by mitochondrial transcription factor A (TFAM), a high-mobility-group box domain protein (Brown et al., 2011Brown T.A. Tkachuk A.N. Shtengel G. Kopek B.G. Bogenhagen D.F. Hess H.F. Clayton D.A. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.Mol. Cell. Biol. 2011; 31: 4994-5010Crossref PubMed Google Scholar, Kukat et al., 2011Kukat C. Wurm C.A. Spåhr H. Falkenberg M. Larsson N.G. Jakobs S. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.Proc. Natl. Acad. Sci. USA. 2011; 108: 13534-13539Crossref PubMed Scopus (360) Google Scholar, Wang et al., 2013Wang Y.E. Marinov G.K. Wold B.J. Chan D.C. Genome-wide analysis reveals coating of the mitochondrial genome by TFAM.PLoS ONE. 2013; 8: e74513Crossref PubMed Scopus (39) Google Scholar). In vivo estimates have determined the concentration of TFAM to one molecule per 15 to 18 bp of mtDNA (Kukat et al., 2011Kukat C. Wurm C.A. Spåhr H. Falkenberg M. Larsson N.G. Jakobs S. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.Proc. Natl. Acad. Sci. USA. 2011; 108: 13534-13539Crossref PubMed Scopus (360) Google Scholar), and TFAM is to date the only protein shown to package and organize mtDNA. Volume calculations suggest that TFAM is the major constituent of the nucleoid, even if other proteins, such as mtDNA replication and transcription factors, can associate with this structure (Kukat and Larsson, 2013Kukat C. Larsson N.G. mtDNA makes a U-turn for the mitochondrial nucleoid.Trends Cell Biol. 2013; 23: 457-463Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). TFAM binds in a cooperative manner to nonspecific DNA sequences and forms stable protein patches (filaments) in which each monomer covers about 30 bp of DNA. TFAM binding leads to partial unwinding of duplex DNA, which in turn causes softening and compaction of the DNA molecule (Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar). TFAM is essential for mtDNA maintenance. Disruption of the Tfam gene in mouse causes loss of mtDNA, whereas overexpression of TFAM leads to increased mtDNA copy number (Ekstrand et al., 2004Ekstrand M.I. Falkenberg M. Rantanen A. Park C.B. Gaspari M. Hultenby K. Rustin P. Gustafsson C.M. Larsson N.G. Mitochondrial transcription factor A regulates mtDNA copy number in mammals.Hum. Mol. Genet. 2004; 13: 935-944Crossref PubMed Scopus (648) Google Scholar, Kanki et al., 2004Kanki T. Ohgaki K. Gaspari M. Gustafsson C.M. Fukuoh A. Sasaki N. Hamasaki N. Kang D. Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA.Mol. Cell. Biol. 2004; 24: 9823-9834Crossref PubMed Scopus (239) Google Scholar, Larsson et al., 1998Larsson N.G. Wang J. Wilhelmsson H. Oldfors A. Rustin P. Lewandoski M. Barsh G.S. Clayton D.A. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.Nat. Genet. 1998; 18: 231-236Crossref PubMed Scopus (1216) Google Scholar). TFAM is also an essential component of the mtDNA transcription machinery (Shi et al., 2012Shi Y. Dierckx A. Wanrooij P.H. Wanrooij S. Larsson N.G. Wilhelmsson L.M. Falkenberg M. Gustafsson C.M. Mammalian transcription factor A is a core component of the mitochondrial transcription machinery.Proc. Natl. Acad. Sci. USA. 2012; 109: 16510-16515Crossref PubMed Scopus (121) Google Scholar). The protein binds in a sequence-specific manner to mitochondrial promoters and induces a stable U-turn in DNA (Ngo et al., 2011Ngo H.B. Kaiser J.T. Chan D.C. The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA.Nat. Struct. Mol. Biol. 2011; 18: 1290-1296Crossref PubMed Scopus (200) Google Scholar, Rubio-Cosials et al., 2011Rubio-Cosials A. Sidow J.F. Jiménez-Menéndez N. Fernández-Millán P. Montoya J. Jacobs H.T. Coll M. Bernadó P. Solà M. Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter.Nat. Struct. Mol. Biol. 2011; 18: 1281-1289Crossref PubMed Scopus (137) Google Scholar). In combination with mitochondrial RNA polymerase (POLRMT) and the mitochondrial transcription factor B2 (TFB2M), TFAM supports transcription from the mitochondrial heavy- and light-strand promoters (HSP and LSP) in vitro (Falkenberg et al., 2002Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA.Nat. Genet. 2002; 31: 289-294Crossref PubMed Scopus (457) Google Scholar, Fisher and Clayton, 1985Fisher R.P. Clayton D.A. A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro.J. Biol. Chem. 1985; 260: 11330-11338PubMed Google Scholar). Mammalian mitochondria also contain specialized DNA replication machinery, which includes DNA polymerase γ (POLγ), the replicative helicase TWINKLE, and mitochondrial single-stranded DNA-binding protein (mtSSB). When combined, these factors can support leading-strand DNA synthesis in vitro (Korhonen et al., 2004Korhonen J.A. Pham X.H. Pellegrini M. Falkenberg M. Reconstitution of a minimal mtDNA replisome in vitro.EMBO J. 2004; 23: 2423-2429Crossref PubMed Scopus (293) Google Scholar). In the nucleus, DNA compaction by histones into nucleosomes negatively regulates DNA transactions, such as DNA replication and transcription (Finkelstein and Greene, 2013Finkelstein I.J. Greene E.C. Molecular traffic jams on DNA.Annu. Rev. Biophys. 2013; 42: 241-263Crossref PubMed Scopus (26) Google Scholar). TFAM-dependent compaction of mtDNA will most likely also have consequences for the activity of DNA-binding molecular machines. Here, we elucidate how the moving replication and transcription machineries react to roadblocks formed by TFAM using a combination of classical biochemical techniques and single-molecule tools. We provide evidence that high TFAM:mtDNA ratios result in the formation of large stable TFAM filaments on the DNA that reduce the progression of replication and transcription complexes. Moreover, we show that at in vivo-relevant concentrations of TFAM, there is a large variation in the amount of compaction among different DNA molecules. At these conditions, small changes in the TFAM concentration have a large impact on the average compaction, as would be expected if TFAM is used as a global regulator for mtDNA transactions. The elongation stages of mtDNA replication and transcription require that the double-stranded DNA (dsDNA) template is partially unwound. We developed a single-molecule assay in which force-induced melting/peeling of dsDNA mimicked strand invasion by a motor protein and monitored the effect TFAM packaging may have on this process (Figure 1A). A dsDNA molecule (lambda DNA ∼48 kbp) was labeled with biotin on both the 5′ and 3′ ends of the same strand and attached between two beads held in a dual optical trap. The DNA molecule was then progressively extended ∼75% into its overstretching plateau (65 pN) and held under this tension during the experiment. Due to both the labeling strategy (the dsDNA molecule has two free ends) and the salt concentration used in this assay (25 mM NaCl), the overstretching of a dsDNA molecule results in base-pair breaking from the free DNA ends that leads to a progressive conversion of the dsDNA into two single-stranded DNA (ssDNA) strands, with only the strand with the biotins still under tension (Candelli et al., 2013Candelli A. Hoekstra T.P. Farge G. Gross P. Peterman E.J. Wuite G.J. A toolbox for generating single-stranded DNA in optical tweezers experiments.Biopolymers. 2013; 99: 611-620Crossref PubMed Scopus (39) Google Scholar, King et al., 2013King G.A. Gross P. Bockelmann U. Modesti M. Wuite G.J. Peterman E.J. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2013; 110: 3859-3864Crossref PubMed Scopus (124) Google Scholar). To monitor what happens to TFAM during DNA strand separation, we incubated the relaxed DNA construct with fluorescently labeled TFAM (Alexa555), overstretched the DNA into the overstretching plateau, and excited the Alexa555 in a continuous fashion while detecting the emitted fluorescence. We first checked the impact of high force on the TFAM-DNA interactions and found that, in the overstretching regime, the dissociation time of TFAM from DNA was not significantly different from those observed previously for forces up to 40 pN (Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar). When keeping the DNA 75% overstretched, we observed a unidirectional displacement of some TFAM molecules along the DNA contour (Figures 1B–1D; Movie S1). We observed this pattern repeatedly and we monitored in total more than 200 fluorescent spots on the DNA. We could also observe a progressive increase of fluorescent intensity as a moving fluorescent spot encountered other stationary fluorescent proteins (Figure 1D, blue arrow). We hypothesized that these events are the result of TFAM molecules being progressively pushed by the junction between the ssDNA and dsDNA during strand separation. To confirm this hypothesis, we needed to follow the peeling process directly, by localizing the single-stranded and double-stranded regions of the DNA molecule. To this end, we performed experiments similar to the one presented in Figure 1 but in the presence of replication protein A (RPA), which binds selectively to ssDNA (King et al., 2013King G.A. Gross P. Bockelmann U. Modesti M. Wuite G.J. Peterman E.J. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2013; 110: 3859-3864Crossref PubMed Scopus (124) Google Scholar, van Mameren et al., 2009van Mameren J. Gross P. Farge G. Hooijman P. Modesti M. Falkenberg M. Wuite G.J. Peterman E.J. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.Proc. Natl. Acad. Sci. USA. 2009; 106: 18231-18236Crossref PubMed Scopus (219) Google Scholar). We first incubated the relaxed dsDNA molecule in a channel containing fluorescent TFAM. We then transferred the DNA molecule into a buffer containing RPA fluorescently labeled with enhanced GFP (eGFP-RPA) and overstretched the DNA to ∼70%. Finally, we excited alternatively with appropriate light to image RPA and TFAM and to obtain an overlay of the two images (Figure 2A). We found that eGFP-RPA binds preferentially to the extremities of the DNA where the strand separation starts, indicating that this part is single stranded. We noticed that eGFP-RPA also binds, but to a lesser extent, to the middle region of the DNA. Under low-ionic-strength conditions and upon extension of a DNA molecule far into its overstretching plateau, we have previously observed scattered RPA binding all through the DNA molecule. This is explained by the presence of RPA (and thus ssDNA) at localized domains where base-pairing is broken (so-called melting bubbles) and the phosphate backbones remain under tension (King et al., 2013King G.A. Gross P. Bockelmann U. Modesti M. Wuite G.J. Peterman E.J. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2013; 110: 3859-3864Crossref PubMed Scopus (124) Google Scholar). At the same time, we observed that most TFAM binding does not overlap with RPA binding. Due to the optical resolution limit (diffraction limit ∼300 nm), we cannot spatially separate RPA and TFAM binding when they are bound closer than ∼750 bps to one another, but the majority of TFAM could unequivocally be assigned to the double-stranded region of the DNA construct. When we extended the DNA molecule far into the overstretching plateau (∼85%), we observed eGFP-RPA binding throughout the whole DNA molecule consistent with the formation of melting bubbles (Figure 2B). The presence of melting bubbles explains why one can observe a “thicker” red line in the middle of the DNA molecule compared to its left side; in a melting bubble (middle of the molecule), the two strands are RPA coated, which results in a higher RPA concentration compared to a peeled ssDNA molecule being coated by RPA (left side of the molecule). However, at the ionic strength conditions used, peeling from the extremities is energetically favorable over the formation of melting bubbles (King et al., 2013King G.A. Gross P. Bockelmann U. Modesti M. Wuite G.J. Peterman E.J. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2013; 110: 3859-3864Crossref PubMed Scopus (124) Google Scholar). Indeed, when we kept the DNA overstretched and imaged the DNA, we could observe that peeling actually progresses on the DNA molecule, albeit rather slow (Figure 2B, the blue arrows show two peeling fronts, one starting on the left from the extremity of the DNA and one from a nick in the DNA on the right). As expected, the slow peeling (left side) is correlated with a TFAM patch localized in the double-stranded region of the DNA molecule that impedes the peeling process. Note also that TFAM is not present in the DNA strand that is free (bulge next to the left blue arrow), staying exclusively on the DNA that is not peeled off. We thus conclude that TFAM moves in a unidirectional way along the dsDNA during DNA peeling. We observed similar patterns several times; however, to obtain quantitative data of the pushing of TFAM on DNA by ssDNA peeling, we chose to use naked DNA molecules to completely exclude possible interferences between RPA and either the unpeeling process or the binding of TFAM on DNA.Figure 2TFAM Localizes on the dsDNA at the Melting Front and Can Prevent PeelingShow full caption(A) Fluorescence images of eGFP-RPA and Alexa555-TFAM and composite fluorescence images displaying the binding of eGFP-RPA (in red) and Alexa555-TFAM (in green).(B) Selection of fluorescence images (time interval 10 s) showing the binding of eGFP-RPA (in red) and Alexa555-TFAM (in green). The arrows indicate the peeling events. For both panels, the dsDNA molecules were incubated with Alexa555-TFAM (20 nM), overstretched, and incubated with eGFP-RPA (2 nM). The relative DNA extension (L/Lc) is indicated in the figure.(C) Kymograph showing a TFAM patch (fluorescence intensity corresponds to four TFAM molecules) moving on a DNA molecule undergoing peeling.(D) The position of the moving patch showed in (C) was tracked and the mean square displacement (MSD) was determined. The MSD plot was fitted (MSD = v2t2 + 2Dt) to determine the velocity (v) of the molecule and the diffusion coefficient (D) (t is time).(E) The obtained velocities for the observed TFAM molecules (n = 221) were binned into five equal-sized bins according to the fraction of TFAM coverage on the DNA. The percentage coverage was calculated using the DNA persistence length (obtained by fitting the DNA force extension curves with the worm like chain model) as described in Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar. Inset: histogram of the distribution of the observed velocities at high TFAM coverage (≥50%, light green bars) and low TFAM coverage (<50%, crossed bars).(F) The velocities at low TFAM coverage (<10%) (n = 53) were binned into five equal-sized bins according to the number of TFAM in a spot and plotted as a function of number of TFAM. The number of TFAMs in each fluorescent spot was determined by single photobleaching steps.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Fluorescence images of eGFP-RPA and Alexa555-TFAM and composite fluorescence images displaying the binding of eGFP-RPA (in red) and Alexa555-TFAM (in green). (B) Selection of fluorescence images (time interval 10 s) showing the binding of eGFP-RPA (in red) and Alexa555-TFAM (in green). The arrows indicate the peeling events. For both panels, the dsDNA molecules were incubated with Alexa555-TFAM (20 nM), overstretched, and incubated with eGFP-RPA (2 nM). The relative DNA extension (L/Lc) is indicated in the figure. (C) Kymograph showing a TFAM patch (fluorescence intensity corresponds to four TFAM molecules) moving on a DNA molecule undergoing peeling. (D) The position of the moving patch showed in (C) was tracked and the mean square displacement (MSD) was determined. The MSD plot was fitted (MSD = v2t2 + 2Dt) to determine the velocity (v) of the molecule and the diffusion coefficient (D) (t is time). (E) The obtained velocities for the observed TFAM molecules (n = 221) were binned into five equal-sized bins according to the fraction of TFAM coverage on the DNA. The percentage coverage was calculated using the DNA persistence length (obtained by fitting the DNA force extension curves with the worm like chain model) as described in Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar. Inset: histogram of the distribution of the observed velocities at high TFAM coverage (≥50%, light green bars) and low TFAM coverage (<50%, crossed bars). (F) The velocities at low TFAM coverage (<10%) (n = 53) were binned into five equal-sized bins according to the number of TFAM in a spot and plotted as a function of number of TFAM. The number of TFAMs in each fluorescent spot was determined by single photobleaching steps. See also Figure S1. We next characterized the movement of TFAM during DNA strand separation as a function of protein concentration. For each fluorescent spot on the DNA (like those on Figure 2C or Figure 1), we recorded a movie and tracked the (possible) movement of TFAM molecules on the DNA undergoing peeling by fitting a 2D Gaussian to the intensity profile in each frame. From the trajectories obtained, we calculated the mean square displacement and fitted it as a function of the time interval (Figure 2D). Under normal conditions, TFAM diffuses or remains stationary on DNA (the later is typically seen at high TFAM concentrations). When DNA is undergoing peeling, we could observe TFAM moving directionally, indicating that strand separation is pushing the TFAM forward. We investigated the dynamics of the peeling front by following the directional motion of TFAM over a wide range of TFAM concentrations. To accurately assess the effective TFAM concentration on the DNA, we determined the percentage of TFAM coverage of the DNA molecule, which was calculated as described previously (Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar). We analyzed in total 221 fluorescent TFAM patches/spots bound to DNA, on DNA molecules ranging from 1% to 100% TFAM coverage. The obtained velocities of the TFAM molecules were binned according to the percentage of TFAM coverage. The results revealed that, at low TFAM coverage, on average TFAM molecules show a significant velocity (Figure 2E). In contrast, at high TFAM coverage, none of the TFAM molecules display any significant velocity, suggesting that DNA peeling is inhibited. It is interesting to note that, at low TFAM coverage, TFAM molecules exhibit a large range of velocities, with some molecules moving fast and some being stationary (Figure 2E, inset). We explain this result by the coexistence, on the same DNA molecule, of regions undergoing peeling (fast velocity) and regions, which are not yet undergoing peeling or cannot be peeled (stationary). TFAM binding to nonspecific DNA can induce local base-pair destabilization of DNA (Farge et al., 2012Farge G. Laurens N. Broekmans O.D. van den Wildenberg S.M. Dekker L.C. Gaspari M. Gustafsson C.M. Peterman E.J. Falkenberg M. Wuite G.J. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.Nat. Commun. 2012; 3: 1013Crossref PubMed Scopus (78) Google Scholar). We thus reasoned that high concentrations of TFAM could stabilize the junctions next to the melting bubbles and, as a consequence, prevent the bubbles from releasing the energy needed for the strand peeling. To exclude this possibility, we decided to consider only the DNA molecules with a low TFAM coverage (<10%) and to investigate the velocities as a function of the number of TFAM molecules in each fluorescent spot. The fluorescence intensity of a single fluorophore was calculated using single photobleaching steps (Figure S1) (Candelli et al., 2013Candelli A. Hoekstra T.P. Farge G. Gross P. Peterman E.J. Wuite G.J. A toolbox for generating single-stranded DNA in optical tweezers experiments.Biopolymers. 2013; 99: 611-620Crossref PubMed Scopus (39) Google Scholar). Our analysis demonstrated that TFAM patches with less than 20 molecules move during peeling, irrespectively of the number of TFAM molecules forming the spots. However, above a threshold of ∼20 TFAM per patch, the molecules do not show any significant movements, demonstrating an inhibition of the peeling process by a 20 TFAM filament on the DNA. Our findings so far demonstrated that TFAM filaments block DNA strand separation. This conclusion prompted us to investigate effects on transcription and mtDNA replication, two processes that involve DNA unwinding. We first investigated if a TFAM compacted DNA molecule could be used as template for mitochondrial DNA replication, a process that can be reconstituted in vitro by combining POLγ, the mitochondrial helicase TWINKLE, and mtSSB on a double-stranded DNA template with a preformed replication fork (Korhonen et al., 2004Korhonen J.A. Pham X.H. Pellegrini M. Falkenberg M. Reconstitution of a minimal mtDNA replisome in vitro.EMBO J. 2004; 23: 2423-2429Crossref PubMed Scopus (293) Google Scholar). We used this system to monitor effects of increasing TFAM concentrations (Figure 3A). We observed a slight decrease in mtDNA replication already at TFAM concentrations of 1 TFAM/40–60 bp. The TFAM concentration at this point was about 15–20 nM, well above the previously measured dissociation constant (KD) for TFAM binding to DNA, which is ∼4 nM (Kaufman et al., 2007Kaufman B.A. Durisic N. Mativetsky J.M. Costantino S. Hancock M.A. Grutter P. Shoubridge E.A. The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures.Mol. Biol. Cell. 2007; 18: 3225-3236Crossref PubMed Scopus (299) Google Scholar). When we increased the ratio to 1 TFAM/20 bp, which is similar to the expected in vivo ratio, we noted a decrease in mtDNA replication. A further increase to a ratio of 1 TFAM per 8 bp abolished DNA replication. We also measured the effects of TFAM levels on DNA binding/compaction (Figure 3B). To this end, we radioactively labeled the DNA template used in the rolling-circle assay and monitored effects of increasing levels of TFAM in an electrophoresis mobility shift assay (EMSA) experiment. At a ratio of 1 TFAM/40 bp, we could not observe nucleoprotein complexes and the template migrated as a naked DNA control (Figure 3B, compare lanes 1 and 2). The formation of a stable nucleoprotein complex was initiated at a ratio of 1 TFAM/20 bp, and full compaction of all DNA was observed when the levels of TFAM were further increased to 1 TFAM/12 bp. Interestingly, this full compaction coincided with a complete blockage of replication (Figure 3C). Our data therefore demonstrated that the DNA replication machinery can function at levels of TFAM similar to those observed in vivo. At higher protein levels, however, TFAM forms a stable nucleoprotein complex with DNA that completely blocks DNA replication. Finally, we investigated effects of TFAM on the DNA replication machinery in a time course experiment (Figure 3D). Before DNA replication was initiated, we preincubated the template with two different TFAM concentrations. At a lower ratio of TFAM to DNA (1 TFAM/60 bp), we observed robust levels of DNA synthesis, even if there was a slight delay in the rate of synthesis compared to a naked DNA template. When the TFAM to DNA ratio was increased to 1 TFAM/12 bp, we observed a strong decrease in DNA synthesis. However, the reactions were not completely blocked, since longer products were produced over time. Our experiments therefore demonstrated that DNA replication can progress also at higher TFAM ratios, but at a substantially reduced rate. Next, we monitored if TFAM compaction also affected transcription, using a reconstituted in vitro transcription system containing TFAM, POLRMT, and TFB2M (Falkenberg et al., 2002Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA.Nat. Genet. 2002; 31: 289-294Crossref PubMed Scopus (457) Google Scholar). We first used the natural HSP1/LSP cassette cloned from mtDNA, in which the two divergent transcription start sites are separated by only 160 nt (Figure 3E). We noted a dramatic drop in transcription when TFAM ratios were increased above the estimated in vivo rates of 1 TFAM per ∼15–18 bp of mt" @default.
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• W2086372007 date "2014-07-01" @default.
• W2086372007 modified "2023-10-10" @default.
• W2086372007 title "In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription" @default.
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• W2086372007 doi "https://doi.org/10.1016/j.celrep.2014.05.046" @default.
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