Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A

Abstract

Mitochondria organize their genome in protein-DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.

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Received 2 Jul 2012 | Accepted 10 Jul 2012 | Published 21 Aug 2012 DOI: 10.1038/ncomms2001

Graldine Farge1,*, Niels Laurens1,*, Onno D. Broekmans1, Siet M.J.L. van den Wildenberg1, Linda C.M. Dekker1, Martina Gaspari2, Claes M. Gustafsson3, Erwin J.G. Peterman1, Maria Falkenberg3 & Gijs J.L. Wuite1

Mitochondria organize their genome in proteinDNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and uorescence microscopy to show the nonspecic DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the exibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specic transcription regulation by TFAM. This apparent connection between transcription and DNA organization claries how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.

1 Department of Physics and Astronomy and LaserLaB, VU University, De Boelelaan 1081, Amsterdam 1081 HV, The Netherlands. 2 Division of Laboratory Medicine, Karolinska Institutet, Novum SE-141 86, Stockholm. 3 Department of Medical Biochemistry and Cell Biology, University of Gothenburg, PO Box 440, Gothenburg SE-405 30, Sweden. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.J.L.W. (email: [email protected]).

NATURE COMMUNICATIONS | 3:1013 | DOI: 10.1038/ncomms2001 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2001

Human mitochondrial DNA (mtDNA) is a 16.6-kb circular double-stranded DNA (dsDNA) molecule present in thousands of copies in the cells mitochondrial network. It

encodes 13 proteins of the respiratory chain, as well as 2 ribosomal RNAs and 22 tRNAs. Mutations in the mitochondrial genome can cause profound problems that are linked to a variety of multisystem disorders1. Similar to chromosomal DNA, which is organized into a highly condensed structure to t within the limited volume of the nucleus, multiple mtDNA molecules are organized into small (~70 nm) proteinDNA complexes called nucleoids2,3. In eukaryotes, compaction is mostly achieved by wrapping DNA around his-tones to form nucleosomes. In bacteria, this is accomplished by a combination of mechanisms, including molecular crowding, super-coiling and the action of architectural proteins that bend, wrap or bridge DNA4,5. To date, little is known about the organization of the mtDNA in mitochondrial nucleoids.

Various constituents of the mitochondrial nucleoid have been identied by immunoprecipitation, most of them being involved in essential processes such as mtDNA replication, transcription and repair6. The major protein component of the nucleoid is the mitochondrial transcription factor A7 (TFAM), which is essential for the maintenance of mtDNA and has been implicated in multiple functions in mtDNA metabolism. TFAM was rst identied as a transcription factor that binds specically to the promoter region of the mtDNA and, together with the transcription factor B2, enhances transcription by the mitochondrial RNA polymerase8,9. The structure of TFAM bound to the promoter region of mtDNA has been solved recently10,11. It reveals that each of the two high-mobility group (HMG) box domains of TFAM causes the DNA to bend nearly 90, resulting in a complete U-turn, a favourable arrangement for transcription initiation. TFAM has also been proposed to have an essential role in the organization of the mitochondrial genome12, because it shows strong non-sequence-specic DNA binding and

is abundant enough to coat the entire mtDNA13. Moreover, uorescence resonance energy transfer and atomic force microscopy (AFM) data suggest that nonspecically bound TFAM and its yeast homologue, Abf2p, can compact DNA by bending the DNA backbone10,14 and/or promoting the formation of loops15,16. However, a clear understanding of the assembly of TFAM on mtDNA and the subsequent organization mechanism is lacking.

In this study, we characterize the nonspecic DNA assembly dynamics of TFAM, as well as the structural role of TFAM in DNA organization, using a combination of single-molecule manipulation and visualization techniques. We show that TFAM induces compaction by modifying the exibility of the DNA. Moreover, we observe in real time that TFAM forms highly stable protein patches on DNA, due to the propensity of TFAM to extensively diuse on DNA before it cooperatively binds on collision with a TFAM patch. On the basis of these observations and, in line with previously reported results10,14, we propose a molecular mechanism to explain the role of TFAM in DNA organization.

ResultsTFAM compacts DNA by increasing its exibility. To investigate the interaction of TFAM with nonspecic DNA molecules under dierent buer conditions, we performed tethered particle motion (TPM) experiments17 (Fig. 1). We recorded the root-mean-square (r.m.s.) motion of the beads over time. Aer addition of TFAM, we observed a lower r.m.s. value, reecting a decrease in the endto-end length of the DNA tethers, that is, compaction (Fig. 1a). To examine the ionic-strength dependency of this phenomenon, the TPM experiments were performed at dierent NaCl and MgCl2

concentrations (Supplementary Fig. S1). A stable reduction of the tether length was observed in buers with low ionic strength (for example, 75 mM NaCl and 0 mM MgCl2). Under these experimental conditions, TFAM caused a gradual decrease of the r.m.s. value,

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Figure 1 | TFAM compacts DNA. (a) Typical TPM data traces showing the DNA end-to-end length shortening by TFAM. The root-mean-square (r.m.s.) amplitude of the Brownian motion of the beads is plotted versus time, in the absence (black trace) and presence (red trace) of 100 nM TFAM. The histograms of the r.m.s. motion are shown on the right. The schematic represents a TPM assay: a single DNA molecule is attached between a glass surface and a bead. The amplitude of the restricted Brownian motion of the bead is related to the end-to-end length of the DNA. In the presence of TFAM (green dots) the DNA becomes shorter. (b) Effect of increasing concentration of wild-type TFAM (black symbols) or C-TFAM (open symbols) on the length of DNA (mean s.e., N = 20). (c) Histograms of r.m.s. motion in the presence of increasing concentration of TFAM. Shown are the r.m.s. motion of DNA tethers with increasing amount of TFAM. The single, well-dened Gaussian distribution of the r.m.s. motion for each of the concentrations indicates that TFAM compacts DNA in the absence of looping.

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Figure 2 | TFAM increases the exibility of DNA and binds to DNA cooperatively. (a) Schematic representation of the multichannel ow cell. Two beads are optically trapped (I); a single DNA molecule is captured between the beads (II); the DNA molecule is moved to a buffer channel where a force-extension curve of the DNA is measured (III); the DNA molecule is incubated with the TFAM protein (IV) and a second force-extension curve is measured in the buffer channel (V). (b) Typical force-extension curve for DNA in the absence (black trace) and presence (red trace) of 50 nM TFAM. The contour length (Lc) and the end-to-end distance of a DNA molecule are schematically depicted. (c) Effect of increasing TFAM concentrations on the persistence length and contour length of DNA (inset; mean s.d., N = 15). The red line represents the Mc-Ghee-von Hippel t to our data and the dashed line the Mc-Ghee-von Hippel t assuming a cooperativity factor of 1.

from 137.9 0.6 nm for bare DNA molecules to 89.3 0.9 nm at saturating TFAM concentration (mean s.e., N = 20; Fig. 1b). From these experiments, we conclude that TFAM can compact DNA, consistent with its presumed role in nucleoid formation.

It has been suggested that, besides having an essential role in the promoter-specic initiation of transcription, the basic C-terminal tail of TFAM is important for nonspecic DNA binding18,19. To test this, we used a truncated version of TFAM lacking the C-terminal domain (C-TFAM) and monitored the r.m.s. motion of the beads at increasing protein concentrations (Fig. 1b). We observed that the truncated protein retained the ability to compact DNA, albeit to a slightly lesser extent than wild-type (WT) TFAM. Hence the C-terminal domain of TFAM is not necessary for the protein to compact DNA.

Does this compaction arise from loop formation, as suggested in previous AFM studies15,16? In that case, on nonspecic DNA, one would expect that, at each TFAM concentration, loops of varying sizes were formed. In TPM, this would result in a broad distribution of r.m.s. levels, and thus a very broad r.m.s. histogram, as was observed for other looping proteins17. In contrast, we observed one well-dened, reproducible, narrow Gaussian distribution at each TFAM concentration (Fig. 1c). This indicates that it is unlikely that compaction takes place by unspecic loop formation.

To understand how TFAM can compact DNA without forming loops, we used optical tweezers to investigate the elastic properties of bare DNA and TFAM-coated DNA. Single DNA molecules of 48 kb (-phage DNA) were end-labelled with biotin and attached between two streptavidin-coated beads20 (Fig. 2a). Aer capture, the DNA molecules were progressively stretched while the force was measured as a function of DNA extension. A typical force-extension curve of a bare DNA molecule is shown in Fig. 2b. On addition of TFAM, we observed that the mechanical response of the DNA changed signicantly. We quantied the TFAM-induced changes of DNA by tting the force-extension curves with the extensible and the twistable worm-like chain models (including forces of up to 30 and 60 pN, respectively)21,22. The persistence length (Lp, a measure for the stiness of the DNA), the contour length (Lc, the total length of the DNA molecule along the backbone), the stretching modulus (K0, the spring constant of the DNA backbone) and the twiststretch coupling of DNA (described by the three parameters g0, g1 and Fc) were obtained for TFAM concentrations ranging from

0.01 to 100 nM (Fig. 2c; Supplementary Table S1). The values of the DNAs stretching modulus and twiststretch coupling did not change on addition of TFAM, indicating that TFAM does not modify either the spring constant or the twiststretch behaviour

of the DNA. For the persistence length, we measured a remarkable decrease, from 45.0 6.8 nm for the bare molecules to 3.9 2 nm at saturating TFAM conditions (mean s.d., N = 13), indicating that the molecule was more exible. Finally, we resolved a slight but signicant increase of the DNA contour length (8%; Supplementary Table S1). Thus, it seems that DNA compaction by TFAM is mediated by its ability to strongly increase the exibility of DNA.

Assembly of TFAM nucleoprotein structures. Next, we studied how TFAM assembles into compact nucleoprotein structures. We started this investigation by determining the footprint of a single TFAM molecule on DNA, using a combination of optical tweezers and uorescence microscopy. The uorescence intensity of single Alexa-555-labelled TFAM monomers was quantied using photo-bleaching (Fig. 3a,b). Thereaer, we determined the total uorescence intensity of a fully TFAM-coated DNA molecule (Fig. 3a) and converted this intensity to the number of TFAM monomers. This way, we found that the footprint of a TFAM monomer was 30.3 0.3 bp (mean s.e., N = 23). To verify this result, we performed a micrococcal nuclease protection assay, which has been used previously to determine the periodic DNA binding pattern of nucleosomes on chromosomal DNA23. Radio-labelled DNA of ~500 bp was treated with micrococcal nuclease in the absence or presence of TFAM. Aer nuclease treatment, an undigested fragment of ~27 bp was observed in the reaction containing TFAM, corresponding to the size of the DNA fragments protected by TFAM (Fig. 3c). Another protected fragment of about 1015 bp could also be observed; however, this fragment was also visible in the control experiment performed in the absence of TFAM, showing that this protected fragment is protein-independent. The size of this footprint conrms the relatively large value determined with our single- molecule assay, and is in agreement with the recently published structural data showing that TFAM makes extensive contact with the DNA10,11.

We analysed the oligomeric state of TFAM bound to DNA, as it has been suggested that TFAM binds in a dimeric form24. We used a low concentration of uorescently labelled TFAM and monitored in real time at the single-protein level the nonspecic TFAM binding to DNA. In this experiment, we always observed the uorescence disappearing in one step, pointing towards a monomeric state of TFAM on DNA. However, we cannot exclude that this phenomenon is due to dissociation of the protein from DNA. To clarify this point, we compared the average uorescence intensity of a protein binding event to DNA (57 3 arbitrary unit (a.u.) averaged over the rst three frames; mean s.e.; N = 66) to the value of the uorescence

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Figure 3 | TFAM has a footprint of 30 bp on DNA. (a) A single uorescent TFAM bound to a DNA molecule (upper image) and a DNA molecule fully coated with TFAM (100 nM; lower image). The DNA, held between two beads in the optical tweezers, is not visible. Scale bar, 1 m. (b) Fluorescence signal of a TFAMAlexa-555 complex as a function of time, showing a single-step photobleaching event characteristic of a single molecule. (c) Intensity histogram of single TFAMAlexa-555 complexes. The uorescence intensity was measured for 20 single complexes and the intensity distribution was tted with a Gaussian, yielding a mean uorescence value for a single TFAMAlexa-555 complex of 142.0 1.5 a.u. (mean s.e., N = 20). (d) Micrococcal nuclease protection assay. A radioactively labelled DNA fragment was digested with micrococcal nuclease for increasing periods of time in the absence or presence of a constant amount of TFAM. An undigested fragment of approximately 27 bp was obtained. SM, size marker (in bp).

intensity of a single Alexa-555 (mean s.e.: 61 2 a.u.; N = 75) and to the value of a single TFAMAlexa-555 (mean s.e.: 63 2 a.u.; N = 75) attached to a glass coverslip. These three values were similar, indicating that TFAM binds to DNA from solution as a monomer. Notably, recent crystal structures showed that specic DNA binding of TFAM also happens in a monomeric manner10,11.

When performing these experiments, we discovered that the single TFAM monomers that bind to the DNA nonspecically started to move rapidly back and forth along the DNA over distances that span several kilobases (Fig. 4a; Supplementary Movie 1). The movement of TFAM on DNA was tracked with nanometre precision by tting a two-dimensional Gaussian to the intensity prole in each frame25 (Fig. 4b). Analysis of the motion of TFAM on DNA revealed a linear mean-squared-displacement (MSD; Fig. 4c), as expected for a protein moving on DNA by one-dimensional diusion25,26. The diusion coefficient obtained from the linear t was D = (8.6) 104 0.5 nm2 s 1 at 25 mM NaCl (mean s.e., N = 66). Proteins use dierent mechanisms to move along DNA, such as hopping, jumping or sliding26. We measured the diusion coefficient of TFAM over a range of salt concentrations and found that it was not salt-dependent (D = (9.1 0.7)104 nm2 s 1 at 75 mM NaCl, N = 44, and (7.4 0.9)104 nm2 s 1 at 150 mM NaCl, N = 37), consistent with a sliding mechanism26,27.

To obtain information on the assembly of TFAM into nucleo-protein structures, we performed a dual-colour uorescence experiment. To this end, we labelled TFAM with two dierent dyes, Alexa-555 and Atto-647N, mixed them and visualized their behaviour on DNA (Fig. 4d). The uorescence intensity of single Alexa-555 and Atto-647N-labelled TFAM monomers was

quantied using photobleaching (Supplementary Fig. S2). In these dual-colour experiments, we observed that TFAM monomers (and occasionally dimers) diused along the DNA, while higher-order multimers formed immobile patches. Moreover, when a diusing TFAM protein encountered a stationary TFAM patch, the diusion stopped and the protein aggregated with this patch (Fig. 4d). Our direct visualization of the assembly of TFAM nucleoprotein structures shows that nonspecic TFAM interaction leads to stable DNA compaction in a two-step mechanism: TFAM monomers rst slide on DNA, before assembling into nucleoprotein laments, by fusing with any encountered patch.

Dynamics of TFAM nucleoprotein assembly. To further investigate the binding parameters of TFAM on DNA, we incubated DNA molecules with a relatively high concentration of uorescent TFAM (50 nM). We noticed that DNA molecules incubated for 30 s and imaged in a protein-free region of our microuidic ow chamber were not uniformly coated. Instead, patches were formed (Fig. 5a), as was also observed at lower TFAM concentration (Fig. 4d). This observation, together with the sharp decrease of the DNAs persistence length as a function of TFAM concentration (Fig. 2c), indicates that TFAM binds to DNA in a cooperative manner. To quantify the cooperative binding of TFAM to DNA, we tted the concentration dependence of the persistence length with the McGhee-von Hippel model28 (Supplementary Fig. S3). In this model, the binding of a protein to DNA is characterized by an equilibrium binding constant K (in M 1), a cooperativity factor and a footprint n. Using our previously obtained footprint of TFAM on DNA as a xed parameter, the t yielded a cooperativity factor of 70 40.

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Figure 4 | Visualizing TFAM diffusion and patches formation on DNA. (a) Kymograph showing the diffusion of TFAMAlexa-555 on DNA. Time (s) and distance (m) are indicated at the bottom and left, respectively. (b) Representative trajectories generated from tracking the motion oftwo single TFAMAlexa-555. (c) Mean-squared displacement (MSD) of TFAMAlexa-555 versus time interval (error bars: s.e., N = 66 diffusion traces). The diffusion coefcient is calculated from the linear t (red line) to the MSD plot. (d) Kymograph showing TFAM patch formation on DNA. TFAM was labelled either with Alexa-555 (green) or Atto-647N (red). The two TFAM preparations were mixed and incubated with the DNA. The uorescence intensity of a single TFAMAlexa-555 and a single Atto-647N were determined by single photobleaching steps. On the basis of these values, we determined that the two green moving spots correspond to TFAM monomers, whereas the upper and lower red signals correspond to patches of, respectively, 4 and 6 TFAM molecules.

This implies that TFAM is ~100 times more likely to stably bind next to an already bound TFAM than to bare DNA. The t also yielded an equilibrium binding constant K between 0.9106 and 2.4106 M 1 (best t: K = 1.6106 M 1), which corresponds to a value of 1/K of approximately 610 7 M (Fig. 2c). This value is within the range of affinities reported earlier for TFAM (approximately 0.610 7 M)15,19 and the (low) affinity of the yeast homologue Abf2p (approximately 2510 7 M)16,29. The dierence between our numbers and the reported ones is likely due to a combination of dierent factors, such as the length of the DNA used and the ionic strength.

Direct visualization of TFAM binding at high concentrations is impossible because of the uorescent background in the protein channel of our microuidic ow chamber. However, TFAM binding decreases the persistence length of the DNA, which, in turn, results in a shortening of the end-to-end length of DNA kept under constant tension (10 pN) (Fig. 5c, green arrow and inset). Therefore, monitoring this change provides a suitable approach to follow TFAM assembly on DNA in real time. We converted the measured DNA end-to-end distance into a fractional coverage of the DNA (Supplementary Fig. S3). The sharp sigmoidal shape of the saturation curve is consistent with a cooperative binding mode (Fig. 5d, black symbols). We performed Monte Carlo simulations to interpret this saturation curve and to extract the nonspecic TFAM binding rate (Fig. 6, and Methods section). In this model, we included the observed diusion and recruitment of TFAM monomers to patches by dening an enlarged target site at the ends of these patches. The enhanced binding at these enlarged target sites is dened by the cooperativity factor we determined previously ( ~100; Fig. 6). By using these assumptions, we could reproduce and t our data (Fig. 5d, red line). This simulation yielded an average association

rate per base pair of (3.5 0.5)102 M 1 s 1 per bp (mean s.d., N = 10). The S-shape of the measured curve could only be reproduced by assuming cooperativity for binding, but not by assuming cooperative dissociation. Furthermore, we found an enlarged target size for binding of at least 200 bp. We interpret this enlarged target site as the minimal distance a TFAM monomer scans while diusing, which is consistent with our diusion experiments, showing that TFAM is able to diuse over distances up to several micrometres.

Finally, we investigated the unbinding of TFAM from DNA in real time by monitoring the uorescence intensity of DNATFAM complexes (Fig. 5a). The decay of intensity allowed a direct measurement of the dissociation time (Fig. 5b). Under our experimental conditions (25 mM NaCl, 10 mM Tris, pH 7.6), the dissociation of TFAM was fairly slow: 315 50 s (mean s.e., N = 15), corresponding to a rate of (3.2 0.6)10 3 s 1. The salt dependence of the dissociation was examined by performing the same experiment at increasing monovalent and divalent salt concentrations (Supplementary Fig. S4). We found that the dissociation rate increases with increasing salt concentration, to reach, in more physiological conditions (150 mM NaCl), a rate of (3.0 1.0)10 2 s 1.

Discussion

In this study, we investigated the mechanisms by which TFAM compacts DNA. We show that TFAM is able to bind and compact DNA under tensions up to 40 pN (Supplementary Fig. S5). In contrast, compaction by nucleosomes through wrapping is slowed down at 5 pN, and even inhibited at tensions exceeding 10 pN (ref. 30). Furthermore, DNA wrapping results in a shorter DNA molecule30, while TFAM actually slightly increases the DNAs length. A wrapping mechanism can therefore be excluded. Similarly, compaction by bridging of DNA molecules, as used by H-NS5,31, is very improbable, because our TPM experiments show that TFAM does not loop DNA. Moreover, in our optical tweezers experiments, when extending a DNA molecule that had been incubated in a completely relaxed conformation with TFAM, two characteristics of DNA bridging5 (that is, an eective decrease in the DNAs contour length; and an increase of force, followed by decreases of force as the bridges break on DNA stretching) were not observed. We can thus exclude DNA looping and/or bridging as mechanisms for DNA compaction by TFAM. It should be noted that an increased DNA exibility due to TFAM binding makes it easier for DNA to coil up on a surface which, in an AFM image, might appear as a looped DNA molecule, as has been reported previously15.

One hypothesis that can explain DNA compaction is a bending mechanism, in which the protein would introduce multiple bends in the DNA backbone. Several non-sequence-specic architectural proteins, such as the eukaryotic HMG and the non-histone HU bacterial protein4,32, have been proposed to compact DNA this way. The introduction of such bends would cause an apparent change of the DNAs exibility, and would thus be consistent with the observed decrease of the DNAs persistence length in the presence of TFAM. AFM and uorescence resonance energy transfer (FRET) experiments have shown that TFAM and Abf2p decrease the end-to-end distance of DNA10,14,16. This is in agreement with our TPM data where we also measured a decreased DNA end-to-end distance in the presence of TFAM. Some of these articles indicated that the decrease of end- to-end distance would result in the formation of rigid bends in the DNA15,16.

Recent crystallographic analyses of TFAM bound to its specic binding site have shown that the two HMG domains of TFAM lock into the DNA minor groove to generate two 90 kinks, resulting in a U-turn of the DNA10,11. If such rigid bends would also be introduced by unspecic binding of TFAM to DNA, exerting a force on the DNA would energetically disfavour protein binding and enhance protein dissociation33. To test this hypothesis, we

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Figure 5 | Unbinding and binding dynamics of TFAM on DNA. (a) Selected frames from a movie recording the unbinding of TFAM from DNA. Scale bar,1 m. (b) Intensity trace of a single DNA molecule covered with TFAM held at constant tension (open square symbols). The data were t as described in the Methods section (red line). The distance between the two beads (inset) increases as the uorescence intensity decreases. (c) Real-time association of TFAM to a single DNA molecule. A force-extension curve was measured before (black symbols) and after (red symbols) incubation of the DNA molecule with 50 nM TFAM. During incubation, the DNA molecule was held at constant tension (10 pN) and the distance between the beads was recorded over time (green symbols and inset). (d) The distance measured in c was converted to a percentage of protein coverage and plotted versus incubation time (black symbols). The experimental data points were tted with a Monte-Carlo simulation (Methods; red trace).

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Figure 6 | Determination of the association rate per site and the enhanced target site of TFAM by Monte-Carlo simulations. (a) One-dimensional lattice of protein binding sites (bp of the DNA). For each time step, there is a certain probability K for a protein to bind to the lattice (left). If, however, there is a protein already bound within a certain distance (enhanced target site), the probability of binding of a new protein is enhanced by a factor

(right). (b,c) Fits of the experimental data to the simulation curves. (b) The r.m.s.e. along the Kon axis of N = 10 traces shows a clear minimum that gives the best t to our data set. (c) The r.m.s.e. along the target site axis, in contrast, presents a very at behaviour, indicating that as soon as the target size

is around a few hundred base pairs long, increasing its size does not result in a better t.

measured the dependence of TFAM association and dissociation on the force exerted on the DNA (Supplementary Fig. S5). We found that both association and dissociation of TFAM from DNA were force-independent. Moreover, we noticed that TFAM binding was stable even aer multiple cycles of DNA extension/relaxation. In a previous study, we investigated the impact of force on DNA bending34 and showed that even mild bending would be hampered by tension on the DNA. It is thus unlikely that DNA compaction by TFAM is achieved by the formation of rigid bends in the DNA backbone. It is also possible that TFAM binds and stabilizes spontaneous

DNA bends. However, again, TFAM binding would then be slowed down under tension.

We instead propose that, in contrast to the ~180 static DNA bend created when TFAM interacts with its specic binding site10,11, nonspecic binding of TFAM makes DNA more exible. In fact, small-angle X-ray scattering (SAXS) experiments have shown that TFAM in solution is highly exiblea exibility that is conferred by the linker region and the C-terminal domain11. The dierence between a xed bend at a specic site, and a broad range of angles at a nonspecic site, could be explained by the fact that, for the specic

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Figure 7 | Model of cooperative patch formation and DNA compaction by TFAM. TFAM binds to DNA and diffuses until it reaches an already formed patch. This nonspecic DNA binding results in an enhancing of the DNAs intrinsic exibility, possibly by a local denaturation of the DNA, and thusa compaction of the DNA. This melting of the DNA occurs when TFAM is stably bound to the DNA, not when it is rapidly moving on the DNA.

DNA site at light strand promoter (LSP), there is a xed optimal distance between the two HMG binding sequences, thus imposing a static bend in the DNA.

The compaction mechanism we thus propose is called a exible hinge model. This model has rst been proposed for the architectural proteins HU4 and HMG32. These proteins, similar to TFAM, induce a large decrease of the DNAs persistence length, with little or no change of the contour length. Also, it was shown in an optical tweezers experiment that HMG did not unbind when high forces (up to 150 pN) were applied to the DNA35. Thus, our results are most consistently explained by a exible hinge model, in which the nonspecic DNA binding of TFAM increases the intrinsic exibility of the DNA, causing the formation of an ensemble of bending angles. Such a exible hinge model can yield an apparent rigid bending angle, because the calculated bending angle is an average of all the possible angles that can be formed on TFAM binding. This would thus explain the observed 78, 72 and 100 20 degrees angles obtained in previous studies1416, but at the same time provide an explanation for the insensitivity of TFAM binding and unbinding to force.

How could TFAM induce DNA exibility? We noticed in the force-extension curves that the so-called overstretching transition (a region where the DNA lengthens by almost a factor of two at a force of ~65 pN) changes on addition of TFAM (Fig. 2b). At low salt concentration, during the overstretching transition, the dsDNA melts and is converted into two single-stranded DNA (ssDNA) strands36. The force uctuations during the overstretching transition for the bare DNA molecule are characteristic of a melting of the DNA molecule via an unpeeling of one of the strands, starting from its extremities or from nicks. In contrast, the overstretching transition in the presence of TFAM is smoother, which could indicate a conversion mode that involves base-pair destabilization that is not initiated at the ends of the molecule or at nicks21. On the basis of this observation, it might be that the binding of TFAM to DNA induces some local denaturation of the DNA. Moreover, the crystal structures of specic TFAM binding show unwinding of the DNA extending from the bound HMG boxes10,11. Thus, TFAM molecules might, on stable binding, induce some denatured or unwound DNA around them (Fig. 7). As ssDNA is much more exible than dsDNA, TFAM binding would thus result in an increase in the DNAs intrinsic exibility, and thereby promote DNA compaction. The small increase in the DNAs contour length observed in our stretching experiments is also in agreement with the hypothesis that some denaturation might take place because ssDNA has a larger contour length than dsDNA. Assuming that this is the mechanism involved, we estimate the number of base pairs (x) that are denatured on TFAM binding, using the value of the contour length obtained in the presence of TFAM (LcTFAM):

L L x L x

cTFAM cdsDNA cssDNA

= +

* ( ) *

48502 (1)(1)

with Lc dsDNA = 0.34 nm per bp, Lc ssDNA = 0.58 nm per bp and Lc TFAM = 17.4 m.

Solving equation (1) yields a value of x = 3.8 0.4 kb converted from dsDNA into ssDNA per DNA molecule. Using our determined footprint of TFAM, this value corresponds to a melting bubble of 23 bp between neighbouring TFAM molecules.

We have shown that the binding of TFAM is enhanced by the presence of TFAM already bound on the DNA. What are the mechanisms responsible for TFAM (cooperative) binding? Our two-colour experiments show that, aer binding to the DNA, TFAM diuses until it reaches a patch, whereupon it binds stably. Cooperativity is thus due to an enhancement of the binding of diusing TFAM molecules next to patches. Our Monte-Carlo modelling further conrms this hypothesis, as we demonstrate that TFAM requires an enhanced target size of at least 200 bp to account for the nucleation and subsequent cooperative binding. An explanation for the stable binding of TFAM aer it reaches a patch is that, once in the vicinity of an already bound TFAM, the diusing protein could sense the structural change in the DNA (that is, melting) induced by the bound protein (Fig. 7). This locally destabilized DNA, in turn, might help the stable binding of TFAM to the DNA. The combination of TFAM sliding and melting could thus explain the observed nucleation behaviour and cooperative binding of TFAM.

Finally, we propose that these features of TFAM bindingsliding and meltingcould also be used by TFAM when acting as a specic transcription factor. First of all, the propensity of TFAM to slide on the DNA before stable binding could facilitate the localization of the specic binding site in the promoter regionsimilar to the lac repressor, for which the nonspecic DNA binding associated with sliding considerably increases the rate of target location37. Secondly, TFAM could locally melt the surrounding DNA when bound to the promoter region, which would permit the mitochondrial RNA polymerase and the transcription factor B2 to bind and transcription to initiate38. This connection between transcription initiation and DNA organization would explain how TFAM can accomplish its two complementary roles in the mitochondrial nucleoid simultaneously.

Methods

TFAM preparation and labelling. WT TFAM and the truncated form of TFAM (C-TFAM) were expressed and puried as previously described for WT TFAM9. To obtain uorescent TFAM, the cysteine residues of the protein were labelled with either maleimide Alexa-555 (Molecular Probes) or Atto-647N dye (Sigma-Aldrich). Unreacted dye was removed from the sample with size-exclusion spin-columns (Sephadex G-25, GE Healthcare). The labelling ratio, determined by single-step photobleaching of labelled TFAM molecules immobilized on a glass surface, was one uorophore per TFAM monomer. The TFAM enzyme preparation used in all experiments was fully active, as determined using a gel-shi assay. All the experiments were done at room temperature (19 C).

DNA substrates. The DNA construct used for the TPM experiments (528 bp) was obtained by PCR amplication of the plasmid pSKFokI, a 2,953-bp derivative of pBluescriptSK()39, using a digoxigenin-modied reverse primer (5-DIG-CGATT TCGGCCTATTGGTTAAAAAATGAGC-3) and a biotin-labelled forward primer (5-BIO-CAGGCTTTACACTTTATGCTTCCGGCTCG-3). The PCR was puried using the QIAquick PCR purication kit (Qiagen). Lambda DNA (48,502 bp) used for the dual-optical tweezers experiments was biotinylated at the 5-ends of both strands using Klenow polymerase, as described in ref. 20.

Micrococcal nuclease digestion assay. A radiolabelled fragment of 530 bp was generated by PCR amplication of the plasmid pUC18 using the oligonucleotides: 5-GTAAAACGACGGCCAGTGCCAAGCTTGC-3 and 5-CGATTTTTGTGAT GCTCGTCAGGGGGG-3 as primers, in the presence of -32P dCTP. The PCR was puried on a QIA quick spin column (Qiagen). The labelled DNA fragment(2 fmol) was incubated with 200 fmol of TFAM for 20 min at 20 C in 10 mM Tris HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mg ml 1 bovine serum albumin, and 1 mM CaCl2 in a nal volume of 15 l. Micrococcal nuclease (1.610 4 U,

Sigma) was added and the reaction was incubated at 37 C and quenched by the addition of EDTA (15 mM nal concentration) at the times indicated in the gure legend. The samples were treated with 0.5% SDS and 0.2 mg ml 1 proteinase-K for 60 min at 42 C, and precipitated by addition of 0.6 ml of ice-cold ethanol.

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The pellets were dissolved in 10 l H2O and 2 l of gel loading buer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll in water) and separated on an 8% native polyacrylamide gel in 1 TBE buer. The gel was dried before exposure.

Tethered particle motion assay. DNA molecules were attached between a glass surface and 440-nm polystyrene beads as described in ref. 17. Flow cell preparation, data acquisition and analysis were performed as previously described17. On average, 40 single DNA tethers were measured simultaneously in a buer containing 10 mM TrisHCl, pH 7.6, 1 mM dithiothreitol and the salt (NaCl or MgCl2) concentration indicated in the text.

Optical trapping and uorescence microscopy. The combined single-molecule uorescence and optical trapping instrument, as well as the custom-built micro-uidic ow system with multiple laminar channels, has been described in detail elsewhere20,36. Before use, the ow cell was coated with casein (100 g ml 1), to decrease the adsorption of the protein to the glass surface. To reduce photo-bleaching of the uorophores, buers were degassed and kept under nitrogen atmosphere, and 1 mM of the reducing agent dithiothreitol was added.

Determination of diffusion coefcients. The diusion of TFAM on DNA was measured using a low concentration of TFAM (20 nM). The acquired movies were analysed with a custom-written tracking program (Labview VIEW, National Instruments). Briey, the position of a moving TFAMAlexa-555 was obtained from a two-dimensional Gaussian t to the intensity prole in each frame25,40. Only traces with a minimal length of 20 frames (10 s) were used. The obtained positions were connected to form a trajectory from which the displacement for the dierent time intervals was determined. The MSD was calculated by averaging the squared displacement per time interval over all trajectories measured40 at a given salt concentration. The diusion constant (D) was calculated from the MSD plot (MSD = 2Dt + oset).

Determination of disassembly times. Single Lambda DNA molecules were incubated in the presence of 50 mM of TFAMAlexa-555 for 30 s. Aer incubation, the DNAprotein complex was rapidly moved to the buer channel (to avoid uorescence background due to the free uorescent protein), held at constant tension and visualized by stroboscopic illumination of the sample (usually 0.5 s every 30 s) with a 532-nm excitation laser (Fig. 5a). The dissociation times (diss) were determined by tting the uorescence intensity traces over time (Fig. 5b, black symbols) with equation (2) (Fig. 5b, red trace):

I=I .

0 diss

diss bleach bleach

e e

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32. Skoko, D., Wong, B., Johnson, R. C. & Marko, J. F. Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals

t t

where tdiss is the total time, tbleach the illumination time and bleach the photobleaching time of the uorophore (obtained for an independent experiment).

Monte-Carlo simulation. The association rate per site (Kon) and the enhanced target site of TFAM were determined by performing Monte-Carlo simulations

(Fig. 6). A one-dimensional lattice of protein binding sites (bp of the DNA) was simulated (Fig. 6a). For each time step dt (with dt chosen sufficiently small), there is a certain probability K for a protein to bind to the lattice (le). If, however, there is a protein already bound within a certain distance (enhanced target site) the probability of binding of a new protein is enhanced by a factor (right). Once bound to DNA, the protein diuses and forms a patch with the already bound proteins. The simulation ends at a given time or when all lattice sites are occupied. The fraction of sites that are occupied is calculated for each time step and gives the simulated curve (Fig. 5d, red line). The simulations do not include an o-rate for proteins, as we showed that the association is much faster than dissociation (Fig. 5b versus Fig. 5d). To t the simulated traces to our experimental TFAM-association curves, a database consisting of 10,000 simulated traces was built by varying the target site linearly between 1 and 1,000 and the binding probability Kon logarithmically between 10 5 and 10 3 s 1. Each trace in the database was obtained by simulating a DNA molecule of 50,000 bp and was averaged over ten individual simulation runs. The traces were shied with a time oset t0 to t against the experimental data, as the binding time of the rst TFAM to the DNA can vary.

The reduced 2 for every trace was recorded; the global minimum and the shape of the well gave the best t and the condence band, respectively.

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Acknowledgements

We thank A. Biebricher and A. Candelli for their input to this study. This work was

supported by: Physics of the Genome of Stichting voor Fundamenteel Onderzoek der

Materie, Nederlandse Organisatie voor Wetenschappelijk Onderzoek, VICI and ECHO

grant as well as an ERC starting grant.

Author contributions

G.F. and G.J.L.W. designed research; G.F., N.L., O.D.B., L.C.M.D. and M.G. performed

research; C.M.G. and M.F. provided proteins and tested their biochemical activity; G.F.,

N.L., O.D.B. and S.M.J.L.vd.W. analysed data; G.F., N.L., O.D.B., M.F., E.J.G.P. and

G.J.L.W. wrote the paper. G.J.L.W. supervised the project.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/

naturecommunicationsCompeting nancial interests: The authors declare no competing nancial interests. Reprints and permission information is available online at http://npg.nature.com/

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How to cite this article: Farge G. et al. Protein sliding and DNA denaturation are

essential for DNA organization by human mitochondrial transcription factor A.

Nat. Commun. 3:1013 doi: 10.1038/ncomms2001 (2012).

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