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DNA-protein interactions explored by atomic force microscopy
Accepted Manuscript Title: DNA-protein interactions explored by Atomic Force Microscopy Authors: S. Kasas, G. Dietler PII: DOI: Reference:
S1084-9521(16)30492-X http://dx.doi.org/doi:10.1016/j.semcdb.2017.07.015 YSCDB 2278
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
23-5-2017 12-7-2017 13-7-2017
Please cite this article as: Kasas S, Dietler G.DNA-protein interactions explored by Atomic Force Microscopy.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.07.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-protein interactions explored by Atomic Force Microscopy
S. Kasas1,2 and G. Dietler1
1
Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2
Plateforme de Morphologie, Faculté de Médecine, Université de Lausanne, Bugnion 9, 1005 Lausanne, Switzerland
Corresponding author email address : [email protected]
Keywords DNA, protein, enzyme, AFM
Abstract DNA-protein interactions play an important role in all living organisms on Earth. The advent of atomic force microscopy permitted for the first time to follow and to characterize interaction forces between these two molecular species. After a short description of the AFM and its imaging modes we review, in a chronological order some of the studies that we think importantly contributed to the field.
1. Introduction The majority of DNA-binding proteins play an important role in the regulation of gene expression. Their interaction with DNA are therefore of prime importance for all living organisms, starting from bacteria up to mammalian cells. Numerous fundamental biological processes such as DNA replication, packing, recombination, DNA repair, RNA transport and translation are controlled by these types of interactions. These mechanisms control the growth, the differentiation and the evolution of living organisms. Most of DNA-binding proteins directly prevent or release the transcription of certain genes according to environmental conditions, stages of development or specific stimuli. Some others such as histones and some high mobility group proteins, pack genomic DNA into compact form. Since only the genes that are accessible to the transcriptional machinery can be decoded, these proteins therefore indirectly control transcription. Any perturbation in DNA-protein interactions can have serious consequences and causes many diseases. Artificially interfering with these interactions with chemicals permits to control the expression of certain genes and opens avenues in the treatment of numerous diseases. Unfortunately, DNA-protein interactions are far from being completely understood despite numerous techniques that
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have been developed. The very first step in such a study is the identification and isolation of DNA interacting proteins. The classical, i.e. biochemical methods include various techniques such as chromatin immunoprecipitation analysis based methods (ChIP), Systematic Evolution of Ligands by EXponential enrichment (SELEX), Electrophoretic mobility shift assays, DNA footprinting, or protein binding microarray. In chromatin immunoprecipitation living cells are incubated with molecules that covalently bind proteins to each other but also to their DNA targets. Once cross linked, the protein-DNA complexes are extracted and the cross-links that bind proteins to DNA are reversed, DNA is purified and amplified to determine if a specific sequence is present (1). The SELEX method consists in preparing a random library of oligonucleotides and incubating it with the target protein. The oligonucleotides that are bound to the target are separated from the others and amplified by Polymerase chain reaction (PCR) (2). DNase I footprinting permits to locate binding sites of specific proteins to DNA. It consists in binding the protein of interest onto a DNA fragment and to conduct a limited digestion. The digestion is accomplished by DNase I and leads to fragments terminating everywhere except in the footprint region which is inaccessible to the nuclease (3). In electrophoretic mobility shift assays proteins that bound to DNA fragments have their electrophoretic mobility reduced in no denaturing polyacrylamide or agarose gels (4). The basic idea of protein binding microarray consists in labelling a protein of interest and allowing it to bound to double stranded DNA microarray. The DNA binding specificity is eventually determined by measuring the fluorescence intensity. For the interested reader a comprehensive review describing the different methods can be found in (5) and (6). Once the protein of interest and its DNA target are identified and isolated different techniques exist to study DNA-enzyme complexes. Basically they can be divided in imaging and force applying categories. Among the imaging techniques optical, fluorescence, electron and atomic force microscopies are the most popular. The highest spatial resolution is obtained by the two last techniques. Electron microscopy is limited to static observations whereas the other 3 also permit to follow the dynamics of the complex. The spatial and temporal resolution varies of course among the techniques but is one of the highest in the case of atomic force microscopy. The second category of techniques are based on the application of a force onto the complex or one of its components and in monitoring the induced deformation. The deforming force can be applied through optical tweezers, magnetic traps or again atomic force microscopy. In terms of force and time resolution, here again atomic force microscopy is very competitive. AFM therefore offers a wide spectrum of investigation possibilities of DNA-enzyme interactions and will therefore constitute the main focus of this short review. After describing the working principle of the instrument we will mention some of the most relevant accomplishments realized with this instrument in the field of DNA-enzyme interaction.
2. AFM and its different imaging modes The AFM consists in a soft cantilever with a very sharp tip that scans the surface of the sample. During the scan, the tip interacts with the sample and its surface topography induces deflections of the cantilever. These deflections are detected by monitoring the reflection angle of a laser beam that illuminates the end of the cantilever. The cantilever deflections are eventually recorded by a computer that reconstructs the 3D topography of the surface and displays it on a screen as depicted in figure 1.
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The microscope can indifferently image samples in vacuum, air or liquid medium. Imaging in vacuum theoretically should give the highest resolution but requires sophisticated equipment that is only available in few laboratories. Imaging in air is the simplest and fastest option, however the thin water layer that covers the sample and the AFM tip generates capillary forces that dramatically increase the tip-sample interaction and importantly deform the imaged molecules. In addition, imaging in air can only be used to explore static molecular conformations. The last capability, i.e. imaging in liquids, made the instrument very popular among life scientists, since it permits the observation of biological samples in nearly physiological conditions although the sample must be adsorbed on flat substrate for observation. It should also be mentioned that it is possible to modify the chemical composition of the liquid medium during the observation and therefore to access the dynamic response of the investigated biological specimen. Despite the simplicity of the instrument, the resolution it can achieve is astonishingly high: up to 0.1 Å for hard and flat samples and ~1 nm for soft or rough biological samples ! In the very beginning the AFM was used exclusively in the so called contact mode in air. In this imaging mode, the tip is permanently touching the sample and applies a vertical and a lateral force on it. The vertical component comes from the upwards deformation of the cantilever that keeps the tip in contact with the sample whereas the lateral component is present because of the lateral displacement of the sample during the scan. In this imaging mode, atomically flat and stiff samples, such as silicon, graphite or mica can be imaged at an atomic resolution. However soft samples and those that are poorly attached to the substrate (i.e. single proteins or DNA molecules) will be deformed and displaced during the scan. Deformation of the sample reduces the resolution of the instrument whereas sample displacement makes it invisible to the microscope. It is the reason why alternative imaging modes have been developed relatively early after the invention of the technique. The imaging mode that permitted for the first time to reproducibly observe poorly attached molecules is the so called tapping mode. In this mode the cantilever is oscillated close to its resonant frequency and approached to the sample. The tip therefore only periodically touches the sample and applies only a vertical force onto it (figure 2). The absence of a lateral force component permits to image poorly attached molecules. The development of this imaging mode (7) and its subsequent derivatives (8) (9) literally revolutionized the field of single molecule imaging. In tapping mode imaging, different parameters can be displayed, such as the amplitude or the phase of the oscillating cantilever. The amplitude signal corresponds to the magnitude of the cantilever’s response to the driving force. This parameter decreases when the cantilever touches the surface whereas the phase signal measures the angle lag between the piezo drive and the motion of the cantilever. This parameter reflects among others the elastic properties of the sample. The phase imaging mode can be useful for highlighting poorly attached DNA molecules on mica surfaces and to record images at a somewhat higher speed then the traditional amplitude imaging mode permits. In attempts to increase resolution some groups developed AFMs that are operating in vacuum and low temperature (CryoAFM) (10) (11). The low temperature stiffens the molecules because of the reduced thermal energy and reduces their deformation by the AFM tip leading to an increased resolution. The vacuum evaporates the thin water film that covers all samples when they are exposed to air. At ambient temperature this water film interacts with the AFM tip and dramatically increases the force with which the tip presses onto the sample as depicted in figure 3.
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In order to prevent water condensation on the surface of the sample and its freezing, low temperature experiments have to be performed under high vacuum or very dry atmosphere. It shouldn't escape to the reader's attention that AFM possesses numerous drawbacks too. Since the sample is explored by a tip, AFM image resolution will be determined (among other factors) by the tip's geometry: sharper tips lead to higher resolution images. Nowadays AFM tips with a radius of curvature lower than 2 nm are commercially available. However, one should keep in mind that sharper tips increase the risk of sample damaging since the pressure they apply is inversely proportional to the surface of contact. Another limitation of the technique resides in the fact that more than 50% of the sample remains invisible to the AFM. This phenomenon is depicted in figure 4 where the dark green part of the sample is invisible to AFM. Another major issue of the technique is the scanning speed. Conventional AFM's require several minutes to scan the sample. Since most biochemical reactions occur in a time frame of ms or less, it is clear that the majority of the reactions are inaccessible to conventional AFM imaging. Since the middle of 90’s some groups spent tremendous efforts to increase AFM’s scanning speed. All these efforts led to instruments that nowadays routinely permit video-rate (30-100 images/sec) AFM imaging. To accomplish such a tour de force numerous changes/improvements were required. Cantilever’s sizes were reduced to a few microns, the scanners were changed to accommodate to the new cantilevers in order to match higher resonant frequencies. In addition, the laser focus system was rebuilt and all the data acquisition electronics changed to allow high data transfer. However, despite the availability of these devices on the market high speed AFMs are not as common as their conventional counterparts are. Among their limitations we can mention their reduced scanning size and small z axis freedom that seriously limit these instruments application envelopes. Nevertheless, these instruments permitted impressive breakthroughs in the field of molecular dynamics and especially in the study of DNA protein interactions. Finally, AFM is a very versatile instrument. In addition to its imaging capabilities the instrument can also be used to explore interaction forces between single molecules. This type of measurements is accomplished by attaching a molecular species onto the AFM tip and a second species onto the substrate as depicted in figure 5. By lowering the tip close to the substrate, the two molecular species bind together. A retraction motion of the tip is then initiated and it quickly induces a downwards bending of the cantilever, since the tip is now attached to the substrate through the two molecules. Once the interaction force between the two molecules is overtaken by the retraction force of the cantilever, the bond between them breaks and the cantilever returns to its resting position. The maximal downwards deflection of the cantilever before the bond ruptures is a measure of the interaction force between the two molecular species (12). Valuable biochemical parameters can be extracted from such a measurement among which the most straightforward one is the dissociation constant.
3. Sample preparation When used in imaging mode, AFM only provides topographical information. It is therefore mandatory to deposit the sample onto an atomically flat surface not to confuse the topography of the sample with the substrate asperities. Among the available substrate candidates, mica is by far the most used one. In order
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to be imaged by AFM, the sample needs not only to lie onto the surface but also to be attached to it. For an exhaustive report on sample preparation and imaging techniques the interested reader can refer to the recent review of Kim et al. (13). Unfortunately, mica possesses a surface that is negatively charged in water and that compromises the attachment of similarly charged molecules such as DNA and proteins. In order to attach such samples onto mica, cross linkers are required. Usually divalent cations such as Mg 2+ or Ni2+ permits to adhere negatively charged samples by bridging them onto the surface. Their presence in the imaging buffer promote adhesion of negatively charged molecules onto the mica surface in a concentration dependent way (14). Sometimes Mg2+ or Ni2+ can interact with the sample or its metabolism and alternative attachment methods are required. One of those is mica surface pretreatment with aminopropyltriethoxy silane (APTES) (15), the deposition of Langmuir-Blodgett films onto the mica surface (16) or other surface treatments. It must also be said that there is also advantages related to the negative charge of the mica’s surface: namely the possibility, by using divalent ions, to modulate the interaction between negatively charged biomolecules and the mica substrate and thus preserving the function of the biomolecule. One example were the experiments carried out by Alonso et al. on the action of Topoisomerase II on knotted circular DNA (17). Real time monitoring of DNA-protein interactions raises additional challenges. As it was previously highlighted, the AFM tip strongly interacts with the sample and removes it of the surface if it is loosely bound to it. Therefore, the stronger the sample’s attachment is and the higher the AFM resolution will be. However, attaching a molecule too strongly to the substrate will compromise its function and inhibits its dynamical changes. Alternatively, attaching the molecule too loosely to the substrate will permit its function but compromise AFM imaging. Therefore, a delicate balance between these two extremes has to be found. It is usually done by finely adjusting the divalent ions concentration in the imaging medium and flowing it through the imaging chamber of the microscope while imaging the sample. Such a gravity driven setup was reported by Thomson et al. 1996 (14) and depicted in figure 6.
4. DNA-protein complex observation in air DNA is among the very first biological molecule that was observed by Scanning Tunneling Microscopy (STM) (18), the ancestor of AFM. As mentioned above, the most straightforward way to observe DNA and its protein complexes with AFM consists in depositing the sample onto an atomically flat substrate such as mica in presence of Mg2+ or Ni2+. The divalent cations promote DNA attachment onto the mica and permit AFM observation. The simplest AFM imaging procedure consists in drying the sample and in imaging it in air in contact or tapping mode. Despite being observed in non-physiological conditions DNA conserves in air its properties such as its flexibility (19), bending (20) or wrapping (21) that can easily be monitored. Along these lines of research of imaging DNA in dry conditions, detailed statistical properties of DNA were extracted and analyzed according to polymer theory and yielded persistence length, critical exponents for the divergence of the end-to-end distance, distributions of end-to-end distance and the related critical exponents as well as topological properties. (22), (23), (24) . Similarly, a wealth of information can be gathered about proteins too. One of those parameters is the volume in AFM images that can be correlated to the molecular weight (25). Morphological information such as the multimeric state of helicases (26) or Rad50 (27) were also studied by AFM in air. Rad50 is involved in the repair of DNA double-strand breaks whereas helicases separate strands of DNA double helixes. Among the numerous DNA-protein complexes that have been observed by AFM in air we can also mention DNA-Fur
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complex observation by Le Cam et al. in 1994 (28). Flur is a bacterial protein that regulates various bacterial genes involved in iron transport. The AFM and electron microscopic observation of the complex permitted to highlight a Flur induced local stiffening of the DNA. Such observation in air have the great advantage to be technically simple and rapid. Chen et al. in 2002 (20) studied in this manner DNA glycosylases. These enzymes preserve DNA genetic integrity by recognizing damaged bases and catalyzing their excision. The AFM images in air of hOGG1-DNA complexes revealed important DNA distortions that are certainly related to the search of damaged bases by the enzyme. SfilI is a restriction enzyme that catalyzes four strand DNA breakage by binding to two distantly separated cognate sites. The recognition sites can be located onto the same (cis) or different (trans) DNA molecules. In order to elucidate the structural properties of the SfiI-DNA complexes Lushnikov et al. (29) imaged them by AFM in air and characterized the DNA path within the complex. The angular distribution between the DNA helices suggested that the DNA binding site forms only weak sequence specific contacts with SfiI. More recently (30), (31) AFM imaging in air was employed to study single stranded DNA binding protein (SSB), its binding to bubbles in circular DNA and/or its interaction with fork DNA and the RecG DNA helicase. These latter enzymes participate in genome duplication and DNA repair machinery. In this work the authors studied the DNA-SSB-RecG complexes and could recognize the different participants according to morphological criteria such as the shape and the volume of the proteins on AFM images. The study permitted, among other results, to assess an increase in the RecG-DNA complex binding in the presence of SSB and to postulate that SSB is required to permit RecG translocation along the duplex DNA. The DNA mismatch (MMR) repairing system is composed of several DNA binding proteins that were extensively investigated by AFM imaging in air. MMR system detects and repairs post-replicative DNA errors and participates therefore to the stability of the genome. MutS proteins family participate in this process and the study of their complex with DNA permitted to Wang et al. to unveil some of the mechanisms by which MutS achieves mismatch repair. By analyzing AFM images the authors noticed that MutS induces a DNA bending that depends on the DNA integrity (homogeneity) and proposed that MutS binds DNA and bends it in the search of mismatches. Once MutS identifies a mismatch it undergoes conformational changes that ultimately result in DNA nicking and unbending (32). Further studies on MutS mutants carried on by the same group permitted to identify the amino acid residue that is involved in the mismatch recognition process (33). MutS affinitiy to mismatched DNA segments was also determined by analyzing AFM images recorded in air. The method, developed by Yang et al. (34) consisted in counting and statistically analyzing, the number of DNA-binding proteins attached onto a specific position of a DNA strand. More recently, the other components of the MMR repairing system were studied by AFM in air by Josephs et al. (35). We previously mentioned that MultS is responsible for replication error identification. In this work the authors investigated the structure and location on DNA of other proteins of the complex namely, MutL and MutH during the initial mismatch repair. It is believed that MutS catalyses the binding of MutL which in turn activates MutH. The authors could not only recognize the different actors of the complex on AFM images but also distinguished MutL sub-domains and their rearrangement upon ADP exposure. This study permitted among others to highlight the role of MutS as a scaffold to anchor MutL and MutH onto DNA.
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Monitoring protein interaction with single stranded DNA (ssDNA) constitute an AFM imaging challenge. If proteins usually aren’t too difficult to image, ssDNA in contrast remains almost invisible to AFM due to its reduced hight. But ssDNA was investigated under dry conditions (36), (37) and the persistence length of ssDNA or the influence of chemicals on the promotion of secondary structure was investigated. Binding of single stranded DNA binding proteins (SSBs) to bubbles in circular DNA was evidenced by a similar method as previously developed (30). However, SSB proteins play a central role during replication since they contribute to separate the two single stranded DNA filaments and prevent the ssDNA from reforming as a double helix. In order to study this type of interaction Lyubchenko’s team developed an original approach to monitor SSBs with ssDNA (38). The authors synthesized double stranded DNA that contain single stranded segments in the middle or the end of the filament as depicted in figure 7 In such constructs the dsDNA was used as a marker that delimits the ssDNA segments. With this technique, the authors observed that SSBs require high Mg2+ cations to bind ssDNA specifically whereas in low salt solutions SSBs can bind to dsDNA too. It suggests that SSBs undergo a conformational change that depends on the ionic strength and that modulates its binding affinity to DNA. For a more exhaustive review about DNA-protein complexes observation in air the interested reader can refer to (39).
5. Measuring Protein – DNA interaction forces The interaction force measurement capability of the AFM was used by Bartels et al. in 2003 to evaluate DNA binding proteins interaction with DNA (40). More specifically, the authors measured ExpG protein interaction with different DNA target sequences of the exp gene cluster of the gram negative soil bacterium Sinorhizobium meliloti. The measurements gave an interaction force in the range of 50 to 165 pN between ExpG and the promoter regions. Later, the same team further explored the same complex (41) but with mutated binding sites and additional dynamic AFM imaging. S. meliloti has a symbiotic relationship with several types of vegetables and fixes molecular nitrogen. These studies were conducted in the frame of the exploration of the symbiosis regulation. In the same period of time the pioneer of the AFM spectroscopy technique H.E. Gaub and co-workers (42) investigated E. coli DNA repair protein LexA interaction with DNA. LexA is a repressor protein that controls several dozens of genes and that is involved in the response of E. coli to DNA damage and in the regulation of transcription. The team characterized by AFM force spectroscopy the dissociation rates as well the widths of the binding potentials of LexA with two different DNA binding domains i.e. recA and yebG. In these experiments the authors used amino silanized cantilevers and substrates. dsDNA and LexA were crosslinked onto the tip and the substrate with carboxymethylamylose. The interested reader can refer to a recent and very well written review article about AFM force spectroscopy that describes all the details and challenges of the technique (13)
6. DNA-protein complex observation in liquids As it has been pointed out in paragraph 2 the development of the tapping mode in liquids literally revolutionized AFM imaging field. The absence of lateral forces permitted for the first time to image poorly
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attached samples in almost physiological conditions and more importantly to monitor online biochemical reactions. This technological breakthrough paved the way to numerous biochemical reactions visualization by AFM. Degradation of DNA by DNAse I was among the first biochemical reaction to be observed by using this new technique. Later E. coli RNA polymerase was among the first complex to be observed with this technique (43). The same year Guthold et al. observed the complex formation between RNA polymerase and DNA (44). Later, transcription of DNA by RNA polymerase could be monitored on line with tapping mode imaging (45). For these experiments the authors used specifically designed DNA molecules that contained a stall site to permit positioning of RNA polymerase at a specific location on the DNA molecule before imaging. The preformed complex was deposited onto the mica substrate and inserted into the imaging chamber of the microscope. Once a potentially functional complex was identified with the AFM, a medium that restarts the transcription was injected into the imaging chamber to resume the process. To permit low speed AFM imaging, the DNA-RNA polymerase interaction was slowed down by reducing the nucleotides concentration in the imaging buffer. Later, RNA polymerase was also monitored during its one dimensional random walk along DNA molecules by using a similar immobilization technique (46). In this work the authors demonstrated that in addition to sliding, RNA polymerase can jump from one site to another. Among other similar studies we can mention p53 interaction with DNA. P53 acts as a transcription factor for a number of genes involved in inhibiting tumor cell growth and transformation. Jiao et al. observed on line numerous types of interactions among the two molecules such as association-dissociation as well as sliding of p53 along the DNA molecule (47). Also in liquid, the action of Topoisomerase II (Topo II) was evidenced when unknotting DNA and transforming it into circular DNA: a delicate balance between attraction of DNA to mica was found that permitted to keep the accessibility of knotted DNA by Topo II (48). Figure 8a depicts Topo II as complexed to two linear DNA molecules whereas figure 8b shows a time-lapse sequence of Topo II unknotting a single DNA molecule.
7. High speed AFM (HSAFM) As pointed out previously one of the most serious drawback of traditional AFM imaging is its slow data acquisition rate. It should be kept in mind that it takes between 1-4 minutes to acquire a typical AFM image whereas RNA polymerase translocation along DNA is completed in a timeframe of seconds. To circumvent this bottleneck very early attempts were undertaken to accelerate the acquisition speed. One of these attempts consisted in using phase imaging instead of amplitude when operating in tapping mode (49). This approach permitted to increase somehow the speed but the ultimate goal, i.e. the real-time imaging of biological processes was still afar. Numerous groups struggled to develop instruments that can achieve a sub-second image acquisition rate and among all of them the probably most successful was the group of T. Ando (50), (51). To increase AFM imaging speed by one or two orders of magnitude required tremendous efforts and modifications. The readers interested in this topic and applications are invited to consult the very complete and comprehensive description of high speed AFM technique that was published in the recent review by Ando et al. (52).
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Biotin binding to streptavidin is one of the most studied and best known protein-protein interaction. The two proteins bind each to other in a 4 :1 fashion (four biotins for one streptavidin) with high affinity and selectivity. In 2007 Kobayashi et al (53) used HSAFM (5-7 frames/sec) to follow the dynamics of short DNA segments on mica. By covalently attaching biotin molecules at the end of a DNA strand the authors could follow DNA-binding - unbinding from streptavidin. The way restriction enzymes find their recognition sites is a highly interesting topic since they have been shown to find their recognition site 100-1000 times faster than what would be expected by random diffusion. In order to investigate this topic Gilmore et al. (54) used HSAFM to follow DNA-EcoRII complex dynamics. EcoRII is a restriction enzyme that belongs to the IIE REases group and that requires to bind several (2 or 3) DNA recognition sites to cleave one of them. The HSAFM movies recorded at a rate of two frames per second highlighted the way the enzyme dissociates off the DNA, how it diffuses and translocate along it. Another application of HSAFM in protein DNA interaction exploration was the study of nucleosomes dynamics. Nucleosomes consist in a DNA segment wound around histone protein cores and constitute the fundamental repeating units of eukaryotic chromatin. They play an important role in gene expression and are thought to carry epigenetically inherited information too. Nucleosomes unwrapping provides accessibility to DNA to bind proteins for replication but up to recently the details of its dynamics were largely unexplored. In 2010 Suzuki et al. (55) used HSAFM to follow DNA dynamics. Importantly the DNA movements were analyzed and permitted to concluded that the motion was thermally induced and not triggered by the scanning. Nucleosomes observation permitted to visualize nucleosome sliding on DNA in a range of 50 nm. In some cases, the authors observed nucleosome disruptions too. One year later Miyagi et al. (56) employed HSAFM to observe nucleosomes dynamics and their study highlighted that nucleosomes spontaneous unwrapping occurs in a sub-second timeframe. The information gathered by HSAFM was fully compatible with the previous model of Widom (57) but HSAFM additionally highlighted a possible and novel alternative to unwrapping i.e. sliding. In this case the nucleosome moves rapidly in one direction, unconvering a given DNA sequence and comes back to its original position, all in a timeframe of about 2 sec. A more recent HSAFM study involving nucleosomes and tetrasomes dynamics has been conducted by Katan et al. (58). Tetrasomes are subnucleosomal structures that contain 4 instead of 8 histones in the protein core. The authors observed at high spatial and temporal resolution nucleosomes and tetrasomes disassembly, sliding, and hopping. High time resolution AFM images of transcription were achieved in 2012 by Suzukiet al. (59) and Endo. Endo et al. (60) used a HSAFM to directly observe single RNA polymerase binding and sliding along dsDNA. RNA synthesis as well dissociation could also be observed. The setup was very original and efficient. It consisted in attaching a 1000 bp template dsDNA onto an 1150 bp long DNA origami platform (61). Between its two extremities the template DNA was free to float, fully accessible RNA polymerase, and still visible to AFM. During transcription the newly produced RNA transcript is very difficult to image by AFM. In order to visualize it the authors used biotinylated UTP and labeled it with streptavidin. This procedure dramatically increased RNA transcripts and made it visible to HSAFM. The same team further developed this HSAFM-DNA origami based DNA immobilization technique (62). In its new version the DNA segment to be studied (DNA template) is incorporated in a 100x80 nm square DNA origami containing a 40x40 nm wide window across which two DNA template are suspended. The 9
system is very convenient since it permits the placement of any sequence of DNA (double or singlestranded) across the frame and the precise control of the relative position of the two templates, their lengths, chemical composition (i.e. modified nucleotides) tension and rotation. The team used this system to follow numerous protein-DNA interactions such as DNA bending (EcoRI methyltransferase), DNA base repair (8-oxoguanine glycosylase and T4 pyrimidyne dimer glycosylase) or to follow DNA structural changes such as G-quadruplex formation or B-Z transition. A similar technology has recently been adapted by Godonoga et al. (63) to study Plasmodium falciparum lactate dehydrogenase (PfLDH) interaction with different DNA aptamers. Aptamers are synthetic single stranded DNA or RNA fragments that are designed to bind specific targets. Once synthetized, intermolecular binding forces induce their folding in particular 3D configurations that is responsible for their affinity. Aptamers are nowadays used as therapeutic agents (64) and biosensors (65). Godonoga et al. integrated PfLDH-binding DNA aptamer in a DNA origami and used HSAFM to monitor in real time attachment-detachment of PfLDH.
8. Conclusion After the invention of the Scanning Tunneling Microscope (STM) by Binning, Rohrer, Gerber and Weibel (66) science has received a tremendous impulse due to the possibility now to investigate nature on a spatial scale nearing the atomic-subatomic scale with a fairly limited effort: this has led to the blooming of a new field in science and technology, namely nanotechnology. This paradigm change was possible because a table top instrument permits to image samples and investigate atomic scale phenomena in direct space. The limitation of the STM, namely the condition that the sample must be a conductor, was quickly surpassed by the invention of Atomic Force Microscope (AFM) by Binnig, Quate and Gerber (67) Now, with this instrument, an almost infinite class of samples could be imaged and investigated down to the atomic scale. The whole realm of biology was opened to AFM when the microscope was expanded to function in liquid environment permitting the study of bio-samples in physiological conditions. From this point on, there were numerous declinations of the microscope, named often Scanning Probe Microscope, where the “probe” could be a simple tip (AFM), an optical fiber (SNOM), a pipette (SICM) or a temperature probe, etc. Not all of the declinations have lived up to the initial hopes, but progress has been steady. For example, nowadays high-speed AFM is a reality and delivers video rate imaging of biological processes like myosin “walking” on actin filaments, an experiment unthinkable only few years ago, and probably be doomed to remain a dream if it were not for the invention of the AFM. The review presented here gives an overview of the possibilities offered by the AFM and should stimulate further ideas and developments beyond the present methods and techniques, especially applications to biological processes should be here emphasized which, according to the personal view of the authors, contains a large potential for new and paradigm changing developments.
Acknowledgments We apologize to our colleagues whose work could not be included in this review due to space limitations. Authors were funded by the Swiss National Grant 200021-144321, 407240_167137 and also acknowledge the support of Gebert Rüf Stiftung GRS-024/14.
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Figure captions Figure 1 Working principle of the AFM. A sharp tip fixed at the end of a soft cantilever scans the surface of the sample. The deflections of the cantilever during the scan are detected and displayed on a computer screen to reconstruct the sample’s topography
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Figure 2 In the so called tapping mode (or intermittent contact) the cantilever is oscillated close to its resonant frequency and the tip periodically hits the sample. The feed-back loop is locked on the cantilever oscillation amplitude.
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Figure 3 A thin layer of water covers the samples to be imaged in air and dramatically increases the interaction force between the tip and the sample (green ball in this illustration) by the formation of a water capillary bridge.’
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Figure 4 AFM images consist in a convolution between the geometry of the tip and the sample (green) therefore only the uppermost parts (light green) of the samples are visible to the AFM.
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Figure 5 Interaction force measurement by AFM. The tip is periodically approached and retracted off the substrate. In case of attachment and dissociation a characteristic peak appears on the force distance curve (red)
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Figure 6. Typical setup for AFM imaging in liquids 1. liquids to be flown in the analysis chamber 2. selector 3. flow speed regulator 4. AFM 5. waste container 6. balance that permits the determination of the moment a new solution arrives into the analysis chamber
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Figure 7 Single stranded DNA inserted in the middle (A) or the end (B) of a double stranded DNA filament
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Figure 8a Topo II bind to DNA cross-overs on linear DNA molecules. Figure from (48) with permission.
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Figure 8b Time-lapse imaging of Topo II unknotting a single DNA molecule. The white bar represents 250 nm. Figure from (48) with permission.
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S1084-9521(16)30492-X http://dx.doi.org/doi:10.1016/j.semcdb.2017.07.015 YSCDB 2278
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
23-5-2017 12-7-2017 13-7-2017
Please cite this article as: Kasas S, Dietler G.DNA-protein interactions explored by Atomic Force Microscopy.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.07.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-protein interactions explored by Atomic Force Microscopy
S. Kasas1,2 and G. Dietler1
1
Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2
Plateforme de Morphologie, Faculté de Médecine, Université de Lausanne, Bugnion 9, 1005 Lausanne, Switzerland
Corresponding author email address : [email protected]
Keywords DNA, protein, enzyme, AFM
Abstract DNA-protein interactions play an important role in all living organisms on Earth. The advent of atomic force microscopy permitted for the first time to follow and to characterize interaction forces between these two molecular species. After a short description of the AFM and its imaging modes we review, in a chronological order some of the studies that we think importantly contributed to the field.
1. Introduction The majority of DNA-binding proteins play an important role in the regulation of gene expression. Their interaction with DNA are therefore of prime importance for all living organisms, starting from bacteria up to mammalian cells. Numerous fundamental biological processes such as DNA replication, packing, recombination, DNA repair, RNA transport and translation are controlled by these types of interactions. These mechanisms control the growth, the differentiation and the evolution of living organisms. Most of DNA-binding proteins directly prevent or release the transcription of certain genes according to environmental conditions, stages of development or specific stimuli. Some others such as histones and some high mobility group proteins, pack genomic DNA into compact form. Since only the genes that are accessible to the transcriptional machinery can be decoded, these proteins therefore indirectly control transcription. Any perturbation in DNA-protein interactions can have serious consequences and causes many diseases. Artificially interfering with these interactions with chemicals permits to control the expression of certain genes and opens avenues in the treatment of numerous diseases. Unfortunately, DNA-protein interactions are far from being completely understood despite numerous techniques that
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have been developed. The very first step in such a study is the identification and isolation of DNA interacting proteins. The classical, i.e. biochemical methods include various techniques such as chromatin immunoprecipitation analysis based methods (ChIP), Systematic Evolution of Ligands by EXponential enrichment (SELEX), Electrophoretic mobility shift assays, DNA footprinting, or protein binding microarray. In chromatin immunoprecipitation living cells are incubated with molecules that covalently bind proteins to each other but also to their DNA targets. Once cross linked, the protein-DNA complexes are extracted and the cross-links that bind proteins to DNA are reversed, DNA is purified and amplified to determine if a specific sequence is present (1). The SELEX method consists in preparing a random library of oligonucleotides and incubating it with the target protein. The oligonucleotides that are bound to the target are separated from the others and amplified by Polymerase chain reaction (PCR) (2). DNase I footprinting permits to locate binding sites of specific proteins to DNA. It consists in binding the protein of interest onto a DNA fragment and to conduct a limited digestion. The digestion is accomplished by DNase I and leads to fragments terminating everywhere except in the footprint region which is inaccessible to the nuclease (3). In electrophoretic mobility shift assays proteins that bound to DNA fragments have their electrophoretic mobility reduced in no denaturing polyacrylamide or agarose gels (4). The basic idea of protein binding microarray consists in labelling a protein of interest and allowing it to bound to double stranded DNA microarray. The DNA binding specificity is eventually determined by measuring the fluorescence intensity. For the interested reader a comprehensive review describing the different methods can be found in (5) and (6). Once the protein of interest and its DNA target are identified and isolated different techniques exist to study DNA-enzyme complexes. Basically they can be divided in imaging and force applying categories. Among the imaging techniques optical, fluorescence, electron and atomic force microscopies are the most popular. The highest spatial resolution is obtained by the two last techniques. Electron microscopy is limited to static observations whereas the other 3 also permit to follow the dynamics of the complex. The spatial and temporal resolution varies of course among the techniques but is one of the highest in the case of atomic force microscopy. The second category of techniques are based on the application of a force onto the complex or one of its components and in monitoring the induced deformation. The deforming force can be applied through optical tweezers, magnetic traps or again atomic force microscopy. In terms of force and time resolution, here again atomic force microscopy is very competitive. AFM therefore offers a wide spectrum of investigation possibilities of DNA-enzyme interactions and will therefore constitute the main focus of this short review. After describing the working principle of the instrument we will mention some of the most relevant accomplishments realized with this instrument in the field of DNA-enzyme interaction.
2. AFM and its different imaging modes The AFM consists in a soft cantilever with a very sharp tip that scans the surface of the sample. During the scan, the tip interacts with the sample and its surface topography induces deflections of the cantilever. These deflections are detected by monitoring the reflection angle of a laser beam that illuminates the end of the cantilever. The cantilever deflections are eventually recorded by a computer that reconstructs the 3D topography of the surface and displays it on a screen as depicted in figure 1.
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The microscope can indifferently image samples in vacuum, air or liquid medium. Imaging in vacuum theoretically should give the highest resolution but requires sophisticated equipment that is only available in few laboratories. Imaging in air is the simplest and fastest option, however the thin water layer that covers the sample and the AFM tip generates capillary forces that dramatically increase the tip-sample interaction and importantly deform the imaged molecules. In addition, imaging in air can only be used to explore static molecular conformations. The last capability, i.e. imaging in liquids, made the instrument very popular among life scientists, since it permits the observation of biological samples in nearly physiological conditions although the sample must be adsorbed on flat substrate for observation. It should also be mentioned that it is possible to modify the chemical composition of the liquid medium during the observation and therefore to access the dynamic response of the investigated biological specimen. Despite the simplicity of the instrument, the resolution it can achieve is astonishingly high: up to 0.1 Å for hard and flat samples and ~1 nm for soft or rough biological samples ! In the very beginning the AFM was used exclusively in the so called contact mode in air. In this imaging mode, the tip is permanently touching the sample and applies a vertical and a lateral force on it. The vertical component comes from the upwards deformation of the cantilever that keeps the tip in contact with the sample whereas the lateral component is present because of the lateral displacement of the sample during the scan. In this imaging mode, atomically flat and stiff samples, such as silicon, graphite or mica can be imaged at an atomic resolution. However soft samples and those that are poorly attached to the substrate (i.e. single proteins or DNA molecules) will be deformed and displaced during the scan. Deformation of the sample reduces the resolution of the instrument whereas sample displacement makes it invisible to the microscope. It is the reason why alternative imaging modes have been developed relatively early after the invention of the technique. The imaging mode that permitted for the first time to reproducibly observe poorly attached molecules is the so called tapping mode. In this mode the cantilever is oscillated close to its resonant frequency and approached to the sample. The tip therefore only periodically touches the sample and applies only a vertical force onto it (figure 2). The absence of a lateral force component permits to image poorly attached molecules. The development of this imaging mode (7) and its subsequent derivatives (8) (9) literally revolutionized the field of single molecule imaging. In tapping mode imaging, different parameters can be displayed, such as the amplitude or the phase of the oscillating cantilever. The amplitude signal corresponds to the magnitude of the cantilever’s response to the driving force. This parameter decreases when the cantilever touches the surface whereas the phase signal measures the angle lag between the piezo drive and the motion of the cantilever. This parameter reflects among others the elastic properties of the sample. The phase imaging mode can be useful for highlighting poorly attached DNA molecules on mica surfaces and to record images at a somewhat higher speed then the traditional amplitude imaging mode permits. In attempts to increase resolution some groups developed AFMs that are operating in vacuum and low temperature (CryoAFM) (10) (11). The low temperature stiffens the molecules because of the reduced thermal energy and reduces their deformation by the AFM tip leading to an increased resolution. The vacuum evaporates the thin water film that covers all samples when they are exposed to air. At ambient temperature this water film interacts with the AFM tip and dramatically increases the force with which the tip presses onto the sample as depicted in figure 3.
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In order to prevent water condensation on the surface of the sample and its freezing, low temperature experiments have to be performed under high vacuum or very dry atmosphere. It shouldn't escape to the reader's attention that AFM possesses numerous drawbacks too. Since the sample is explored by a tip, AFM image resolution will be determined (among other factors) by the tip's geometry: sharper tips lead to higher resolution images. Nowadays AFM tips with a radius of curvature lower than 2 nm are commercially available. However, one should keep in mind that sharper tips increase the risk of sample damaging since the pressure they apply is inversely proportional to the surface of contact. Another limitation of the technique resides in the fact that more than 50% of the sample remains invisible to the AFM. This phenomenon is depicted in figure 4 where the dark green part of the sample is invisible to AFM. Another major issue of the technique is the scanning speed. Conventional AFM's require several minutes to scan the sample. Since most biochemical reactions occur in a time frame of ms or less, it is clear that the majority of the reactions are inaccessible to conventional AFM imaging. Since the middle of 90’s some groups spent tremendous efforts to increase AFM’s scanning speed. All these efforts led to instruments that nowadays routinely permit video-rate (30-100 images/sec) AFM imaging. To accomplish such a tour de force numerous changes/improvements were required. Cantilever’s sizes were reduced to a few microns, the scanners were changed to accommodate to the new cantilevers in order to match higher resonant frequencies. In addition, the laser focus system was rebuilt and all the data acquisition electronics changed to allow high data transfer. However, despite the availability of these devices on the market high speed AFMs are not as common as their conventional counterparts are. Among their limitations we can mention their reduced scanning size and small z axis freedom that seriously limit these instruments application envelopes. Nevertheless, these instruments permitted impressive breakthroughs in the field of molecular dynamics and especially in the study of DNA protein interactions. Finally, AFM is a very versatile instrument. In addition to its imaging capabilities the instrument can also be used to explore interaction forces between single molecules. This type of measurements is accomplished by attaching a molecular species onto the AFM tip and a second species onto the substrate as depicted in figure 5. By lowering the tip close to the substrate, the two molecular species bind together. A retraction motion of the tip is then initiated and it quickly induces a downwards bending of the cantilever, since the tip is now attached to the substrate through the two molecules. Once the interaction force between the two molecules is overtaken by the retraction force of the cantilever, the bond between them breaks and the cantilever returns to its resting position. The maximal downwards deflection of the cantilever before the bond ruptures is a measure of the interaction force between the two molecular species (12). Valuable biochemical parameters can be extracted from such a measurement among which the most straightforward one is the dissociation constant.
3. Sample preparation When used in imaging mode, AFM only provides topographical information. It is therefore mandatory to deposit the sample onto an atomically flat surface not to confuse the topography of the sample with the substrate asperities. Among the available substrate candidates, mica is by far the most used one. In order
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to be imaged by AFM, the sample needs not only to lie onto the surface but also to be attached to it. For an exhaustive report on sample preparation and imaging techniques the interested reader can refer to the recent review of Kim et al. (13). Unfortunately, mica possesses a surface that is negatively charged in water and that compromises the attachment of similarly charged molecules such as DNA and proteins. In order to attach such samples onto mica, cross linkers are required. Usually divalent cations such as Mg 2+ or Ni2+ permits to adhere negatively charged samples by bridging them onto the surface. Their presence in the imaging buffer promote adhesion of negatively charged molecules onto the mica surface in a concentration dependent way (14). Sometimes Mg2+ or Ni2+ can interact with the sample or its metabolism and alternative attachment methods are required. One of those is mica surface pretreatment with aminopropyltriethoxy silane (APTES) (15), the deposition of Langmuir-Blodgett films onto the mica surface (16) or other surface treatments. It must also be said that there is also advantages related to the negative charge of the mica’s surface: namely the possibility, by using divalent ions, to modulate the interaction between negatively charged biomolecules and the mica substrate and thus preserving the function of the biomolecule. One example were the experiments carried out by Alonso et al. on the action of Topoisomerase II on knotted circular DNA (17). Real time monitoring of DNA-protein interactions raises additional challenges. As it was previously highlighted, the AFM tip strongly interacts with the sample and removes it of the surface if it is loosely bound to it. Therefore, the stronger the sample’s attachment is and the higher the AFM resolution will be. However, attaching a molecule too strongly to the substrate will compromise its function and inhibits its dynamical changes. Alternatively, attaching the molecule too loosely to the substrate will permit its function but compromise AFM imaging. Therefore, a delicate balance between these two extremes has to be found. It is usually done by finely adjusting the divalent ions concentration in the imaging medium and flowing it through the imaging chamber of the microscope while imaging the sample. Such a gravity driven setup was reported by Thomson et al. 1996 (14) and depicted in figure 6.
4. DNA-protein complex observation in air DNA is among the very first biological molecule that was observed by Scanning Tunneling Microscopy (STM) (18), the ancestor of AFM. As mentioned above, the most straightforward way to observe DNA and its protein complexes with AFM consists in depositing the sample onto an atomically flat substrate such as mica in presence of Mg2+ or Ni2+. The divalent cations promote DNA attachment onto the mica and permit AFM observation. The simplest AFM imaging procedure consists in drying the sample and in imaging it in air in contact or tapping mode. Despite being observed in non-physiological conditions DNA conserves in air its properties such as its flexibility (19), bending (20) or wrapping (21) that can easily be monitored. Along these lines of research of imaging DNA in dry conditions, detailed statistical properties of DNA were extracted and analyzed according to polymer theory and yielded persistence length, critical exponents for the divergence of the end-to-end distance, distributions of end-to-end distance and the related critical exponents as well as topological properties. (22), (23), (24) . Similarly, a wealth of information can be gathered about proteins too. One of those parameters is the volume in AFM images that can be correlated to the molecular weight (25). Morphological information such as the multimeric state of helicases (26) or Rad50 (27) were also studied by AFM in air. Rad50 is involved in the repair of DNA double-strand breaks whereas helicases separate strands of DNA double helixes. Among the numerous DNA-protein complexes that have been observed by AFM in air we can also mention DNA-Fur
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complex observation by Le Cam et al. in 1994 (28). Flur is a bacterial protein that regulates various bacterial genes involved in iron transport. The AFM and electron microscopic observation of the complex permitted to highlight a Flur induced local stiffening of the DNA. Such observation in air have the great advantage to be technically simple and rapid. Chen et al. in 2002 (20) studied in this manner DNA glycosylases. These enzymes preserve DNA genetic integrity by recognizing damaged bases and catalyzing their excision. The AFM images in air of hOGG1-DNA complexes revealed important DNA distortions that are certainly related to the search of damaged bases by the enzyme. SfilI is a restriction enzyme that catalyzes four strand DNA breakage by binding to two distantly separated cognate sites. The recognition sites can be located onto the same (cis) or different (trans) DNA molecules. In order to elucidate the structural properties of the SfiI-DNA complexes Lushnikov et al. (29) imaged them by AFM in air and characterized the DNA path within the complex. The angular distribution between the DNA helices suggested that the DNA binding site forms only weak sequence specific contacts with SfiI. More recently (30), (31) AFM imaging in air was employed to study single stranded DNA binding protein (SSB), its binding to bubbles in circular DNA and/or its interaction with fork DNA and the RecG DNA helicase. These latter enzymes participate in genome duplication and DNA repair machinery. In this work the authors studied the DNA-SSB-RecG complexes and could recognize the different participants according to morphological criteria such as the shape and the volume of the proteins on AFM images. The study permitted, among other results, to assess an increase in the RecG-DNA complex binding in the presence of SSB and to postulate that SSB is required to permit RecG translocation along the duplex DNA. The DNA mismatch (MMR) repairing system is composed of several DNA binding proteins that were extensively investigated by AFM imaging in air. MMR system detects and repairs post-replicative DNA errors and participates therefore to the stability of the genome. MutS proteins family participate in this process and the study of their complex with DNA permitted to Wang et al. to unveil some of the mechanisms by which MutS achieves mismatch repair. By analyzing AFM images the authors noticed that MutS induces a DNA bending that depends on the DNA integrity (homogeneity) and proposed that MutS binds DNA and bends it in the search of mismatches. Once MutS identifies a mismatch it undergoes conformational changes that ultimately result in DNA nicking and unbending (32). Further studies on MutS mutants carried on by the same group permitted to identify the amino acid residue that is involved in the mismatch recognition process (33). MutS affinitiy to mismatched DNA segments was also determined by analyzing AFM images recorded in air. The method, developed by Yang et al. (34) consisted in counting and statistically analyzing, the number of DNA-binding proteins attached onto a specific position of a DNA strand. More recently, the other components of the MMR repairing system were studied by AFM in air by Josephs et al. (35). We previously mentioned that MultS is responsible for replication error identification. In this work the authors investigated the structure and location on DNA of other proteins of the complex namely, MutL and MutH during the initial mismatch repair. It is believed that MutS catalyses the binding of MutL which in turn activates MutH. The authors could not only recognize the different actors of the complex on AFM images but also distinguished MutL sub-domains and their rearrangement upon ADP exposure. This study permitted among others to highlight the role of MutS as a scaffold to anchor MutL and MutH onto DNA.
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Monitoring protein interaction with single stranded DNA (ssDNA) constitute an AFM imaging challenge. If proteins usually aren’t too difficult to image, ssDNA in contrast remains almost invisible to AFM due to its reduced hight. But ssDNA was investigated under dry conditions (36), (37) and the persistence length of ssDNA or the influence of chemicals on the promotion of secondary structure was investigated. Binding of single stranded DNA binding proteins (SSBs) to bubbles in circular DNA was evidenced by a similar method as previously developed (30). However, SSB proteins play a central role during replication since they contribute to separate the two single stranded DNA filaments and prevent the ssDNA from reforming as a double helix. In order to study this type of interaction Lyubchenko’s team developed an original approach to monitor SSBs with ssDNA (38). The authors synthesized double stranded DNA that contain single stranded segments in the middle or the end of the filament as depicted in figure 7 In such constructs the dsDNA was used as a marker that delimits the ssDNA segments. With this technique, the authors observed that SSBs require high Mg2+ cations to bind ssDNA specifically whereas in low salt solutions SSBs can bind to dsDNA too. It suggests that SSBs undergo a conformational change that depends on the ionic strength and that modulates its binding affinity to DNA. For a more exhaustive review about DNA-protein complexes observation in air the interested reader can refer to (39).
5. Measuring Protein – DNA interaction forces The interaction force measurement capability of the AFM was used by Bartels et al. in 2003 to evaluate DNA binding proteins interaction with DNA (40). More specifically, the authors measured ExpG protein interaction with different DNA target sequences of the exp gene cluster of the gram negative soil bacterium Sinorhizobium meliloti. The measurements gave an interaction force in the range of 50 to 165 pN between ExpG and the promoter regions. Later, the same team further explored the same complex (41) but with mutated binding sites and additional dynamic AFM imaging. S. meliloti has a symbiotic relationship with several types of vegetables and fixes molecular nitrogen. These studies were conducted in the frame of the exploration of the symbiosis regulation. In the same period of time the pioneer of the AFM spectroscopy technique H.E. Gaub and co-workers (42) investigated E. coli DNA repair protein LexA interaction with DNA. LexA is a repressor protein that controls several dozens of genes and that is involved in the response of E. coli to DNA damage and in the regulation of transcription. The team characterized by AFM force spectroscopy the dissociation rates as well the widths of the binding potentials of LexA with two different DNA binding domains i.e. recA and yebG. In these experiments the authors used amino silanized cantilevers and substrates. dsDNA and LexA were crosslinked onto the tip and the substrate with carboxymethylamylose. The interested reader can refer to a recent and very well written review article about AFM force spectroscopy that describes all the details and challenges of the technique (13)
6. DNA-protein complex observation in liquids As it has been pointed out in paragraph 2 the development of the tapping mode in liquids literally revolutionized AFM imaging field. The absence of lateral forces permitted for the first time to image poorly
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attached samples in almost physiological conditions and more importantly to monitor online biochemical reactions. This technological breakthrough paved the way to numerous biochemical reactions visualization by AFM. Degradation of DNA by DNAse I was among the first biochemical reaction to be observed by using this new technique. Later E. coli RNA polymerase was among the first complex to be observed with this technique (43). The same year Guthold et al. observed the complex formation between RNA polymerase and DNA (44). Later, transcription of DNA by RNA polymerase could be monitored on line with tapping mode imaging (45). For these experiments the authors used specifically designed DNA molecules that contained a stall site to permit positioning of RNA polymerase at a specific location on the DNA molecule before imaging. The preformed complex was deposited onto the mica substrate and inserted into the imaging chamber of the microscope. Once a potentially functional complex was identified with the AFM, a medium that restarts the transcription was injected into the imaging chamber to resume the process. To permit low speed AFM imaging, the DNA-RNA polymerase interaction was slowed down by reducing the nucleotides concentration in the imaging buffer. Later, RNA polymerase was also monitored during its one dimensional random walk along DNA molecules by using a similar immobilization technique (46). In this work the authors demonstrated that in addition to sliding, RNA polymerase can jump from one site to another. Among other similar studies we can mention p53 interaction with DNA. P53 acts as a transcription factor for a number of genes involved in inhibiting tumor cell growth and transformation. Jiao et al. observed on line numerous types of interactions among the two molecules such as association-dissociation as well as sliding of p53 along the DNA molecule (47). Also in liquid, the action of Topoisomerase II (Topo II) was evidenced when unknotting DNA and transforming it into circular DNA: a delicate balance between attraction of DNA to mica was found that permitted to keep the accessibility of knotted DNA by Topo II (48). Figure 8a depicts Topo II as complexed to two linear DNA molecules whereas figure 8b shows a time-lapse sequence of Topo II unknotting a single DNA molecule.
7. High speed AFM (HSAFM) As pointed out previously one of the most serious drawback of traditional AFM imaging is its slow data acquisition rate. It should be kept in mind that it takes between 1-4 minutes to acquire a typical AFM image whereas RNA polymerase translocation along DNA is completed in a timeframe of seconds. To circumvent this bottleneck very early attempts were undertaken to accelerate the acquisition speed. One of these attempts consisted in using phase imaging instead of amplitude when operating in tapping mode (49). This approach permitted to increase somehow the speed but the ultimate goal, i.e. the real-time imaging of biological processes was still afar. Numerous groups struggled to develop instruments that can achieve a sub-second image acquisition rate and among all of them the probably most successful was the group of T. Ando (50), (51). To increase AFM imaging speed by one or two orders of magnitude required tremendous efforts and modifications. The readers interested in this topic and applications are invited to consult the very complete and comprehensive description of high speed AFM technique that was published in the recent review by Ando et al. (52).
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Biotin binding to streptavidin is one of the most studied and best known protein-protein interaction. The two proteins bind each to other in a 4 :1 fashion (four biotins for one streptavidin) with high affinity and selectivity. In 2007 Kobayashi et al (53) used HSAFM (5-7 frames/sec) to follow the dynamics of short DNA segments on mica. By covalently attaching biotin molecules at the end of a DNA strand the authors could follow DNA-binding - unbinding from streptavidin. The way restriction enzymes find their recognition sites is a highly interesting topic since they have been shown to find their recognition site 100-1000 times faster than what would be expected by random diffusion. In order to investigate this topic Gilmore et al. (54) used HSAFM to follow DNA-EcoRII complex dynamics. EcoRII is a restriction enzyme that belongs to the IIE REases group and that requires to bind several (2 or 3) DNA recognition sites to cleave one of them. The HSAFM movies recorded at a rate of two frames per second highlighted the way the enzyme dissociates off the DNA, how it diffuses and translocate along it. Another application of HSAFM in protein DNA interaction exploration was the study of nucleosomes dynamics. Nucleosomes consist in a DNA segment wound around histone protein cores and constitute the fundamental repeating units of eukaryotic chromatin. They play an important role in gene expression and are thought to carry epigenetically inherited information too. Nucleosomes unwrapping provides accessibility to DNA to bind proteins for replication but up to recently the details of its dynamics were largely unexplored. In 2010 Suzuki et al. (55) used HSAFM to follow DNA dynamics. Importantly the DNA movements were analyzed and permitted to concluded that the motion was thermally induced and not triggered by the scanning. Nucleosomes observation permitted to visualize nucleosome sliding on DNA in a range of 50 nm. In some cases, the authors observed nucleosome disruptions too. One year later Miyagi et al. (56) employed HSAFM to observe nucleosomes dynamics and their study highlighted that nucleosomes spontaneous unwrapping occurs in a sub-second timeframe. The information gathered by HSAFM was fully compatible with the previous model of Widom (57) but HSAFM additionally highlighted a possible and novel alternative to unwrapping i.e. sliding. In this case the nucleosome moves rapidly in one direction, unconvering a given DNA sequence and comes back to its original position, all in a timeframe of about 2 sec. A more recent HSAFM study involving nucleosomes and tetrasomes dynamics has been conducted by Katan et al. (58). Tetrasomes are subnucleosomal structures that contain 4 instead of 8 histones in the protein core. The authors observed at high spatial and temporal resolution nucleosomes and tetrasomes disassembly, sliding, and hopping. High time resolution AFM images of transcription were achieved in 2012 by Suzukiet al. (59) and Endo. Endo et al. (60) used a HSAFM to directly observe single RNA polymerase binding and sliding along dsDNA. RNA synthesis as well dissociation could also be observed. The setup was very original and efficient. It consisted in attaching a 1000 bp template dsDNA onto an 1150 bp long DNA origami platform (61). Between its two extremities the template DNA was free to float, fully accessible RNA polymerase, and still visible to AFM. During transcription the newly produced RNA transcript is very difficult to image by AFM. In order to visualize it the authors used biotinylated UTP and labeled it with streptavidin. This procedure dramatically increased RNA transcripts and made it visible to HSAFM. The same team further developed this HSAFM-DNA origami based DNA immobilization technique (62). In its new version the DNA segment to be studied (DNA template) is incorporated in a 100x80 nm square DNA origami containing a 40x40 nm wide window across which two DNA template are suspended. The 9
system is very convenient since it permits the placement of any sequence of DNA (double or singlestranded) across the frame and the precise control of the relative position of the two templates, their lengths, chemical composition (i.e. modified nucleotides) tension and rotation. The team used this system to follow numerous protein-DNA interactions such as DNA bending (EcoRI methyltransferase), DNA base repair (8-oxoguanine glycosylase and T4 pyrimidyne dimer glycosylase) or to follow DNA structural changes such as G-quadruplex formation or B-Z transition. A similar technology has recently been adapted by Godonoga et al. (63) to study Plasmodium falciparum lactate dehydrogenase (PfLDH) interaction with different DNA aptamers. Aptamers are synthetic single stranded DNA or RNA fragments that are designed to bind specific targets. Once synthetized, intermolecular binding forces induce their folding in particular 3D configurations that is responsible for their affinity. Aptamers are nowadays used as therapeutic agents (64) and biosensors (65). Godonoga et al. integrated PfLDH-binding DNA aptamer in a DNA origami and used HSAFM to monitor in real time attachment-detachment of PfLDH.
8. Conclusion After the invention of the Scanning Tunneling Microscope (STM) by Binning, Rohrer, Gerber and Weibel (66) science has received a tremendous impulse due to the possibility now to investigate nature on a spatial scale nearing the atomic-subatomic scale with a fairly limited effort: this has led to the blooming of a new field in science and technology, namely nanotechnology. This paradigm change was possible because a table top instrument permits to image samples and investigate atomic scale phenomena in direct space. The limitation of the STM, namely the condition that the sample must be a conductor, was quickly surpassed by the invention of Atomic Force Microscope (AFM) by Binnig, Quate and Gerber (67) Now, with this instrument, an almost infinite class of samples could be imaged and investigated down to the atomic scale. The whole realm of biology was opened to AFM when the microscope was expanded to function in liquid environment permitting the study of bio-samples in physiological conditions. From this point on, there were numerous declinations of the microscope, named often Scanning Probe Microscope, where the “probe” could be a simple tip (AFM), an optical fiber (SNOM), a pipette (SICM) or a temperature probe, etc. Not all of the declinations have lived up to the initial hopes, but progress has been steady. For example, nowadays high-speed AFM is a reality and delivers video rate imaging of biological processes like myosin “walking” on actin filaments, an experiment unthinkable only few years ago, and probably be doomed to remain a dream if it were not for the invention of the AFM. The review presented here gives an overview of the possibilities offered by the AFM and should stimulate further ideas and developments beyond the present methods and techniques, especially applications to biological processes should be here emphasized which, according to the personal view of the authors, contains a large potential for new and paradigm changing developments.
Acknowledgments We apologize to our colleagues whose work could not be included in this review due to space limitations. Authors were funded by the Swiss National Grant 200021-144321, 407240_167137 and also acknowledge the support of Gebert Rüf Stiftung GRS-024/14.
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Figure captions Figure 1 Working principle of the AFM. A sharp tip fixed at the end of a soft cantilever scans the surface of the sample. The deflections of the cantilever during the scan are detected and displayed on a computer screen to reconstruct the sample’s topography
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Figure 2 In the so called tapping mode (or intermittent contact) the cantilever is oscillated close to its resonant frequency and the tip periodically hits the sample. The feed-back loop is locked on the cantilever oscillation amplitude.
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Figure 3 A thin layer of water covers the samples to be imaged in air and dramatically increases the interaction force between the tip and the sample (green ball in this illustration) by the formation of a water capillary bridge.’
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Figure 4 AFM images consist in a convolution between the geometry of the tip and the sample (green) therefore only the uppermost parts (light green) of the samples are visible to the AFM.
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Figure 5 Interaction force measurement by AFM. The tip is periodically approached and retracted off the substrate. In case of attachment and dissociation a characteristic peak appears on the force distance curve (red)
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Figure 6. Typical setup for AFM imaging in liquids 1. liquids to be flown in the analysis chamber 2. selector 3. flow speed regulator 4. AFM 5. waste container 6. balance that permits the determination of the moment a new solution arrives into the analysis chamber
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Figure 7 Single stranded DNA inserted in the middle (A) or the end (B) of a double stranded DNA filament
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Figure 8a Topo II bind to DNA cross-overs on linear DNA molecules. Figure from (48) with permission.
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Figure 8b Time-lapse imaging of Topo II unknotting a single DNA molecule. The white bar represents 250 nm. Figure from (48) with permission.
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