Single-molecule studies of high-mobility group B architectural DNA bending proteins

Abstract

Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

Keywords: Bending; Binding; DNA; HMGB; Kinetics; Protein.

Fig. 1

Schematic illustrations (not to scale) depicting single-molecule techniques used to investigate HMGB architectural protein binding to DNA. Optical tweezers, magnetic tweezers and atomic force microscopy are used. a In an optical tweezers setup, DNA tethered between labeled beads is extended and released. A glass micropipette tip is used to extend the DNA molecule, while on the other extremity, the deflection of the laser beam during extension is recorded and the signal is then translated into force. (From Murugesapillai et al. 2014). b In a magnetic tweezers setup, DNA tethered between a labeled paramagnetic bead and a functionalized cover slip is held at constant magnetic force and the extension is recorded using a CCD camera. Magnetic tweezers combined with fluorescently labeled proteins (green) allows visualization as well as quantification of protein binding. (Adapted from Skoko et al. and Xiao et al. 2010). c In a dual trap optical tweezers setup, DNA tethered between labeled polystyrene beads is extended and released. Fluorescently-labeled molecules (green) interact with the DNA and their binding can be visualized. (Adapted from Heller et al. 2014). d Atomic force microscopy is used to visualize protein–DNA complexes. The reflection of the laser beam off the cantilever to detector is then converted into an imaging signal. (Adapted from Murugesapillai et al. 2014)

Fig. 2

Extension and release of a bacteriophage λ DNA. a Measured extension (solid black) and release (dotted black) curves of bacteriophage λ DNA (48,500 base pairs). (Adapted from McCauley et al. ; Murugesapillai et al. 2014)

Fig. 3

Binding of Nhp6A and HMO1 proteins to λ DNA characterized by optical tweezers. a Solution structure of the yeast single box Nhp6A protein bound to DNA with intercalating amino acid side chains shown as gray space-filled atoms (PDB code: 1J5N). b Force–extension curves are shown for phage λ DNA in the absence (black) and presence (red) of the single box Nhp6A protein. c Fits to the WLC model in the absence (black) and presence (red) of Nhp6A. d Solution structure of a double box HMGB protein bound to DNA (PDB code: 2GZK). e Force–extension curves are shown for phage λ DNA in the absence (black) and presence (blue) of the double box HMO1 protein. f Fits to the WLC model in the absence (black) and presence (blue) of HMO1. (Adapted from McCauley et al. ; Murugesapillai et al. 2014)

Fig. 4

Equilibrium analysis of Nhp6A and HMO1 protein binding to DNA. a Persistence length of the DNA in the presence of Nhp6A (red) and HMO1 (blue) as a function of concentration is fitted to Eqs. 2 and 4 to obtain K _D = 71 ± 14 nM and ω = 20 for Nhp6A, and K _D = 2.1 ± 0.8 nM and ω = 20 ± 7 for HMO1. b Contour length of DNA in the presence of Nhp6A (red) and HMO1 (blue) as a function of concentration is fitted to Eqs. 2 and 6 to obtain K _D = 71 ± 14 nM and ω = 20 for Nhp6A, and K _D = 1.9 ± 0.7 nM and ω = 18 ± 5 for HMO1. c The DNA overstretching region with extensions only is shown for DNA in the absence (black circles) and presence of Nhp6A (red triangles) and HMO1 (blue triangle). (Adapted from McCauley et al. ; Murugesapillai et al. 2014). d Overstretching force is fitted to the site exclusion binding isotherm of Eqs. 2 and 3, yielding measurements of K _D = 160 ± 20 nM and ω = 20 for Nhp6A, and K _D = 2.8 ± 0.6 nM and ω = 80 ± 15 for HMO1

Fig. 5

Global flexibility. Binding of double box HMO1 to pBR322 DNA characterized by atomic force microscopy (AFM). a Schematic of the AFM instrument used to image DNA–protein interactions. b A two-dimensional image illustrates linearized pBR322 DNA on a mica surface (scale bar 300 nm). c Schematic diagram showing local DNA bend. The angle is calculated from two adjacent line segments (gold) drawn between three agacent points, separated by a distance L (green dots). d A fit to the two-dimensional WLC model (Eq. 7) enables the calculation of DNA persistence length. Red and blue curves correspond to 0.11 nM DNA in the absence (lower right; scale bar 300 nm) or presence (upper left inset, white dots are bound protein; scale bar 200 nm) of 3 nM HMO1 protein. (Adapted from Murugesapillai et al. 2014)

Fig. 6

Binding of the double box HMO1 to pBR322 DNA characterized by AFM, illustrating the analysis of local DNA flexibility. a A three-dimensional AFM image of HMO1 protein bound to linearized plasmid pBR322 DNA (4361 bp). The vertical color gradient bar represents the sample height ranging from 0.0 to 2.0 nm. b Schematic diagram showing protein-bound locations from DNA only. The angle is calculated from two adjacent line segments (in gold) drawn at the location of the protein-bound site (green dots are the three equidistant points used to draw the line segments). c The measured angle could be either clockwise (positive) or counterclockwise (negative). Both directions are taken into account resulting in a bi-Gaussian fit (red), where β is the mean bend angle and σ gives the width of the distribution. d Histogram of measured local protein-induced DNA bend angles for the double box HMO1 and fit. The average measured angle is 38 ± 2.0° with σ = 33 ± 3° (Murugesapillai et al. 2014)

Fig. 7

Models describing the nature of local flexibility induced by HMGB proteins upon binding DNA. a In the static kink model, the protein binds to DNA and induces a bend angle, β. While the protein remains electrostatically bound in the vicinity of the DNA, it can dissociate and associate and each binding event induces the same bend angle, β. b Measured local protein-induced DNA bend angles for the single box protein human HMGB2 (Box A) and fit (red). The average measured angle peaks at 64.5 ± 2.0° with σ = 26.0 ± 1.7°. c Model describing the average bend angle and the standard deviation. The narrow standard deviation is indicative of a static kink model. d In the flexible hinge model, the protein induces a different bend angle at each binding event, and β ₂′ (purple) represents a binding event after some time. e Measured local protein-induced DNA bend angles for HU proteins. The distribution of angles is very broad. f Model describing the average bend angle and the standard deviation. The broad standard deviation is indicative of a flexible hinge model. (Adapted from Zhang et al. and van Noort et al. 2004)

Fig. 8

Average bend angle as a function of standard deviation. a Model depicting average induced DNA bend angle and its associated standard deviation. b The bending nature of HMGB proteins can be explained by the static kink model and a model between static kink and flexible hinge, which we refer to as intermediate

Fig. 9

Protein-DNA off-rate in single molecule experiments is bimolecular. a The DNA is compacted in the presence of Nhp6A, the extension is reduced from 13.5 to 7 μm. No return of the extension was observed when buffer solution was flowed at ~600 s (transition from light to dark symbols on the graph), but after flowing competitor DNA fragments at ~2400 s, the compacted DNA recovered its initial length. b Fluorescence images of Fis exchange illustrates that not all proteins have exchanged. See text for description of individual frames. c Fluorescent protein is exchanged for non-fluorescent wild-type HU, a decrease in fluorescence intensity is observed. d Exchange rate obtained from each fit in (c) is proportional to concentration of wild-type HU in solution (Adapted from Skoko et al. and Graham et al. , with permission)

Fig. 10

Protein–DNA loop formation as a mechanism for DNA compaction. a–d Schematic illustrating the formation and breaking of loops. When the DNA is held at low forces, HMO1 proteins are able to mediate and stabilize loops and the force–extension curve is relatively flat (b). Here, the blue line represents the force–extension curve. In contrast, when the DNA is extended further, the force–extension curve shows jumping events, revealing the breaking of loops mediated by HMO1 (c). As the DNA is further extended, unlooped DNA with proteins bound is stretched (d). e Force–extension curves for phage λ DNA in the presence of 0.3 nM HMO1. Each jump illustrates a loop-breaking event. Fitting is to the WLC model (solid red lines). Loop size is estimated by measuring the contour length change over the force jump. f Loop sizes. The most probable loop size is between 400 and 600 bp. g Loop breaking forces. The most probable loop breaking force is between 10 and 15 pN at this pulling rate of 950 nm/s. (Adapted from Murugesapillai et al. 2014)

Fig. 11

HMO1 bridges, loops and compacts DNA. a Two-dimensional representation of bridges and loops mediated by 3 nM HMO1 in the presence of 0.11 nM pBR322 DNA (scale bar 200 nm). b Three-dimensional representation of a looped single DNA molecule; the cross-sections of DNA only, DNA with protein-bound, protein bridging two DNA double helices, and two DNA double helices held close to each other by protein on its ends are shown in green, red, purple and blue, respectively. The top right inset displays a two-dimensional representation of locally probed HMO1–DNA complexes (scale bar 100 nm). Graphs of the heights (bottom-left inset) are shown for each cross-section on the image (c) Two-dimensional representation of HMO1 looping (protein-bound at the intersection of a loop) and bridging (protein-bound holding two strands close together) a DNA molecule. Traced loops are shown in blue. Inset Original AFM image without traces (scale bar 100 nm). d DNA loop sizes mediated by HMO1. The color bar in each panel represents the sample height ranging from 0.0 to 2.0 nm. (Adapted from Murugesapillai et al. 2014)

Fig. 12

Constant force measurement. a A single DNA molecule is kept at a constant force of 10 pN. In the absence of proteins, the distance between beads (red arrow) does not change in time. b While keeping the force constant at 10 pN and in the presence of proteins (red circles), the distance between the beads decreases (red arrow) at a later time t₂. c As the exposure time of proteins to DNA increases, the DNA molecule is further compacted and the distance between beads has further decreased and has reached a constant value (red arrow). The difference between the initial position and the final position of the bead indicates the total amount compacted (green arrow). d When DNA is exposed to 10 nM HMO1, constant force measurements at 10 pN and 100 nM NaCl indicate that HMO1 compacts DNA (red arrow pointing to the left). The compaction force is ∆F_c = 1.7 ± 0.3 pN and the rate constant for compaction, k, is 0.64 ± 0.10 s⁻¹ (τ = 1.6 ± 0.2 s), obtained by fitting the change in extension as a function of time (inset, green curve) to a single exponential (red line). (Adapted from Murugesapillai et al. 2014). e Similarly, when DNA is exposed to 50 nM double box HMGB protein TFAM at 150 mM NaCl, the rate constant for compaction, k, is (3.0 ± 1.0) × 10⁻² s⁻¹ (From Farge et al. , with permission)

Fig. 13

Study of 50 nM TFAM binding and unbinding events on DNA held at 10 pN. a The frames illustrate the unbinding events of TFAM from DNA. b Fluorescence intensity as a function of time for a molecule held at a constant force and covered with TFAM. The red line represents a fit to obtain the dissociation time. The distance between beads increases as a function of time (inset). (From Farge et al. , with permission)

Fig. 14

DNA torsionally constrained overstretching transition and the kinetic analysis of Nhp6A protein binding to DNA. a Torsionally constrained DNA is characterized by an overstretching force at 110 pN whereas torsionally relaxed DNA experiences the overstretching force at 65 pN. At high pulling rates, DNA in the presence of HMGB proteins displays an overstretching transition at forces comparable to those expected for torsionally constrained DNA. This result is interpreted as evidence that bound HMGB proteins block DNA unwinding. At low pulling rate the increase in overstretching transition due to protein binding occurs at ∼75 pN. b Schematic of torsionally constrained DNA (1 unable to rotate), and torsionally unconstrained DNA (2 able to rotate and unwind). c In the presence of HMGB proteins, DNA becomes torsionally constrained at pulling rates higher than the protein dissociation rate. d DNA overstretching force in the absence (black) or presence (red) of Nhp6A versus pulling rate, fitted to Eq. (8) to estimate the dissociation rate, k _off. (Adapted from McCauley et al. 2013)

Fig. 15

Schematic interpretation of fast microscopic dissociation versus slow macroscopic dissociation. While remaining electrostatically bound, HMG proteins locally dissociate from bent DNA at a rate described by k _off,micro and then diffuse along DNA before rebinding (not shown) or fully escaping from DNA at a rate described by k _off,macro