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. 2011 Jan;37(1):117-31.
doi: 10.1007/s10867-010-9203-7. Epub 2010 Oct 9.

DNA condensation by TmHU studied by optical tweezers, AFM and molecular dynamics simulations

Carolin Wagner, Carsten Olbrich, Hergen Brutzer, Mathias Salomo, Ulrich Kleinekathöfer, Ulrich F Keyser, Friedrich Kremer

DNA condensation by TmHU studied by optical tweezers, AFM and molecular dynamics simulations

Carolin Wagner et al. J Biol Phys. 2011 Jan.

Abstract

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.

Keywords: Force clamp; Optical tweezers; Protein–DNA interaction; Single-molecule study; Steered molecular dynamics; Thermotoga maritima.

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Figures

Fig. 1
Fig. 1
Condensation reaction at 2 pN. Three typical results for the condensation of DNA by TmHU at a force level of 2 pN with a protein concentration of 100 formula imageg/ml (blue, red, green) and a control trace of DNA in the absence of TmHU (orange) are displayed. The DNA duplex is hold at a constant force level (±0.4 pN) while the protein solution is flushed in. The change in length of the DNA under the action of TmHU is recorded, which is defined as the difference to the plateau at t = 0–10 s. The moment of t = 0 was located about 10 s before the reaction starts. We distinguish two regimes during the reaction, the initial one, where shortening happens linearly with a rate of (70 ± 30) nm/s until a shortening of (0.77 ± 0.12) formula imagem is reached, and the subsequent condensation (for t > 10 s) that proceeds at a much slower rate and is less continuous. The reaction comes to a halt at approximately 1 formula imagem shortening. Inset shows the scheme of the experiment. (a) A single double-stranded DNA is immobilised between two microparticles. One of them is held by the optical trap with a constant force while the other is fixed at the micropipette tip. (b) After flushing the protein solution into the sample chamber the condensation of the DNA can be observed
Fig. 2
Fig. 2
Force dependence of the condensation reaction. The TmHU-induced condensation of the DNA at different force levels is displayed. With increasing force, the shortening decreases significantly, until at 40 pN almost no condensation is taking place. In contrast to the case for 2 pN, above 10 pN no slow shortening is observed. Inset shows mean shortening at different forces. The error bars indicate minimal and maximal values
Fig. 3
Fig. 3
Concentration dependence of the condensation reaction. The condensation of DNA by TmHU at a force of 2 pN is compared for concentrations between 250 and 20 formula imageg/ml. At smaller protein concentrations the reduction in length proceeds slower and less linearly. Insets show the mean values of at least five single measurements of (a) the total reduction in length and (b) the condensation rate to a shortening of 0.5 formula imagem. The error bars are given by minimal and maximal values. Both rate and final extension increase with increasing concentration. At 100 formula imageg/ml, a saturation is observed
Fig. 4
Fig. 4
Condensation of DNA (250 bp) by TmHU observed by AFM at TmHU concentrations of 0 mg/ml, 20 formula imageg/ml and 100 formula imageg/ml. The results presented here are representative for the 30–100 individual complexes that were analysed for each concentration. Insets show zooming in and height profile along the blue line. Left AFM image of double-stranded DNA (250 bp). The DNA shows an extended shape with mean end-to-end distance of 70 nm. The height profile displays a value around 0.5 nm. Middle at 20 formula imageg/ml, the complexes still show an extended shape with a mean end-to-end distance of 42 nm. However, the height of the complex is here almost 2 nm. Right the structure of the complexes at 100 formula imageg/ml is highly condensed and has a globular shape with a diameter of 38 nm
Fig. 5
Fig. 5
Force–distance dependence of the SMD-simulated stretching of the TmHU–DNA complex. The 35 bp DNA is stretched with a velocity of 0.5 m/s with a force constant of 70 pN/nm. The snapshots A–D show the rupture of bonds between the DNA and the protein. The corresponding parts of the force–distance trace are indicated. A Snapshot at t = 7.5 ns. The arrows indicate the two resulting forces pointing in opposite directions. The force increases almost linearly with the distance of the DNA ends to the protein. The DNA sticks to both sides of the protein. B Snapshot at t = 11.0 ns. The DNA is disrupted from the left side of the helical body of the protein, which leads to an increase in distance of about 2 nm. Simultaneously, an unwinding of both DNA tails takes place, which yields an additional extension of ~1 nm each. C Snapshot at t = 20.25 ns. At around 500 pN, the right part of the DNA unbinds from the body of the protein and relaxes for another 2 nm. D Snapshot at t = 25.0 ns. Both DNA ends are released from the protein and are stretched further
Fig. 6
Fig. 6
Visualisation of the supposed structural formation of the TmHU/DNA complex. The condensation starts with a primary TmHU binding until a shortening of around 0.77 formula imagem is reached. Henceforward, the final complex builds up in higher levels of structural organisation. The total reduction in length compared to the extension of the bare DNA reaches around 1 formula imagem, which corresponds to about 70% of the initial DNA length
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References

    1. Dame RT. The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Mol. Microbiol. 2005;56(4):858–870. doi: 10.1111/j.1365-2958.2005.04598.x. - DOI - PubMed
    1. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM.Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima Nature 1999399323–329.10.1038/206011999Natur.399..323N - DOI - PubMed
    1. Huber R, Langworthy TA, König H, Thomm M, Woese CR, Sleytr W, Stetter KO. Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°. Arch. Microbiol. 1986;144:324–333. doi: 10.1007/BF00409880. - DOI
    1. Mukherjee A, Sokunbi AO, Grove A. DNA protection by histone-like protein HU from the hyperthermophilic eubacterium Thermotoga maritima. Nucleic Acids Res. 2008;36:3956–3968. doi: 10.1093/nar/gkn348. - DOI - PMC - PubMed
    1. Esser D, Rudolph R, Jaenicke R, Böhm G. The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein. J. Mol. Biol. 1999;291:1135–1146. doi: 10.1006/jmbi.1999.3022. - DOI - PubMed

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