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Nanotribology results show that DNA forms a mechanically resistant 2D network in metaphase chromatin plates - PubMed

  • ️Fri Jan 01 2010

Nanotribology results show that DNA forms a mechanically resistant 2D network in metaphase chromatin plates

Isaac Gállego et al. Biophys J. 2010.

Abstract

In a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+) have a relatively high friction coefficient (μ≈0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ≈0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (μ≈0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ≈0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis.

Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Direct AFM visualization of plate denaturation. (a) Two consecutive images of a plate before the addition of a concentrated solution of NaCl (final concentration ∼1.3 M). The image of the denatured structure (lower) was obtained 43 min after the addition of NaCl. (b) The image at right was obtained 13 min after the addition of a concentrated solution of EDTA (final concentration ∼6 mM). (c) Plates were completely denatured (see image at right) 53 min after the addition of trypsin (final concentration ∼0.7 mg/mL). (d) The image at right was obtained 5 min after the addition of micrococcal nuclease (final concentration ∼42 units/mL). In all images the plates show their characteristic smooth surface; the large amorphous aggregates are seen as intense white spots. Scale bars, 500 nm (a, b, and d) and 200 nm (c).

Figure 2
Figure 2

Nanotribology analysis of metaphase chromosome plates in the presence of NaCl and EDTA. (a) Images corresponding to a native plate before and after the measurement that produced the friction-versus-vertical force curve shown in this figure. Images of plates treated with ∼2.9 M NaCl (b) and ∼0.3 M EDTA (c) before and after the friction measurement that produced the curves shown in these figures. Scale bars, 250 nm.

Figure 3
Figure 3

Nanotribology analysis of metaphase chromosome plates treated with nuclease and protease enzymes. (a, left and middle) The plate was treated with micrococcal nuclease (∼6 units/mL) and imaged (middle) after the measurement that produced the orange curve in the friction-versus-vertical force plot (inset). After the readdition of nuclease (final concentration ∼13 units/mL), the red curve was obtained; the image obtained after this friction measurement is shown at right. (b) Images corresponding to plates treated with 1/20 and 1/5 dilutions of a concentrated solution of pronase (∼1 mg/mL) after the friction measurements that produced the black and green curves, respectively (inset). Scale bars, 250 nm (a) and 500 nm (b).

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