Abstract
Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.
Highlights
The pairing of homologous chromosomes in the absence of DNA breakage is an essential step in many biological processes [1]
Though the size of the extension difference varied with buffer conditions and temperature, dsDNA containing the homologous 10 kb regions always showed significantly shorter extension than the controls at forces approximately 1–5 pN, with many buffer conditions showing a deviation over a wider range of forces
The initial increase is attributable to the force-dependent factor that sits in front of Lloop in equation (3.6); this is due to worm-like chain (WLC) behaviour of the un-looped part of the DNA
Summary
The pairing of homologous chromosomes in the absence of DNA breakage is an essential step in many biological processes [1]. In solutions containing multivalent ions, there can be strong attractive homology insensitive electrostatic interactions between dsDNA molecules [17,18]; explanations include correlation effects between fluctuations in the local counterion charge densities [19] and ordering of multivalent ions near the surfaces of DNA duplexes [9,20]. Both possibilities create a negative–positive charge motif that depends on the counterion valence and salt concentration. The temperature was controlled by a thermoelectric cooler that allowed us to do experiments at temperatures from 20◦C to 45◦C
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