Evidence of protein-free homology recognition in magnetic bead force–extension experiments
D. J. (O’) Lee, C. Danilowicz, C. Rochester, A. A. Kornyshev, M. Prentiss
Published 20 July 2016.DOI: 10.1098/rspa.2016.0186
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.
Data supporting this article are included in the electronic supplementary material. S1 contains additional supporting experimental data; S2 contains details on the background of Model 1; S3 discusses the approximations behind the equations for Lloop; S4 presents plots of the free energy for model 1 as well as a plot showing how Lloop varies with the parameter b in that model; S5 shows the fitted the values of the model parameters; S6 specifies exactly the DNA text that was added to the end of the λ DNA in the constructs.
D.J.(O’)L. had a major role in the writing of the paper and development of the theoretical models, as well as helping with the data analysis and curve fitting. C.D. designed and performed the experiments. C.R. helped with the data analysis and performed the curve fitting. A.A.K. participated in the development of the theory, discussion of experimental results and writing of the paper. M.P. designed the experiments, analysed data, as well as participated in writing the paper and the development of the theory.
The authors have no competing interests.
This work was supported by the National Institutes of Health to M.P. (grant no. R01 GM044794), the Human Frontier Science Program to A.A.K. (grant no. RG0049/2010-C102) and the grant of the Engineering and Physical Sciences Research Council, EP/H010106/1.
The authors acknowledge useful discussions with Nancy Kleckner and Tim Albrecht.
Received March 15, 2016. Accepted June 17, 2016.
© 2016 The Authors.
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Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.
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