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. 2014 Sep 2;42(17):11218–11232. doi: 10.1093/nar/gku782

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — MBD4_MBD exchanges between methylated sites in the same molecule of DNA on the fast NMR timescale. (a) A diagram depicting slow intermolecular and fast intramolecular exchange by MBD4_MBD. (b) 2D ¹H-¹⁵N TROSY HSQC spectra of MBD4_MBD bound to methylated wild-type and inverted (10 bp) DNA (red), tandem (30 bp) DNA (blue) and tandem nicked (30 bp) DNA (green) show that MBD4_MBD more rapidly exchanges between sites in the same molecule of DNA. The spectra show only a single crosspeak for Arg¹⁰⁵ (left panel) when bound to DNA containing both wild-type and inverted sites, which is consistent with fast intramolecular exchange. A similar comparison for Phe¹⁰⁶ (right panel) shows marked broadening of the intramolecular crosspeaks consistent with intermediate exchange on the NMR timescale which likely reflects line-broadening from additional internal dynamic motions. Incorporating a single-strand defect does significantly alter rapid intramolecular exchange. (c) In contrast, increasing NaCl concentration accelerates both intermolecular and intramolecular exchange. The two separate peaks reflective of intermolecular exchange (red) for both Arg¹⁰⁵ (upper panels) and Phe¹⁰⁶ (lower panels) coalesce into a single peak with increasing NaCl concentration. This change indicates that the intermolecular exchange rate has increased from the slow to fast NMR timescale. Likewise the crosspeaks reflective of intramolecular exchange (blue and green) sharpen with increasing NaCl concentration, which is consistent with accelerated intramolecular exchange. For comparison, 2D¹H-¹⁵N TROSY HSQC spectra of MBD4_MBD bound to methylated wild-type (gray) and inverted (purple) DNA are shown at 100 mM NaCl.