The Double Helix in Motion: New Insights into Sequence-specific, FunctionalDNA Dynamics Using NMR Spectroscopy

[摘要] DNA is a highly flexible molecule that undergoes a variety of structural transitions in response to cellular cues. Sequence-directed variations in the canonical double helix structure that retain Watson-Crick base-pairing play important roles in DNA recognition, topology, and nucleosome positioning. Here, we use NMR relaxation methods to study sequence-directed dynamics occurring at picosecond to millisecond timescales in variable size DNA duplexes. Traditionally, atomic-level spin relaxation studies of DNA dynamics have been limited to short duplexes, in which sensitivity to biologically relevant nanosecond fluctuations is often inadequate. We introduce a method for preparing residue-specific 13C/15N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply it towards probing fast inter-helical bending motions induced by an adenine tract. Our results suggest the presence of elevated A-tract independent end-fraying and/or bending internal nanosecond motions, which evade detection in short constructs and that penetrate deep within the helix and gradually fade away towards its interior. By studying picosecond-nanosecond dynamics in short DNA dodecamers with variable length A-tracts, we discover that A-tracts are relatively rigid and can modulate the flexibility of their junctions in a length-dependent manner. We identify the presence of large-amplitude deoxyribose internal motions in CA/TG and CG steps placed in different sequences that likely represent rapid sugar repuckering. Moreover, by using NMR relaxation dispersion in concert with steered molecular dynamics simulations, we observe transient sequence-specific excursions away from Watson-Crick base-pairing at CA/TG and TA steps inside DNA dodecamers towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. We show that their populations and lifetimes can be modulated by environmental factors like acidity, monovalent and divalent ions as well as intrinsic sequence and chemical modifications. The observation of Hoogsteen base pairs in duplexes specifically bound to transcription factors and in damaged sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.

[发布机构] University of Michigan

[效力级别] NMR Spectroscopy

[关键词] DNA Dynamics;NMR Spectroscopy;Hoogsteen Base Pair;Relaxation Dispersion;A-tract DNA;Biological Chemistry;Science;Chemical Biology