Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

Abstract

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.

Differential repair enzyme-substrate selection within dynamic DNA energy landscapes

“…Since 2Ap and pdC fluorescence increases when these bases become solvent-exposed, clearly bulge loop formation leads to a decrease in the degree of order/ stacking encountered by both fluorophores relative to the single strand structure. A high degree of single-strand order in the CNG repeat domains has been widely reported in the literature and is generally considered the basis for the propensities of CNG repeats to miss-fold (Amrane et al, 2005;Figueroa, Cattie, and Delaney, 2011;Gacy, Goellner, Juranic, Macura, and McMurray, 1995;Hu et al, 2021;Huang et al, 2017;Paiva and Sheardy, 2004;Petruska, Arnheim, and Goodman, 1996;Xu et al, 2020). Prior to bulge loop formation this high degree of single-strand order is also inferred by the strong CD signal of the two isolated single strands at low temperature, as well as by the single strand melting curves shown in the supplementary material.…”

“…The magnitude of the activation energy barriers between dynamically interconverting bulge loops is as yet unknown but one can posit that the maximum barrier height is simply the free energy cost of unfolding the entire loop plus 3 base pairs needed to allow movement. One can also posit, based on recent single-molecule measurements (Hu, Morten, and Magennis, 2021;Xu, Pan, Roland, Sagui, and Weninger, 2020) and our own unpublished data that a minimum barrier height is given by a sliding/migrating mini bulge of 3 nucleotides moving across the larger repeat bulge to allow migration. Conceptually, in a dynamic landscape, each macrostate consists of a 'family' of more rapidly interconverting/fluctuating, energetically similar conformations; a framework similar to that recently described for a different DNA construct as 'microstates' by von Hippel and co-workers (Israels et al, 2021).…”

Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy

“…Another question is why longer oligomeric products were not observed on the longer r(CUG) 90 template. The structure of r(CUG) 90 is not fully understood, but it is likely a dynamic structure containing multiple hairpins of varied length, rather than one long hairpin [42–45] …”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…Dynamics intrinsic to HJ were analyzed to find cognate sequence and achieve site-specific cleavage [88] Kinetic details of GEN1 dimers decomposed HJ were explained [91] Strand sequence A model of reversible branch migration in mobile 3WJ with trinucleotide repeats was proposed, which may help the treatment of diseases [93] RNA secondary structure Metal cation Na + and K + were proved to facilitate the formation of RNA tetraloop-tetraloop receptor tertiary motif [101] Small molecules in solution…”

“…Moreover, replication slippage is a frequent reason for the emergence of 3WJs. Hu et al [ 93 ] designed 3WJs with slip-outs of between 2 and 30 CTG or CAG repeats, and the FRET signals showed that repeats of slip-out led to a two-state behavior. Based on previous works and existing data, the authors proposed a model of reversible branch migration in mobile 3WJs with trinucleotide repeats, which may help the treatment of diseases.…”