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.
Highlights
Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease
The presence of 3 complementary CAG-CTG triplets in the duplex allows, in principle, four possible Watson-Crick positional isomers to exist, with identical numbers of base pairs; we refer to these as mobile 3WJs. We reveal that these secondary structures undergo two distinct types of dynamics, which has implications for understanding and treating trinucleotide repeat expansion diseases (REDs), and their development as drug targets[23,24]
We designed four static 3WJs with slip-outs of 10 repeats of either CAG or CTG that form a stable intrastrand hairpin[12]; these mimic the possible positional isomers that could form for mobile 3WJs having a 10-repeat slip-out
Summary
Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 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. While isolated hairpins have received most attention, less is known about the structures formed when the hairpin extrudes from the duplex[9] These branched structures, which are DNA three-way junctions (3WJs), can form during replication, repair, and recombination, and are involved in all current models of repeat expansion. We reveal that these secondary structures undergo two distinct types of dynamics, which has implications for understanding and treating trinucleotide REDs, and their development as drug targets[23,24]
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