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DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as “rollamers”. Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain (“rollamerization”). We demonstrate that such “rollameric” migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor “rollamers” with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic “universal hinge” relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.

We have measured at 25 degrees C the relative specific sound velocity increment, [u], and the partial specific volume, v degrees, of cytochrome c as a function of pH. Our data reveal that the base-induced native to unfolded transition of the protein is accompanied by a volume decrease of 0.014 cm3 g-1 and a compressibility decrease of 3.8 x 10(-6) cm3 g-1 bar-1. These results allow us to conclude that, relative to a fully unfolded conformation, the base-denatured state of cytochrome c has only 70 to 80% of its surface area exposed to the solvent. Recently, we reported a similar result for the acid-denatured state of cytochrome c. Thus, insofar as solvent exposure is concerned, both the base- and the acid-induced unfolded states of cytochrome c retain some order, with 20 to 30% of their surface areas remaining solvent-inaccessible. We discuss the implications of this result in terms of defining potential intermediate states in protein folding pathways.

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, non-canonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype-phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes.

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