DNA energy landscapes via calorimetric detection of microstate ensembles of metastable macrostates and triplet repeat diseases

Abstract

Biopolymers exhibit rough energy landscapes, thereby allowing biological processes to access a broad range of kinetic and thermodynamic states. In contrast to proteins, the energy landscapes of nucleic acids have been the subject of relatively few experimental investigations. In this study, we use calorimetric and spectroscopic observables to detect, resolve, and selectively enrich energetically discrete ensembles of microstates within metastable DNA structures. Our results are consistent with metastable, ''native'' DNA states being composed of an ensemble of discrete and kinetically stable microstates of differential stabilities, rather than exclusively being a single, discrete thermodynamic species. This conceptual construct is important for understanding the linkage between biopolymer conformational/configurational space and biological function, such as in protein folding, allosteric control of enzyme activity, RNA and DNA folding and function, DNA structure and biological regulation, etc. For the specific DNA sequences and structures studied here, the demonstration of discrete, kinetically stable microstates potentially has biological consequences for understanding the development and onset of DNA expansion and triplet repeat diseases.calorimetry ͉ nucleic acid conformations T he concept of rough biopolymer energy landscapes is important for understanding biopolymer properties and function. Complex landscapes allow partial population of discrete, kinetically stable (metastable) species (1-12). These substates, in turn, may serve as biologically significant ligands, beyond the traditional, thermodynamically most stable, time-averaged, ''native'' macrostrate. Although it is well recognized that macroscopic states are composed of microscopic substates, it has been customary for experimentalists to think of a biologically relevant native state as a singular macrostate with a unique, well defined structure characterized by a homogeneous/smooth energy profile. Many biophysical measurements probe properties that are averaged over the ensemble of microstates that collectively correspond to a macroscopic native state. However, experimental evidence for the presence of, and dynamic interchange between, multiple states that constitute a global macrostate is provided by direct time-resolved measurements, and by structural dispersions in crystallographic and NMR studies (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Such results underscore the potential importance as ligands or receptors in biological mechanisms of kinetically stable, trapped microstates embedded within the rough energy landscapes of purportedly singular macrostates. For example, the detection of transiently populated structural states in the native unbound protein ensemble that resemble those of the proteins in their bound state provides evidence for the so-called ''conformational selection model'' of protein binding, thereby suggesting significant biological functions for microstates in proteins (23)(24)(25)(26).By contrast with proteins, relatively few stu...

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

“…There is no significant gain in the thermodynamic driving forces for larger repeat bulge loops relative to the duplex state compared to smaller bulge loops. There may, however, be a more pronounced activation energy barrier/ kinetic trap that makes larger repeat bulge loops persist more easily than smaller repeat bulge loops . There may also be potential higher order interactions within larger bulge loops; such as, branching or loops folded back on themselves.…”

“…Using oligonucleotide based models, we and others have shown that repeat sequences can form bulge loop ensembles of closely related structures stabilized by intramolecular interactions between the loop bases. Reversion of these structures to the more stable duplex state is inhibited at ambient temperature but occurs at elevated temperature . In other words, these bulge loops form metastable complexes that require input of external energy in order to revert to the more thermodynamically stable Watson‐Crick duplex state.…”

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

“…23 Others have reported various structural forms of trinucleotide repeats and suggested the impact that such variations might have upon instability. 24 The slower migration of these extra species suggested that multiple structural conformations may be formed by each oligo; when denatured with formamide prior to native electrophoresis or when run on denaturing gels, each oligo ran as a single species (Figure S3 of the Supporting Information). Notably, gel purification of each of the J4/5 species resulted in a mixture of the two species, suggesting that they may be interconverting.…”

Energetic coupling between clustered lesions modulated by intervening triplet repeat bulge loops: Allosteric implications for DNA repair and triplet repeat expansion

“…To assess potential energetic origins of such coupled biological events, we have evaluated the impact of such proximal pairs of lesions/repair intermediates on the thermodynamic properties of triplet repeat DNAs. To this end, we have site specifically incorporated 8oxodG (O) and/or tetrahydrofuran (F), a stable abasic site analog, in place of guanidine residues located in opposite strands within oligonucleotide models of slipped DNA structures we previously have described 20, 49, 50. Our choice of 8oxodG is dictated by the observation that deletion of OGG1, the main glycosylase involved in 8oxodG lesion repair, significantly reduces the likelihood of DNA expansion in mouse models of Huntington's disease 17.…”