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Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states.

Völker J, Gindikin V, Klump HH, Plum GE, Breslauer KJ - J. Am. Chem. Soc. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, New Jersey 08854, USA.

ABSTRACT
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.

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Fluorescence excitation spectra at 25 °C (native)of the 2Ap-and PC-labeled constructs: (A) excitation at 305 nm, the 2Ap excitationmaximum; (B) excitation at 340 nm, the PC excitation maximum. Shownare the steady-state emission spectra of constructs with an eight-CAGrepeat loop in a fixed position (red), a six-CAG repeat loop in threepossible loop positions (purple), a four-CAG repeat loop in five possibleloop positions (blue), and a two-CAG repeat loop in seven possibleloop positions (green). Also shown are the emission spectra of ourconstruct when the repeat is fully base-paired (light-brown) and fullysingle-stranded (black). It should be noted that the PC fluorescencesignal (460 nm peak) in (A) may be due in part to energy transferfrom either 2Ap or the other bases, as PC excitation at this wavelengthis at a minimum.
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fig3: Fluorescence excitation spectra at 25 °C (native)of the 2Ap-and PC-labeled constructs: (A) excitation at 305 nm, the 2Ap excitationmaximum; (B) excitation at 340 nm, the PC excitation maximum. Shownare the steady-state emission spectra of constructs with an eight-CAGrepeat loop in a fixed position (red), a six-CAG repeat loop in threepossible loop positions (purple), a four-CAG repeat loop in five possibleloop positions (blue), and a two-CAG repeat loop in seven possibleloop positions (green). Also shown are the emission spectra of ourconstruct when the repeat is fully base-paired (light-brown) and fullysingle-stranded (black). It should be noted that the PC fluorescencesignal (460 nm peak) in (A) may be due in part to energy transferfrom either 2Ap or the other bases, as PC excitation at this wavelengthis at a minimum.

Mentions: The two reporter fluorophores we chose are minimallyperturbingmimics of their respective natural base analogues, adenine and cytosine,in terms of their base-pairing properties while being sensitive toDNA secondary structure.72−79 A comparison of CD spectra as a function of temperature, with andwithout these fluorescent bases, for all the [CAG]8·[CTG]n constructs (data not shown) revealed onlyminor differences in spectral and thermal properties, subtleties thatcan be attributed primarily to the spectral differences between fluorescentbases and their natural nonfluorescent analogues.80−82 The CD dataare consistent with the presence of the fluorescent bases not appreciablyaltering the conformational and thermal properties of the repeat bulgeloop construct, for all constructs examined. A similar lack of impactof fluorescent base analogues on global repeat loop properties hasbeen reported previously.45,47,49,50,70,83 In contrast, the fluorescence emission spectraof 2Ap and PC excited at their respective excitation maxima (Figure 3) show significant differences depending on whetherthe loop can exist in multiple loop arrangements (e.g., [CAG]8·[CTG]4) or in a fixed loop position (e.g.,[CAG]8·[CTG]0) or when the repeat sequenceis fully base-paired (e.g., [CAG]8·[CTG]8). Clearly, the fluorescence intensity is sensitive to the natureof the loop, reflecting changes in the environment surrounding thefluorophores due to loop size and positional distribution within therepeat sequence domain. The fluorescence intensity also may dependon potential energy transfer between the fluorophores. The steady-statefluorescence data confirmed that the loop was not located exclusivelynear either the upstream or downstream 11-mer arm, as the fluorescenceintensities of 2Ap and PC were consistent with neither of these basesbeing fully base-paired. If one of these two positional isomers hadbeen the exclusively occupied state (see Table 1), then either the 2Ap fluorescence or the PC fluorescence wouldhave been strongly quenched, similar to what was observed for [CAG]8·[CTG]8, while the other base would have beenhighly fluorescent.


Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states.

Völker J, Gindikin V, Klump HH, Plum GE, Breslauer KJ - J. Am. Chem. Soc. (2012)

Fluorescence excitation spectra at 25 °C (native)of the 2Ap-and PC-labeled constructs: (A) excitation at 305 nm, the 2Ap excitationmaximum; (B) excitation at 340 nm, the PC excitation maximum. Shownare the steady-state emission spectra of constructs with an eight-CAGrepeat loop in a fixed position (red), a six-CAG repeat loop in threepossible loop positions (purple), a four-CAG repeat loop in five possibleloop positions (blue), and a two-CAG repeat loop in seven possibleloop positions (green). Also shown are the emission spectra of ourconstruct when the repeat is fully base-paired (light-brown) and fullysingle-stranded (black). It should be noted that the PC fluorescencesignal (460 nm peak) in (A) may be due in part to energy transferfrom either 2Ap or the other bases, as PC excitation at this wavelengthis at a minimum.
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Related In: Results  -  Collection

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fig3: Fluorescence excitation spectra at 25 °C (native)of the 2Ap-and PC-labeled constructs: (A) excitation at 305 nm, the 2Ap excitationmaximum; (B) excitation at 340 nm, the PC excitation maximum. Shownare the steady-state emission spectra of constructs with an eight-CAGrepeat loop in a fixed position (red), a six-CAG repeat loop in threepossible loop positions (purple), a four-CAG repeat loop in five possibleloop positions (blue), and a two-CAG repeat loop in seven possibleloop positions (green). Also shown are the emission spectra of ourconstruct when the repeat is fully base-paired (light-brown) and fullysingle-stranded (black). It should be noted that the PC fluorescencesignal (460 nm peak) in (A) may be due in part to energy transferfrom either 2Ap or the other bases, as PC excitation at this wavelengthis at a minimum.
Mentions: The two reporter fluorophores we chose are minimallyperturbingmimics of their respective natural base analogues, adenine and cytosine,in terms of their base-pairing properties while being sensitive toDNA secondary structure.72−79 A comparison of CD spectra as a function of temperature, with andwithout these fluorescent bases, for all the [CAG]8·[CTG]n constructs (data not shown) revealed onlyminor differences in spectral and thermal properties, subtleties thatcan be attributed primarily to the spectral differences between fluorescentbases and their natural nonfluorescent analogues.80−82 The CD dataare consistent with the presence of the fluorescent bases not appreciablyaltering the conformational and thermal properties of the repeat bulgeloop construct, for all constructs examined. A similar lack of impactof fluorescent base analogues on global repeat loop properties hasbeen reported previously.45,47,49,50,70,83 In contrast, the fluorescence emission spectraof 2Ap and PC excited at their respective excitation maxima (Figure 3) show significant differences depending on whetherthe loop can exist in multiple loop arrangements (e.g., [CAG]8·[CTG]4) or in a fixed loop position (e.g.,[CAG]8·[CTG]0) or when the repeat sequenceis fully base-paired (e.g., [CAG]8·[CTG]8). Clearly, the fluorescence intensity is sensitive to the natureof the loop, reflecting changes in the environment surrounding thefluorophores due to loop size and positional distribution within therepeat sequence domain. The fluorescence intensity also may dependon potential energy transfer between the fluorophores. The steady-statefluorescence data confirmed that the loop was not located exclusivelynear either the upstream or downstream 11-mer arm, as the fluorescenceintensities of 2Ap and PC were consistent with neither of these basesbeing fully base-paired. If one of these two positional isomers hadbeen the exclusively occupied state (see Table 1), then either the 2Ap fluorescence or the PC fluorescence wouldhave been strongly quenched, similar to what was observed for [CAG]8·[CTG]8, while the other base would have beenhighly fluorescent.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, New Jersey 08854, USA.

ABSTRACT
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.

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