Spectroscopic approaches for studies of site-specific DNA base and backbone 'breathing' using exciton-coupled dimer-labeled DNA.

DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule Förster Resonance Energy Transfer.

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)n] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.