Biology subjects

Heussman, D.

Publications and source records attributed to Heussman, D..

3 recordsLinked to original sources

bioRxiv2023

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 test 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 anti-symmetric (-) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave linearly polarized laser with polarization direction continuously rotated at the frequency 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and the anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and 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.

biophysics↗

bioRxiv2023

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of internally-labeled (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

biophysics↗

bioRxiv2021

Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded -- double-stranded DNA junctions by 2D fluorescence spectroscopy

DNA replication, and the related processes of genome expression, require binding, assembly, and function of protein complexes at and near single-stranded (ss) - double-stranded (ds) DNA junctions. These central protein-DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss-dsDNA junctions is likely critical for understanding the molecular mechanisms of these central biological processes. Here we describe spectroscopic studies of model ss-dsDNA fork constructs that contain dimers of internally labeled cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar-phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism (CD) and two-dimensional fluorescence spectroscopy (2DFS) permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar-phosphate backbones undergo abrupt successive changes in their local conformations - initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation - as the dimer probes are moved across the ss-dsDNA junction. Our results suggest that the sugar-phosphate backbones at and near ss-dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson-Crick structure. We suggest that some of these conformations are likely to function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

biophysics↗