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

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

Authors :
Maurer J
Albrecht CS
Herbert P
Heussman D
Chang A
von Hippel PH
Marcus AH
Source :
The journal of physical chemistry. B [J Phys Chem B] 2023 Dec 21; Vol. 127 (50), pp. 10730-10748. Date of Electronic Publication: 2023 Dec 07.
Publication Year :
2023

Abstract

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) <subscript>2</subscript> ] 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) <subscript>2</subscript> 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) <subscript>2</subscript> 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) <subscript>2</subscript> probe. We find that at most construct positions that we examined, the (iCy3) <subscript>2</subscript> 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.

Details

Language :
English
ISSN :
1520-5207
Volume :
127
Issue :
50
Database :
MEDLINE
Journal :
The journal of physical chemistry. B
Publication Type :
Academic Journal
Accession number :
38060691
Full Text :
https://doi.org/10.1021/acs.jpcb.3c06463