Unraveling DNA Breathing: Advancing Spectroscopic Methods to Probe Nucleobase Conformational Dynamics and Protein-DNA Interactions

Abstract

Thermally driven fluctuations of the DNA double-helix provide transient openings for proteins to access the genetic information encoded in the base sequence. Such DNA `breathing' instabilities are important regulators of many biological processes, but the molecular-level mechanism is not well understood. Here, we present developments of two techniques that permit new insights into the conformational dynamics and protein binding mechanisms that depend on DNA `breathing.’ These methodologies include: (i) microsecond resolved single-molecule FRET and (ii) fluorescence detected two-photon excitation (2PE) Fourier transform spectroscopy.

Extending the time resolution of single-molecule (sm) FRET experiments to the microsecond regime revealed new insights into the conformational dynamics of primer/template (p/t)-DNA lattices as they are subject to DNA `breathing’ fluctuations. A follow up study revealed the sensitivity of the binding mechanism of the T4 bacteriophage single-stranded DNA binding proteins (ssbs) to the polarity of ssDNA lattices. These insights can be leveraged in future experiments investigating how the ssbs regulate and coordinate the activities of the major protein subassemblies of the DNA replication complex.

Developing 2PE Fourier transform spectroscopies, which we have applied to fluorescent base analogue probes, establishes a new class of experiments to directly measure the local conformations and dynamics of nucleobases within model DNA constructs. We demonstrate 2PE one-dimensional Fourier transform experiments, which suggest the feasibility of future single-molecule studies of nucleobase dynamics. Furthermore, 2PE two-dimensional Fourier transform spectroscopy experiments are carried out in the intermediate- and low-fluorescence (photon-counting) detection regimes. We obtain very good agreement between our theoretical model and the experimental spectra, thus setting the stage for future experiments to investigate 6-MI substituted DNA constructs at the ensemble and single-molecule levels.

The new experiments described in this dissertation set the stage for future studies that will advance our molecular-level understanding about the mechanisms of DNA ‘breathing’ and its role in protein binding and recognition.

This dissertation contains previously published, unpublished and co-authored material.