Stopped-flow fluorescence resonance energy transfer for analysis of nucleosome dynamics

Abstract

Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis of millisecond or longer timescale processes, with concentrations as low as a few nanomolar and volumes of just a few hundred microliters. One can now place specific dyes anywhere desired on a nucleic acid, and often on a protein as well. This ability allows the use of fluorescence resonance energy transfer experiments for detailed conformational analyses, even as the system is evolving rapidly over time following the initiation of a reaction. This approach is ideally suited for analysis of intrinsic properties of chromatin and of the machines that control chromatin assembly, disassembly, and function.

The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes

“…This suggests that DNA breathing may only marginally increase accessibility to occluded uracils by capturing the transient exposed states. This is not surprising given that only 1-10% of the time occluded sites are exposed through this mechanism (61). This is in agreement with the findings reported previously on the effects of rotational and translational positioning on uracil removal efficiency (32).…”

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

“…This time scale is very close to that of unwrapping-rewrapping event observed in single-molecule FRET experiments [23,24]. It has been shown that wrapping-unwrapping of DNA is an intrinsic property of nucleosome, and, nucleosomes are partially unwrapped about 2-10% of the time [23].…”

“…Single-molecule measurement using optical trap has shown that the wrapping and unwrapping of DNA between 1 and 1.75 turns occur reversibly [21,22]. Single-molecule fluorescence resonance energy transfer (FRET) techniques have detected a local dissociation of DNA from histone core particle occurring in sub-second time scale [23,24]. These investigations indicate that thermal fluctuation is possibly responsible for the nucleosome dynamics.…”

Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair

“…Similarly, it was shown that differences in NCP stability occur in DNA positioning sequences around promoter regions of Mouse Mammary Tumor Virus, where the nucleosome at the promoter is inherently more dynamic/unstable compared to another nucleosome nearby, possibly for targeting transcriptional activators (35). Additionally, DNA sequence plays an important role in regulating transient dynamic states of DNA on the surface of the histone octamer in which the DNA transiently lifts off the surface of the histone octamer to allow regulatory proteins access to buried target sites (36-38). Although these ‘site exposure’ or ‘DNA unwrapping’ events were originally considered to occur only in vitro , in vivo results also point to the existence of such a mechanism.…”