Optical tweezers experiments resolve distinct modes of DNA‐protein binding

Abstract

Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double‐stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double‐stranded structure. Proteins binding to both double‐ and single‐stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single‐stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double‐stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 265–282, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Peeling back the mystery of DNA overstretching

“…Later experiments measuring overstretching as a function of salt, pH, and temperature quantitatively verified this prediction and showed that solution conditions that inhibit DNA annealing also induced strong hysteresis in the DNA stretching curves, as expected for melting (7). Later studies on DNA stretched in the presence of binding ligands were also consistent with this model (8).…”

“…For example, whereas SSB proteins will most likely favor end-melting, intercalators and other binding ligands that stabilize dsDNA will lead to DNA melting in the middle, similar to that observed for torsionally constrained DNA in this study. These details will be important for studies of the kinetics of protein-DNA interactions (8,18).…”

Correlation Force Spectroscopy for Single Molecule Measurements

“…One interesting case to be studied is interactions between DNA and DNA-binding proteins. A motivation for this study comes from the important role of specific proteins in regulation and catalysis of multiple DNA tasks, such as replication, transcription, compaction, unwinding, chemical modification, and sequence repair [154]. In cells, DNA is constantly twisted, bent and stretched by numerous proteins mediating genome transactions.…”

“…Protein binding to DNA alters the conformation of DNA and thus function. For example, the high mobility group B protein (HMGB) when binds to DNA enhances its flexibility by decreasing DNA persistence length [154]. The affinity of protein-DNA binding, that we describe using mechanical terms of stiffness and friction, are vital in cancer treatment studies [154].…”

“…For example, the high mobility group B protein (HMGB) when binds to DNA enhances its flexibility by decreasing DNA persistence length [154]. The affinity of protein-DNA binding, that we describe using mechanical terms of stiffness and friction, are vital in cancer treatment studies [154]. Using CFS the dynamics of protein binding and unbinding and its interpretation in terms of mechanical properties can be obtained.…”