Single-Molecule Twist and Stretch of Mononucleosome using Optical Torque Wrench

Abstract

The fundamental chromatin packing unit in eukaryotes is the nucleosome, where ∼147 base pairs of DNA are wrapped in ∼1.7 turns around a core histone octamer. A crucial question in biology is to explain how proteins are able to access DNA which is tightly bound in. For example, RNA polymerase must navigate through the nucleosome while transcribing DNA. Hence, the DNA-histone interactions play a key role in gene regulation. Single-molecule force spectroscopy is a powerful tool to probe this system. Prior studies have exerted linear tension to stretch both chromatin fibers and mononucleosome molecules, which gave information on the nature of the free-energy barrier for a particular disruption pathway. Theoretical studies have suggested that the disruption pathway may be strongly sensitive to the torsional loading of the nucleosome. This is of interest because helicases, polymerases, or other motor proteins may use a combination of force and torque to disrupt chromatin. Here we simultaneously apply force and torque to a mononucleosome structure using an optical torque wrench. By determining the influence of supercoiling density on the disruption barrier we obtain more detailed information on nucleosome unwrapping dynamics.