A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins.

Roberto Fabian,Raghabendra Adhikari,Ian Pegg,Christopher Tyson,Abhijit Sarkar,Santosh Gaire

doi:10.3390/molecules26164781

Roberto Fabian, Raghabendra Adhikari + Show 4 more

Open Access

https://doi.org/10.3390/molecules26164781

Copy DOI

Abstract

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

Highlights

Histones are the basic protein unit of the nucleosome core particle
The basic principle of our single molecule experiments was to (1) allow the binding of histone octamers onto a naked DNA tether held under tension less than 4 pN and (2) to raise the force to destabilize the DNA-histone complexes and map the step-wise decompaction events
The binding of the core histones to the DNA tether was observed via the visible compaction of the DNA tether

Summary

Introduction

Histones are the basic protein unit of the nucleosome core particle. The core particle allows for the first stage of DNA compaction in eukaryotic cells, which involves the formation of a linear array of nucleosomes along the DNA [1]. The four types of histones, H2A, H2B, H3, and H4, combine to form an octameric complex comprising two copies of each type of histone. The interaction between the DNA and the histones are non-specific, with the DNA wrapped approximately 1.75 times [2] around the octameric complex. Electrostatic attraction is responsible for the binding of histones to the DNA. DNA has a net negative charge on its phosphate backbone, while histones have a net positive charge in their amino terminal tails, which results in a strong electrostatic attraction. The formation of a single nucleosome results in a relatively large decrease in free energy of ~20 kBT [4]

Objectives

Methods

Results