molecules Article A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins Roberto Fabian, Jr. 1,*, Santosh Gaire 1, Christopher Tyson 2, Raghabendra Adhikari 3, Ian Pegg 1 and Abhijit Sarkar 1 1 Department of Physics and Vitreous State Laboratory, The Catholic University of America, Washington, DC 20064, USA; [email protected] (S.G.); [email protected] (I.P.); [email protected] (A.S.) 2 Biomedical Engineering Department and Vitreous State Laboratory, The Catholic University of America, Washington, DC 20064, USA; [email protected] 3 Department of Biology, The Catholic University of America, Washington, DC 20064, USA; [email protected] * Correspondence: [email protected] 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 Citation: Fabian, R., Jr.; Gaire, S.; force-mediated DNA-core histone assembly and disassembly processes. Tyson, C.; Adhikari, R.; Pegg, I.; Sarkar, A. A Horizontal Magnetic Tweezers for Studying Single DNA Keywords: single molecule micromanipulation techniques; horizontal magnetic tweezers; DNA- Molecules and DNA-Binding histone interactions; nucleosomes Proteins. Molecules 2021, 26, 4781. https://doi.org/10.3390/ molecules26164781 1. Introduction Academic Editor: Francesco Histones are the basic protein unit of the nucleosome core particle. The core particle Simone Ruggeri 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, Received: 30 June 2021 H2A, H2B, H3, and H4, combine to form an octameric complex comprising two copies of Accepted: 3 August 2021 each type of histone. The interaction between the DNA and the histones are non-specific, Published: 7 August 2021 with the DNA wrapped approximately 1.75 times [2] around the octameric complex. The fifth type of histone, H1 or H5, serves as a link between the adjacent nucleosomes. Publisher’s Note: MDPI stays neutral Electrostatic attraction is responsible for the binding of histones to the DNA. DNA has with regard to jurisdictional claims in a net negative charge on its phosphate backbone, while histones have a net positive charge published maps and institutional affil- in their amino terminal tails, which results in a strong electrostatic attraction. Additionally, iations. the helix dipoles in H2B, H3, and H4 contribute to the interaction with DNA [3]. As a result, the formation of a single nucleosome results in a relatively large decrease in free energy of ~20 kBT[4]. The strong electrostatic interactions between DNA and histones present a great chal- Copyright: © 2021 by the authors. lenge to the cell’s genetic machinery. Genetic processes like DNA transcription, DNA Licensee MDPI, Basel, Switzerland. repair, and DNA replication, which involve specialized protein machinery, require access to This article is an open access article the sections of the DNA stored in the nucleosomes but that is rendered inaccessible by the distributed under the terms and strong binding between the DNA and the histone core particles. The application of force conditions of the Creative Commons and torque by DNA-bound protein complexes may alter the stability of the nucleosome Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and may play a key role in providing access to the DNA. Interestingly, several molecular 4.0/). machines such as RNA polymerase and DNA polymerase have been observed to apply [5] Molecules 2021, 26, 4781. https://doi.org/10.3390/molecules26164781 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4781 2 of 12 large amounts of force in the range of ~10 pN to single DNA templates. The theoretical estimate of the minimum force, f*, needed to mechanically destabilize a nucleosome is ~2 pN [4]. This suggests that in vivo protein machines may be able to apply forces large enough to locally affect the stability of the nucleosomes. Optical traps and magnetic tweezers have been used to investigate force-mediated nucleosome dynamics. Several studies have been performed using different procedures for histone purification and nucleosome reconstitution [6–9]. The results depend on (a) the experimental technique used, i.e., optical or magnetic tweezers, (b) the protocols used for nucleosome reconstitution, (c) the degree of control of the posttranslational modifications, (d) the absence or presence of linker histones, (e) the ionic conditions, (f) the buffer com- positions, and (g) other experimental factors [6]. In Sections 1.1 and 1.2, we summarize results from selected studies on nucleosomes using optical trapping and magnetic tweez- ers and briefly discuss how they have contributed to our understanding of the structure and dynamics of nucleosomes and chromatin. In Section 1.3, we introduce and motivate our study. 1.1. Single Molecule Studies on Nucleosomes Using Optical Traps Optical traps and magnetic tweezers have been used to probe the structure and dynamics of chromatin. In this section, we summarize selected references on the study of single molecule experiments on nucleosomes using optical tweezers. Cui and Bustamante [10] pioneered single molecule micromanipulation studies on nucleosomes using optical tweezers. In their experiments, they stretched and relaxed single chromatin fibers extracted from chicken erythrocytes at three different NaCl concentrations: 5 mM, 40 mM, and 150 mM. Their results established that the force versus extension curves of single chromatin fibers are reversible at forces less than 6 pN and that are irreversible at forces greater than 20 pN in all three NaCl concentrations. Nucleosome disassembly events were not reported in their experiments. Following this study, Bennink et al. [11] performed optical trapping experiments on single chromatin fibers reconstituted from λ-DNA and Xenopus laevis egg extract. They were able to detect nucleosome disruption events starting at 20 pN and up to 40 pN. These were associated with three disruption lengths 65 nm, 130 nm, and 195 nm, which were interpreted as the signatures of one, two, and three nucleosome disassembly events, respectively. Brower-Toland et al. [12] investigated single nucleosomes using optical tweezers with nucleosomes reconstituted from single DNA molecules and core histones (without linker histones) purified using the salt dialysis method. The DNA tethers included 17 tandem repeats of 5S positioning sequences. They found a ~25 nm disruption length of DNA per nucleosome at forces less than 15 pN and ~27 nm disruption lengths for higher forces. Using optical tweezers, Gemmen et al. [13] investigated nucleosome arrays formed from DNA tethers with random sequences, core histones (without linker histones), the histones chaperone NAP1, and the remodeling factor ACF. They reported that the length released per nucleosome unbinding event changed from ~18 nm to ~31 nm from 5 pN to 65 pN. They also found the average force needed to unravel a single nucleosome was dependent on the NaCl concentration of the buffer, which they suggested reflected variations in the binding affinity of the histone core particle to different sequences of the DNA. Claudet et al. [14] used optical tweezers to study nucleosome arrays reconstituted us- ing salt dialysis. A DNA template with 12 tandem repeats of the 5S nucleosome positioning sequence and core histones (without linker histones) was used. They also conducted com- plementary studies using nucleosome arrays extracted from chicken erythrocytes. A total of two DNA disruption lengths of 25 nm and 50 nm were reported in their studies. Disruption events were observed to occur at forces ≥ 20 pN in both types of nucleosome arrays. Pope et al. [15] used an optical tweezers technique to stretch single chromatin fibers assembled from λ-DNA and Xenopus laevis egg extract at different loading rates. They Molecules 2021, 26, 4781 3 of 12 reported three disruption lengths: 30 nm, 59 nm, and 117 nm. All three disruption lengths occurred at forces ≥ 20 pN. Mihardja et al. [16] investigated single mononucleosomal fibers using optical tweez- ers. The mononucleosomal DNA was assembled from 2582 bp DNA containing a single 601 positioning sequences and core histones extracted from chicken erythrocytes. In their experiments, they observed two disruption lengths: 21 nm at forces starting at 3 pN and 22 nm at forces between 8 and 9 pN. They interpreted these as two stages of nucleosome disassembly. The first stage represented the release of the outer wrap of the nucleosomal DNA while the second stage represented the unwrapping of the remaining inner wrap of the nucleosomal DNA. Bussiek et al. [17] studied single chromatin fibers reconstituted from two types of DNA templates, core histones (no linker histones), and the NAP1/ACF system. The two DNA templates were heterogenous DNA, i.e., DNA with a random sequence, and tandem repeat alpha satellite DNA. They extended the individual fibers using optical tweezers. They found that the disruption length in both DNA templates were almost same, ~23 nm for heterogenous DNA and ~24 nm for alpha satellite DNA. However, the disruption force in the alpha satellite DNA was 26 pN, higher than the heterogenous DNA, which showed a disruption force of 22 pN. 1.2. Single Molecule Studies on Nucleosomes Using Magnetic Tweezers Leuba et al. [18] were the first to study nucleosome arrays using vertical magnetic tweezers.