Protocol for effective surface passivation for single-molecule studies of chromatin and topoisomerase II.

For single-molecule studies requiring surface anchoring of biomolecules, poorly passivated surfaces can result in alterations of biomolecule structure and function that lead to artifacts. Here, we present a surface passivation assay for single-molecule studies of chromatin and topoisomerase II. We detail steps for preparing a nucleosome array and hydrophobic nitrocellulose-coated flow cell.

Tunable elliptical cylinders for rotational mechanical studies of single DNA molecules.

The angular optical trap (AOT) is a powerful technique for measuring the DNA topology and rotational mechanics of fundamental biological processes. Realizing the full potential of the AOT requires rapid torsional control of these processes. However, existing AOT quartz cylinders are limited in their ability to meet the high rotation rate requirement while minimizing laser-induced photodamage.

Chromatin Buffers Torsional Stress During Transcription.

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA.

Human Topoisomerase IIα Promotes Chromatin Condensation Via a Phase Transition.

Topoisomerase II (topo II) enzymes are essential enzymes known to resolve topological entanglements during DNA processing. Curiously, while yeast expresses a single topo II, humans express two topo II isozymes, topo IIα and topo IIβ, which share a similar catalytic domain but differ in their intrinsically disordered C-terminal domains (CTDs). During mitosis, topo IIα and condensin I constitute the most abundant chromosome scaffolding proteins essential for chromosome condensation.

Torsion is a Dynamic Regulator of DNA Replication Stalling and Reactivation.

The inherent helical structure of DNA dictates that a replisome must rotate relative to DNA during replication, presenting inevitable topological challenges to replication. However, little is known about how the replisome progresses against torsional stress. Here, we developed a label-free, high-resolution, real-time assay to monitor replisome movement under torsion.

Torsional Mechanics of Circular DNA.

Circular DNA found in the cell is actively regulated to an underwound state, with their superhelical density close to . While this underwound state is essential to life, how it impacts the torsional mechanical properties of DNA is not fully understood. In this work, we performed simulations to understand the torsional mechanics of circular DNA and validated our results with single-molecule measurements and analytical theory.

DNA Polymerase Locks Replication Fork Under Stress.

Replication of DNA requires the parental DNA to be unwound to allow the genetic information to be faithfully duplicated by the replisome. While this function is usually shared by a host of proteins in the replisome, notably DNA polymerase (DNAP) and helicase, the consequence of DNAP synthesizing DNA while decoupled from helicase remains not well understood. The unwinding of downstream DNA poses significant stress to DNAP, and the interaction between DNAP and the replication fork may affect replication restart.

An Effective Surface Passivation Assay for Single-Molecule Studies of Chromatin and Topoisomerase II.

Unlabelled: For complete details on the use and execution of this protocol, please refer to Le et al. (2019).

Summary: A.

Tunable Elliptical Cylinders for Rotational Mechanical Studies of Single DNA Molecules.

The angular optical trap (AOT) is a powerful technique for measuring the DNA topology and rotational mechanics of fundamental biological processes. Realizing the full potential of the AOT requires rapid torsional control of these processes. However, existing AOT quartz cylinders are limited in their ability to meet the high rotation rate requirement while minimizing laser-induced photodamage.

Optical torque calculations and measurements for DNA torsional studies.

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance.

Optical Torque Calculations and Measurements for DNA Torsional Studies.

Unlabelled: The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping, but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance.

Chromatinization modulates topoisomerase II processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin.

Chromatinization Modulates Topoisomerase II Processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress . While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II).

Etoposide promotes DNA loop trapping and barrier formation by topoisomerase II.

Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and cytotoxicity. How etoposide perturbs topoisomerase dynamics is not known. Here we investigated the action of etoposide on yeast topoisomerase II, human topoisomerase IIα and human topoisomerase IIβ using several sensitive single-molecule detection methods.

Polarity of the CRISPR roadblock to transcription.

CRISPR (clustered regularly interspaced short palindromic repeats) utility relies on a stable Cas effector complex binding to its target site. However, a Cas complex bound to DNA may be removed by motor proteins carrying out host processes and the mechanism governing this removal remains unclear. Intriguingly, during CRISPR interference, RNA polymerase (RNAP) progression is only fully blocked by a bound endonuclease-deficient Cas (dCas) from the protospacer adjacent motif (PAM)-proximal side.

Angular Optical Trapping to Directly Measure DNA Torsional Mechanics.

Angular optical trapping (AOT) is a powerful technique that permits direct angular manipulation of a trapped particle with simultaneous measurement of torque and rotation, while also retaining the capabilities of position and force detection. This technique provides unique approaches to investigate the torsional properties of nucleic acids and DNA-protein complexes, as well as impacts of torsional stress on fundamental biological processes, such as transcription and replication. Here we describe the principle, construction, and calibration of the AOT in detail and provide a guide to the performance of single-molecule torque measurements on DNA molecules.

Resonator nanophotonic standing-wave array trap for single-molecule manipulation and measurement.

Nanophotonic tweezers represent emerging platforms with significant potential for parallel manipulation and measurements of single biological molecules on-chip. However, trapping force generation represents a substantial obstacle for their broader utility. Here, we present a resonator nanophotonic standing-wave array trap (resonator-nSWAT) that demonstrates significant force enhancement.

Torsional Stiffness of Extended and Plectonemic DNA.

DNA torsional elastic properties play a crucial role in DNA structure, topology, and the regulation of motor protein progression. However, direct measurements of these parameters are experimentally challenging. Here, we present a constant-extension method integrated into an angular optical trap to directly measure torque during DNA supercoiling.

Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity.

DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology.

High-Performance Image-Based Measurements of Biological Forces and Interactions in a Dual Optical Trap.

Optical traps enable the nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection; however, successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive.

Helicase promotes replication re-initiation from an RNA transcript.

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle.

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive.

Tunable nanophotonic array traps with enhanced force and stability.

A nanophotonic trapping platform based on on-chip tunable optical interference allows parallel processing of biomolecules and holds promise to make single molecule manipulation and precision measurements more easily and broadly available. The nanophotonic standing wave array trap (nSWAT) device [Nat. Nanotechnol.