Chromatinization Modulates Topoisomerase II Processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. 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). We observed that topo II was at least ~ 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a ~100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive (~ 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.

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. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin's predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

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 IIß using several sensitive single-molecule detection methods. Unexpectedly, we found that etoposide induces topoisomerase to trap DNA loops, compacting DNA and restructuring DNA topology. Loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme. Although etoposide decreases the innate stability of topoisomerase dimers, it increases the ability of the enzyme to act as a stable roadblock. Interestingly, the three topoisomerases show similar etoposide-mediated resistance to dimer separation and sliding along DNA but different abilities to compact DNA and chirally relax DNA supercoils. These data provide unique mechanistic insights into the functional consequences of etoposide on topoisomerase II dynamics.

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. By mapping dCas-DNA interactions at high resolution, we discovered that the collapse of the dCas R-loop allows Escherichia coli RNAP read-through from the PAM-distal side for both Sp-dCas9 and As-dCas12a. This finding is not unique to RNAP and holds for the Mfd translocase. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the guide RNAs. This work highlights the importance of the R-loop in dCas-binding stability and provides valuable mechanistic insights for broad applications of CRISPR technology.

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. We include the constant-force method and, notably, a new constant-extension method that enables measurement of the twist persistence length of both extended DNA, under an extremely low force, and plectonemic DNA. This chapter can assist in the implementation and application of this technique for general researchers in the single-molecule field.

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. This platform integrates a critically-coupled resonator design to the nSWAT and incorporates a novel trap reset scheme. The nSWAT can now perform standard single-molecule experiments, including stretching DNA molecules to measure their force-extension relations, unzipping DNA molecules, and disrupting and mapping protein-DNA interactions. These experiments have realized trapping forces on the order of 20 pN while demonstrating base-pair resolution with measurements performed on multiple molecules in parallel. Thus, the resonator-nSWAT platform now meets the benchmarks of a table-top precision optical trapping instrument in terms of force generation and resolution. This represents the first demonstration of a nanophotonic platform for such single-molecule experiments.

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. We measured the twist persistence length of extended DNA to be 22 nm under an extremely low force (∼0.02 pN) and the twist persistence length of plectonemic DNA to be 24 nm. In addition, we implemented a rigorous data analysis scheme that bridged our measurements with existing theoretical models of DNA torsional behavior. This comprehensive set of torsional parameters demonstrates that at least 20% of DNA supercoiling is partitioned into twist for both extended DNA and plectonemic DNA. This work provides a new experimental methodology, as well as an analytical and interpretational framework, which will enable, expand, and enhance future studies of DNA torsional properties.

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. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.

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. Here, we develop a camera-based detection platform that enables accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision. We then use the DNA unzipping technique to localize bound proteins with sub-base-pair precision, revealing how thermal DNA "breathing" fluctuations allow an unzipping fork to detect and respond to the presence of a protein bound downstream. This work advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

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. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

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. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

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. 9, 448 (2014); Nano Lett. 16, 6661 (2016)] represents such a platform and can trap a large array of beads by the evanescent field of the standing wave of a nanophotonic waveguide and reposition them using an integrated microheater. In this paper, by taking a systematic design approach, we present a new generation of nSWAT devices with significant enhancement of the optical trapping force, stiffness, and stability, while the quality of the standing wave trap is resistant to fabrication imperfections. The device is implemented on a silicon nitride photonic platform and operates at 1064 nm wavelength which permits low optical absorption by the aqueous solution. Such performance improvements open a broader range of applications based on these on-chip optical traps.

Biocompatible and High Stiffness Nanophotonic Trap Array for Precise and Versatile Manipulation.

The advent of nanophotonic evanescent field trapping and transport platforms has permitted increasingly complex single molecule and single cell studies on-chip. Here, we present the next generation of nanophotonic Standing Wave Array Traps (nSWATs) representing a streamlined CMOS fabrication process and compact biocompatible design. These devices utilize silicon nitride (Si3N4) waveguides, operate with a biofriendly 1064 nm laser, allow for several watts of input power with minimal absorption and heating, and are protected by an anticorrosive layer for sustained on-chip microelectronics in aqueous salt buffers. In addition, due to Si3N4's negligible nonlinear effects, these devices can generate high stiffness traps while resolving subnanometer displacements for each trapped particle. In contrast to traditional table-top counterparts, the stiffness of each trap in an nSWAT device scales linearly with input power and is independent of the number of trapping centers. Through a unique integration of microcircuitry and photonics, the nSWAT can robustly trap, and controllably position, a large number of nanoparticles along the waveguide surface, operating in an all-optical, constant-force mode without need for active feedback. By reducing device fabrication cost, minimizing trapping laser specimen heating, increasing trapping force, and implementing commonly used trapping techniques, this new generation of nSWATs significantly advances the development of a high performance, low cost optical tweezers array laboratory on-chip.

T7 replisome directly overcomes DNA damage.

Cells and viruses possess several known 'restart' pathways to overcome lesions during DNA replication. However, these 'bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

DNA Y structure: a versatile, multidimensional single molecule assay.

Optical trapping is a powerful single molecule technique used to study dynamic biomolecular events, especially those involving DNA and DNA-binding proteins. Current implementations usually involve only one of stretching, unzipping, or twisting DNA along one dimension. To expand the capabilities of optical trapping for more complex measurements would require a multidimensional technique that combines all of these manipulations in a single experiment. Here, we report the development and utilization of such a novel optical trapping assay based on a three-branch DNA construct, termed a "Y structure". This multidimensional assay allows precise, real-time tracking of multiple configurational changes. When the Y structure template is unzipped under both force and torque, the force and extension of all three branches can be determined simultaneously. Moreover, the assay is readily compatible with fluorescence, as demonstrated by unzipping through a fluorescently labeled, paused transcription complex. This novel assay thus allows for the visualization and precision mapping of complex interactions of biomechanical events.

Nanophotonic trapping for precise manipulation of biomolecular arrays.

Optical trapping is a powerful manipulation and measurement technique widely used in the biological and materials sciences. Miniaturizing optical trap instruments onto optofluidic platforms holds promise for high-throughput lab-on-a-chip applications. However, a persistent challenge with existing optofluidic devices has been achieving controlled and precise manipulation of trapped particles. Here, we report a new class of on-chip optical trapping devices. Using photonic interference functionalities, an array of stable, three-dimensional on-chip optical traps is formed at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (∼30 kHz) with nanometre precision. We demonstrate sorting and manipulation of individual DNA molecules. In conjunction with laminar flows and fluorescence, we also show precise control of the chemical environment of a sample with simultaneous monitoring. Such a controllable trapping device has the potential to achieve high-throughput precision measurements on chip.

Electro-optofluidics: achieving dynamic control on-chip.

A vital element in integrated optofluidics is dynamic tuning and precise control of photonic devices, especially when employing electronic techniques which are challenging to utilize in an aqueous environment. We overcome this challenge by introducing a new platform in which the photonic device is controlled using electro-optical phase tuning. The phase tuning is generated by the thermo-optic effect using an on-chip electric microheater located outside the fluidic channel, and is transmitted to the optofluidic device through optical waveguides. The microheater is compact, high-speed (> 18 kHz), and consumes low power (~mW). We demonstrate dynamic optical trapping control of nanoparticles by an optofluidic resonator. This novel electro-optofluidic platform allows the realization of high throughput optofluidic devices with switching, tuning, and reconfiguration capability, and promises new directions in optofluidics.