Zero-order free holographic optical tweezers.

Holographic optical tweezers (HOTs) use spatial light modulators (SLM) to modulate light beams, thereby enabling the dynamic control of optical trap arrays with complex intensity and phase distributions. This has provided exciting new opportunities for cell sorting, microstructure machining, and studying single molecules. However, the pixelated structure of the SLM will inevitably bring up the unmodulated zero-order diffraction possessing an unacceptably large fraction of the incident light beam power. This is harmful to optical trapping because of the bright, highly localized nature of the errant beam. In this paper and to address this issue, we construct a cost-effective, zero-order free HOTs apparatus, thanks to a homemade asymmetric triangle reflector and a digital lens. As there is no zero-order diffraction, the instrument performs excellently in generating complex light fields and manipulating particles.

Insight into the biochemical mechanism of DNA helicases provided by bulk-phase and single-molecule assays.

There are multiple assays available that can provide insight into the biochemical mechanism of DNA helicases. For the first 22 years since their discovery, bulk-phase assays were used. These include gel-based, spectrophotometric, and spectrofluorometric assays that revealed many facets of these enzymes. From 2001, single-molecule studies have contributed additional insight into these DNA nanomachines to reveal details on energy coupling, step size, processivity as well as unique aspects of individual enzyme behavior that were masked in the averaging inherent in ensemble studies. In this review, important aspects of the study of helicases are discussed including beginning with active, nuclease-free enzyme, followed by several bulk-phase approaches that have been developed and still find widespread use today. Finally, two single-molecule approaches are discussed, and the resulting findings are related to the results obtained in bulk-phase studies.

Single-molecule insight into stalled replication fork rescue in Escherichia coli.

DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

The Biochemical Mechanism of Fork Regression in Prokaryotes and Eukaryotes-A Single Molecule Comparison.

The rescue of stalled DNA replication forks is essential for cell viability. Impeded but still intact forks can be rescued by atypical DNA helicases in a reaction known as fork regression. This reaction has been studied at the single-molecule level using the Escherichia coli DNA helicase RecG and, separately, using the eukaryotic SMARCAL1 enzyme. Both nanomachines possess the necessary activities to regress forks: they simultaneously couple DNA unwinding to duplex rewinding and the displacement of bound proteins. Furthermore, they can regress a fork into a Holliday junction structure, the central intermediate of many fork regression models. However, there are key differences between these two enzymes. RecG is monomeric and unidirectional, catalyzing an efficient and processive fork regression reaction and, in the process, generating a significant amount of force that is used to displace the tightly-bound E. coli SSB protein. In contrast, the inefficient SMARCAL1 is not unidirectional, displays limited processivity, and likely uses fork rewinding to facilitate RPA displacement. Like many other eukaryotic enzymes, SMARCAL1 may require additional factors and/or post-translational modifications to enhance its catalytic activity, whereas RecG can drive fork regression on its own.

Atomic force microscopy-based characterization of the interaction of PriA helicase with stalled DNA replication forks.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA-binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

Super-resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage.

The E. coli single-stranded DNA-binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100-150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.

Multi-view object topography measurement with optical sectioning structured illumination microscopy.

Various optical instruments have been developed for three-dimensional (3D) surface topography, including the white light interference, reflectance confocal microscopes, and digital holographic microscopes, etc. However, the steep local slope of objects may cause the light to be reflected in a way that it will not be captured by the objective lens because of the finite collection angle of the objective. To solve this "shadow problem," we report a method to enlarge the collection angle range of optical sectioning structured illumination microscopy by capturing sectioned images of the objects from multiple angle of views. We develop a multi-view image fusion algorithm to reconstruct a single 3D image. Using this method, we detect previously invisible details whose slopes are beyond the collection angle of the objective. The proposed approach is useful for height map measurement and quantitative analyses in a variety of fields, such as biology, materials science, microelectronics, etc.

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks.

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of Escherichia coli RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks.

Aberration correction in holographic optical tweezers using a high-order optical vortex: publisher's note.

This publisher's note identifies an error in the author affiliations of Appl. Opt.57, 3618 (2018)APOPAI0003-693510.1364/AO.57.003618.

Aberration correction in holographic optical tweezers using a high-order optical vortex.

Holographic optical tweezers are a powerful optical trapping and manipulation tool in numerous applications such as life science and colloidal physics. However, imperfections in the spatial light modulator and optical components of the system will introduce detrimental aberrations to the system, thereby degrading the trapping performance significantly. To address this issue, we develop an aberration correction technique by using a high-order vortex as the correction metric. The optimal Zernike polynomial coefficients for quantifying the system aberrations are determined by comparing the distorted vortex and the ideal one. Efficiency of the proposed method is demonstrated by comparing the optical trap intensity distribution, trap stiffness, and particle dynamics in a Gaussian trap and an optical vortex trap, before and after aberration corrections.

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 m ammonium sulfate must be present, bringing into question the validity of these results as these are nonphysiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using the purification of tagged complexes, our results show that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the 'storage form' of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB, which, by virtue of its high affinity for single-stranded DNA, allows these helicases to outcompete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

Characterization of the ATPase activity of RecG and RuvAB proteins on model fork structures reveals insight into stalled DNA replication fork repair.

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To clarify the roles of these proteins in fork regression, we used a coupled spectrophotometric ATPase assay to determine how these helicases act on two groups of model fork substrates: the first group mimics nascent stalled forks, whereas the second mimics regressed fork structures. The results show that RecG is active on the substrates in group 1, whereas these are poor substrates for RuvAB. In addition, in the presence of group 1 forks, the single-stranded DNA-binding protein (SSB) enhances the activity of RecG and enables it to compete with excess RuvA. In contrast, SSB inhibits the activity of RuvAB on these substrates. Results also show that the preferred regressed fork substrate for RuvAB is a Holliday junction, not a forked DNA. The active form of the enzyme on the Holliday junction contains a single RuvA tetramer. In contrast, although the enzyme is active on a regressed fork structure, RuvB loading by a single RuvA tetramer is impaired, and full activity requires the cooperative binding of two forked DNA substrate molecules. Collectively, the data support a model where RecG is responsible for stalled DNA replication fork regression. SSB ensures that if the nascent fork has single-stranded DNA character RuvAB is inhibited, whereas the activity of RecG is preferentially enhanced. Only once the fork has been regressed and the DNA is relaxed can RuvAB bind to a RecG-extruded Holliday junction.

PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA.

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.

Facile Conversion and Optimization of Structured Illumination Image Reconstruction Code into the GPU Environment.

Superresolution, structured illumination microscopy (SIM) is an ideal modality for imaging live cells due to its relatively high speed and low photon-induced damage to the cells. The rate-limiting step in observing a superresolution image in SIM is often the reconstruction speed of the algorithm used to form a single image from as many as nine raw images. Reconstruction algorithms impose a significant computing burden due to an intricate workflow and a large number of often complex calculations to produce the final image. Further adding to the computing burden is that the code, even within the MATLAB environment, can be inefficiently written by microscopists who are noncomputer science researchers. In addition, they do not take into consideration the processing power of the graphics processing unit (GPU) of the computer. To address these issues, we present simple but efficient approaches to first revise MATLAB code, followed by conversion to GPU-optimized code. When combined with cost-effective, high-performance GPU-enabled computers, a 4- to 500-fold improvement in algorithm execution speed is observed as shown for the image denoising Hessian-SIM algorithm. Importantly, the improved algorithm produces images identical in quality to the original.

Rapid, artifact-reduced, image reconstruction for super-resolution structured illumination microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is finding increasing application in biomedical research due to its superior ability to visualize subcellular dynamics in living cells. However, during image reconstruction artifacts can be introduced and when coupled with time-consuming postprocessing procedures, limits this technique from becoming a routine imaging tool for biologists. To address these issues, an accelerated, artifact-reduced reconstruction algorithm termed joint space frequency reconstruction-based artifact reduction algorithm (JSFR-AR-SIM) was developed by integrating a high-speed reconstruction framework with a high-fidelity optimization approach designed to suppress the sidelobe artifact. Consequently, JSFR-AR-SIM produces high-quality, super-resolution images with minimal artifacts, and the reconstruction speed is increased. We anticipate this algorithm to facilitate SR-SIM becoming a routine tool in biomedical laboratories.

OB-fold Families of Genome Guardians: A Universal Theme Constructed From the Small ß-barrel Building Block.

The maintenance of genome stability requires the coordinated actions of multiple proteins and protein complexes, that are collectively known as genome guardians. Within this broadly defined family is a subset of proteins that contain oligonucleotide/oligosaccharide-binding folds (OB-fold). While OB-folds are widely associated with binding to single-stranded DNA this view is no longer an accurate depiction of how these domains are utilized. Instead, the core of the OB-fold is modified and adapted to facilitate binding to a variety of DNA substrates (both single- and double-stranded), phospholipids, and proteins, as well as enabling catalytic function to a multi-subunit complex. The flexibility accompanied by distinctive oligomerization states and quaternary structures enables OB-fold genome guardians to maintain the integrity of the genome via a myriad of complex and dynamic, protein-protein; protein-DNA, and protein-lipid interactions in both prokaryotes and eukaryotes.

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Laminar flow cells for single-molecule studies of DNA-protein interactions.

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.

Type I restriction endonucleases are true catalytic enzymes.

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.