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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.

The single-stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism in bacteria. This protein performs two distinct, but closely intertwined and indispensable functions in the cell. SSB binds to single-stranded DNA (ssDNA) and at least 20 partner proteins resulting in their regulation. These partners comprise a family of genome guardians known as the SSB interactome. Essential to interactome regulation is the linker/OB-fold network of interactions. This network of interactions forms when one or more PXXP motifs in the linker of SSB bind to an OB-fold in a partner, with interactome members involved in competitive binding between the linker and ssDNA to their OB-fold. Consequently, when linker-binding occurs to an OB-fold in an interactome partner, proteins are loaded onto the DNA. When linker/OB-fold interactions occur between SSB tetramers, cooperative ssDNA-binding results, producing a multi-tetrameric complex that rapidly protects the ssDNA. Within this SSB-ssDNA complex, there is an extensive and dynamic network of linker/OB-fold interactions that involves multiple tetramers bound contiguously along the ssDNA lattice. The dynamic behavior of these tetramers which includes binding mode changes, sliding as well as DNA wrapping/unwrapping events, are likely coupled to the formation and disruption of linker/OB-fold interactions. This behavior is essential to facilitating downstream DNA processing events. As OB-folds are critical to the essence of the linker/OB-fold network of interactions, and they are found in multiple interactome partners, the SSB interactome is classified as the first family of prokaryotic, oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians.

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.

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.

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.

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.

The RecG DNA helicase is a guardian of the bacterial genome where it dominates stalled DNA replication fork rescue. The single-stranded DNA binding protein (SSB) is involved in this process and promotes the binding of RecG to stalled replication forks. Atomic force microscopy (AFM) was used to investigate the interaction of RecG and SSB on a mobile fork substrate capable of being regressed. In the absence of proteins, the fork undergoes spontaneous dynamics between two states defined by the length of the DNA complementarity at the fork. Binding of SSB does not affect these dynamics as it binds to single-stranded regions as expected. In contrast, RecG interacts with the two states quite differently. We demonstrate that RecG has two modes of interaction with fork DNA in the presence of SSB and ATP. In the first mode, RecG translocates over the duplex region and this activity is defined by SSB-mediated remodeling of the helicase. In the second mode, RecG utilizes its helicase activity to regress the fork, in an ATP-dependent manner, displacing SSB on the ssDNA. Overall, our results highlight two functions of RecG that can be employed in the regulation of stalled DNA replication fork rescue.

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.

The DNA helicase PriA is a key protein for restarting stalled DNA replication forks in bacteria. With 3′ to 5′ helicase activity, PriA is important in primosome assembly. We used atomic force microscopy (AFM) and specifically employed time-lapse AFM to visualize the interaction of PriA with two DNA substrates. The results show that most of the PriA molecules are observed bound at the fork. However, PriA is capable of translocating over distances of about 400 bp. There is a preference for the long-range translocation of PriA depending on the fork type. For a fork with the nascent leading strand as single-stranded DNA (ssDNA; F4 substrate), PriA translocates preferentially on the parental arm of the fork. For the substrate F14, which contains an additional ssDNA segment between the parental and lagging arms (5 nt gap), PriA translocates on both the parental and lagging strand arms. These data suggest that transient formation of the single-stranded regions during the DNA replication can change the selection of the DNA duplex by PriA. Translocation of the helicase was directly visualized by time-lapse AFM imaging, which revealed that PriA can switch strands during translocation. These novel features of PriA shed new light on the mechanisms of PriA interaction with stalled replication forks. [Image: see text]

The RecG DNA helicase plays a crucial role in stalled replication fork rescue as the guardian of the bacterial genome. We have recently demonstrated that single-strand DNA binding protein (SSB) promotes binding of RecG to the stalled replication fork by remodeling RecG, enabling the helicase to translocate ahead of the fork. We also hypothesized that mispairing of DNA could limit such translocation of RecG, which plays the role of roadblocks for the fork movement. Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. We found that a C-C mismatch at a distance of 30 bp away from the fork position prevents translocation of RecG over this mispairing. A G-bulge placed at the same distance also has a similar roadblock efficiency. However, a C-C mismatch 10 bp away from the fork does not prevent RecG translocation, as 10 bp from fork is within the distance of footprint of RecG on fork DNA. Our findings suggest that retardation of RecG translocation ahead of the replication fork can be a mechanism for the base pairing control for DNA replication machinery.

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.

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. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

PriA is a member of the SuperFamily 2 helicase family. Its rolein vivois to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriAin vitroin the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for theE.coliprotein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3’-OH placed at the end of the nascent leading strand at the fork. When both the 3’-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

In bacteria, the restart of stalled DNA replication forks requires the PriA DNA helicase. PriA recognizes and remodels abandoned DNA replication forks performing the DNA unwinding in 3’ to 5’-direction and facilitates loading of the DnaB helicase onto the DNA to restart replication. The single stranded DNA binding protein (SSB) is typically present at the abandoned forks, but there is gap in the knowledge on the interaction between SSB and PriA protein. Here, we used atomic force microscopy (AFM) to visualize the interaction of PriA with DNA substrates in the absence or presence of SSB. Results show that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Preferential binding occurs only on forked DNA structures as 5’- and 3’-tailed duplexes were bound equally well. Furthermore, in the absence of SSB, PriA bound exclusively to the fork regions of substrates. In contrast, fork bound SSB loads PriA onto the duplex DNA arms of forks. When the fork has a gap in the leading strand, PriA localizes to both the parental and lagging strand arms. When the gap is present in the lagging strand, PriA is loaded preferentially onto the leading strand arm of the fork. Remodeling of PriA requires a functional C-terminal domain of SSB.

The E. coli single stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism. Here, it has two seemingly disparate but equally important roles: it binds rapidly and cooperatively to single stranded DNA (ssDNA) and it binds to partner proteins that constitute the SSB interactome. These two roles are not disparate but are instead, intimately linked. A model is presented wherein the intrinsically disordered linker (IDL) is directly responsible for mediating protein-protein interactions. It does this by binding, via PXXP motifs, to the OB-fold (aka SH3 domain) of a nearby protein. When the nearby protein is another SSB tetramer, this leads to a highly efficient ssDNA binding reaction that rapidly and cooperatively covers and protects the exposed nucleic acid from degradation. Alternatively, when the nearby protein is a member of the SSB interactome, loading of the enzyme onto the DNA takes places.

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif-containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.

The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.

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.

TheE. colisingle 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 perhaps 100-150 associated with genome at any one time, either at DNA replication forks or at sites of DNA 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 such as DnaA and RecA. To visualize SSB directly and to ascertain spatiotemporal changes in tetramer localization in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for visualization of prokaryotic cells. Results show that in the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is observed 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. In the process, it may also deliver interactome partners such as RecG or PriA to sites where their repair functions are required.

Quantitative measurement of small forces and small displacement using holographic optical tweezers (HOTs) is finding increasing applications due to the features of non-contact and high accuracy manipulation. Although hologram optimization algorithms have been widely reported, the holographic optical trapping performance relying on the algorithms has not been studied systematically. In this paper, we investigated the force measuring the performance of various types of HOTs generated with six different hologram algorithms (GSW, GAA, GS, SR, S, and RM). To do this, we built up a HOT instrument and compared the light fields’ intensity distribution, trap stiffness, efficiency, and calculation time of multi-point trap arrays generated by six hologram algorithms with this setup. Our work will provide a better understanding of the performance of different hologram algorithms in HOTs.

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 data as these are non-physiological 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 purification of tagged complexes, our results demonstrate 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 out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

The Rep and UvrD DNA helicases are proposed to act at stalled DNA replication forks to facilitate replication restart when RNA polymerase stalls forks. To clarify the role of these DNA helicases in fork rescue, we used a coupled spectrophotometric ATPase assay to determine how they act on model fork substrates. For both enzymes, activity is low on regressed fork structures, suggesting that they act prior to the regression step that generates a Holliday junction. In fact, the preferred cofactors for both enzymes are forks with a gap in the nascent leading strand, consistent with the 3′–5′ direction of translocation. Surprisingly, for Rep, this specificity is altered in the presence of stoichiometric amounts of a single-strand DNA-binding protein (SSB) relative to a fork with a gap in the nascent lagging strand. Even though Rep and UvrD are similar in structure, elevated concentrations of SSB inhibit Rep, but they have little to no effect on UvrD. Furthermore, Rep and UvrD antagonize one another at a fork. This is surprising given that these helicases have been shown to form a heterodimer and are proposed to act together to rescue an RNA polymerase-stalled fork. Consequently, the results herein indicate that although Rep and UvrD can act on similar fork substrates, they cannot function on the same fork simultaneously.

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

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.

RecG is a potent, atypical, monomeric DNA helicase. It simultaneously couples ATP hydrolysis to duplex unwinding and rewinding, and to the displacement of proteins bound to the DNA. A model is presented for the localization of the enzyme to the inner membrane via its binding to SSB. Upon fork stalling, SSB targets the enzyme to the fork where it can act. RecG displays a strong preference for processing the fork in the regression direction, that is, away from the site of damage that initially led to fork arrest. Regression is mediated by strong binding of the wedge domain to the fork arms as well as to parental duplex DNA by the helicase domains. Once RecG has regressed the fork, it will dissociate leaving the now relaxed, Holliday junction-like DNA, available for further processing by enzymes such as RuvAB.

A wide-field fluorescence microscope with a double-helix point spread function (PSF) is constructed to obtain the specimen’s three-dimensional distribution with a single snapshot. Spiral-phase-based computer-generated holograms (CGHs) are adopted to make the depth-of-field of the microscope adjustable. The impact of system aberrations on the double-helix PSF at high numerical aperture is analyzed to reveal the necessity of the aberration correction. A modified cepstrum-based reconstruction scheme is promoted in accordance with properties of the new double-helix PSF. The extended depth-of-field images and the corresponding depth maps for both a simulated sample and a tilted section slice of bovine pulmonary artery endothelial (BPAE) cells are recovered, respectively, verifying that the depth-of-field is properly extended and the depth of the specimen can be estimated at a precision of 23.4nm. This three-dimensional fluorescence microscope with a framerate-rank time resolution is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field.

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif‐containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.

During DNA replication, forks often stall and require restart. One mechanism for restart requires that the fork be moved in a direction opposite to that of replication. This reaction is known as fork regression. For this reaction to occur, the enzyme must couple unwinding of the nascent heteroduplex fork arms to the rewinding of nascent strands ahead of itself and to the parental duplex in its wake. As the arms of the fork are complementary, this reaction is isoenergetic making it challenging to study. To overcome this, a novel adaptation of magnetic tweezers was developed by the Croquette group. Here, a 1200 bp hairpin was attached at opposite ends to a flow cell surface and a magnetic bead. By manipulating the bead with the magnets, force can be applied to unwind the hairpin or alternatively, released to allow the hairpin to rewind. This adaptation was used to study fork regression by RecG. The results show that this is an efficient regression enzyme, able to work against a large opposing force. Critically, it couples DNA unwinding to duplex rewinding and in the process, can displace bound proteins from fork arms.

Structural mimicry of DNA is utilized in nature as a strategy to evade molecular defences mounted by host organisms. One such example is the protein Ocr – the first translation product to be expressed as the bacteriophage T7 infects E. coli. The structure of Ocr reveals an intricate and deliberate arrangement of negative charges that endows it with the ability to mimic ∼24 base pair stretches of B-DNA. This uncanny resemblance to DNA enables Ocr to compete in binding the type I restriction modification (R/M) system, and neutralizes the threat of hydrolytic cleavage of viral genomic material. Here, we report the de novo design and biophysical characterization of DNA mimicking peptides, and describe the inhibitory action of the designed helical bundles on a type I R/M enzyme, EcoR124I. This work validates the use of charge patterning as a design principle for creation of protein mimics of DNA, and serves as a starting point for development of therapeutic peptide inhibitors against human pathogens that employ molecular camouflage as part of their invasion stratagem.

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.

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.

Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn2+>Mg2+>Ca2+. Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (-)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.

Rad54 is a key component of the eukaryotic recombination machinery. Its presence in DNA strand-exchange reactions in vitro results in a significant stimulation of the overall reaction rate. Using untagged Rad54, we show that this stimulation can be attributed to enhancement of the formation of a key reaction intermediate known as DNA networks. Using a novel, single DNA molecule, dual-optical tweezers approach we show how Rad54 stimulates DNA network formation. We discovered that Rad54 oligomers possess a unique ability to cross-bridge or bind double-stranded DNA molecules positioned in close proximity. Further, Rad54 oligomers rapidly translocate double-stranded DNA while simultaneously inducing topological loops in the DNA at the locus of the oligomer. The combination of the cross-bridging and double-stranded DNA translocation activities of Rad54 stimulates the formation of DNA networks, leading to rapid and efficient DNA strand exchange by Rad51.