Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines.

The paradigm of cellular systems as deterministic machines has long guided our understanding of biology. Advancements in technology and methodology, however, have revealed a world of stochasticity, challenging the notion of determinism. Here, we explore the stochastic behavior of multi-protein complexes, using the DNA replication system (replisome) as a prime example. The faithful and timely copying of DNA depends on the simultaneous action of a large set of enzymes and scaffolding factors. This fundamental cellular process is underpinned by dynamic protein-nucleic acid assemblies that must transition between distinct conformations and compositional states. Traditionally viewed as a well-orchestrated molecular machine, recent experimental evidence has unveiled significant variability and heterogeneity in the replication process. In this review, we discuss recent advances in single-molecule approaches and single-particle cryo-EM, which have provided insights into the dynamic processes of DNA replication. We comment on the new challenges faced by structural biologists and biophysicists as they attempt to describe the dynamic cascade of events leading to replisome assembly, activation, and progression. The fundamental principles uncovered and yet to be discovered through the study of DNA replication will inform on similar operating principles for other multi-protein complexes.

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques.

The link between the chemical stability of G-quadruplex (qDNA) structures and their roles in eukaryotic genomic maintenance processes has been an area of interest now for several decades. This Review seeks to demonstrate how single-molecule force-based techniques can provide insight into the mechanical stabilities of a variety of qDNA structures as well as their ability to interconvert between different conformations under conditions of stress. Atomic force microscopy (AFM) and magnetic and optical tweezers have been the primary tools used in these investigations and have been used to examine both free and ligand-stabilized G-quadruplex structures. These studies have shown that the degree of stabilization of G-quadruplex structures has a significant effect on the ability of nuclear machinery to bypass these roadblocks on DNA strands. This Review will illustrate how various cellular components including replication protein A (RPA), Bloom syndrome protein (BLM), and Pif1 helicases are capable of unfolding qDNA. Techniques such as single-molecule fluorescence resonance energy transfer (smFRET), often in conjunction with the aforementioned force-based techniques, have proven extremely effective at elucidating the factors underpinning the mechanisms by which these proteins unwind qDNA structures. We will provide insight into how single-molecule tools have facilitated the direct visualization of qDNA roadblocks and also showcase results obtained from experiments designed to examine the ability of G-quadruplexes to limit the access of specific cellular proteins normally associated with telomeres.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism.

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA.

Observing protein dynamics during DNA-lesion bypass by the replisome.

Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example of a roadblock to replication is chemical modifications in the DNA caused by exposure to ultraviolet light. Protein dynamics are thought to play a crucial role to the molecular pathways that occur in the presence of such DNA lesions, including potential damage bypass. Therefore, many assays have been developed to study these dynamics. In this review, we discuss three methods that can be used to study protein dynamics during replisome-lesion encounters in replication reactions reconstituted from purified proteins. Specifically, we focus on ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy. We discuss two key model DNA replication systems, derived from Escherichia coli and Saccharomyces cerevisiae. The main methods of choice to study replication over the last decades have involved biochemical assays that rely on ensemble averaging. While these assays do not provide a direct readout of protein dynamics, they can often be inferred. More recently, single-molecule techniques including single-molecule fluorescence microscopy have been used to visualize replisomes encountering lesions in real time. In these experiments, individual proteins can be fluorescently labeled in order to observe the dynamics of specific proteins during DNA replication. Finally, cryo-electron microscopy can provide detailed structures of individual replisome components, which allows functional data to be interpreted in a structural context. While classic cryo-electron microscopy approaches provide static information, recent developments such as time-resolved cryo-electron microscopy help to bridge the gap between static structures and dynamic single-molecule techniques by visualizing sequential steps in biochemical pathways. In combination, these techniques will be capable of visualizing DNA replication and lesion encounter dynamics in real time, whilst observing the structural changes that facilitate these dynamics.

Production of long linear DNA substrates with site-specific chemical lesions for single-molecule replisome studies.

Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome-lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.

Rapid Exchange of Stably Bound Protein and DNA Cargo on a DNA Origami Receptor.

Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multivalent interactions to form stable complexes that are also capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multisite competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo, an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to benefit future studies that use DNA origami structures to exploit multivalent interactions for the design and synthesis of a wide range of possible kinetic behaviors.

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies.

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.

Single-Molecule Fluorescence Methods to Study Protein Exchange Kinetics in Supramolecular Complexes.

Recent single-molecule studies have demonstrated that the composition of multi-protein complexes can strike a balance between stability and dynamics. Proteins can dynamically exchange in and out of the complex depending on their concentration in solution. These exchange dynamics are a key determinant of the molecular pathways available to multi-protein complexes. It is therefore important that we develop robust and reproducible assays to study protein exchange. Using DNA replication as an example, we describe three single-molecule fluorescence assays used to study protein exchange dynamics. In the chase exchange assay, fluorescently labeled proteins are challenged by unlabeled proteins, where exchange results in the disappearance of the fluorescence signal. In the FRAP exchange assay, fluorescently labeled proteins are photobleached before exchange is measured by an increase in fluorescence as non-bleached proteins exchange into the complex. Finally, in the two-color exchange assay, proteins are labeled with two different fluorophores and exchange is visualized by detecting changes in color. All three assays compliment in their ability to elucidate the dynamic behavior of proteins in large biological systems.

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates.

Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein-DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

Replisome bypass of a protein-based R-loop block by Pif1.

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies.

Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a diverse range of single-molecule approaches.

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication.

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

When proteins play tag: the dynamic nature of the replisome.

DNA replication, or the copying of DNA, is a fundamental process to all life. The system of proteins that carries out replication, the replisome, encounters many roadblocks on its way. An inability of the replisome to properly overcome these roadblocks will negatively affect genomic integrity which in turn can lead to disease. Over the past decades, efforts by many researchers using a broad array of approaches have revealed roles for many different proteins during the initial response of the replisome upon encountering roadblocks. Here, we revisit what is known about DNA replication and the effect of roadblocks during DNA replication across different organisms. We also address how advances in single-molecule techniques have changed our view of the replisome from a highly stable machine with behavior dictated by deterministic principles to a dynamic system that is controlled by stochastic processes. We propose that these dynamics will play crucial roles in roadblock bypass. Further single-molecule studies of this bypass will, therefore, be essential to facilitate the in-depth investigation of multi-protein complexes that is necessary to understand complicated collisions on the DNA.

Recycling of single-stranded DNA-binding protein by the bacterial replisome.

Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.

Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging.

Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ±â€¯4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic.

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome.

The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1-Tof1-Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication.

Single-molecule visualization of fast polymerase turnover in the bacterial replisome.

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the ß2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.