Bojk A. Berghuis

Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts.

A force calibration standard for magnetic tweezers

To study the behavior of biological macromolecules and enzymatic reactions under force, advances in single-molecule force spectroscopy have proven instrumental. Magnetic tweezers form one of the most powerful of these techniques, due to their overall simplicity, non-invasive character, potential for high throughput measurements, and large force range. Drawbacks of magnetic tweezers, however, are that accurate determination of the applied forces can be challenging for short biomolecules at high forces and very time-consuming for long tethers at low forces below ∼1 piconewton. Here, we address these drawbacks by presenting a calibration standard for magnetic tweezers consisting of measured forces for four magnet configurations. Each such configuration is calibrated for two commonly employed commercially available magnetic microspheres. We calculate forces in both time and spectral domains by analyzing bead fluctuations. The resulting calibration curves, validated through the use of different algorithms that yield close agreement in their determination of the applied forces, span a range from 100 piconewtons down to tens of femtonewtons. These generalized force calibrations will serve as a convenient resource for magnetic tweezers users and diminish variations between different experimental configurations or laboratories.

Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments

Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.

Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments

Single-molecule experiments provide a unique means for real-time observation of the activity of individual biomolecular machines. Through such techniques, insights into the mechanics of for example, polymerases, helicases, and packaging motors have been gleaned. Here we describe the recent advances in single-molecule force spectroscopy instrumentation that have facilitated high-throughput acquisition at high spatiotemporal resolution. The large datasets attained by such methods can capture rare but important events, and contain information regarding stochastic behaviors covering many orders of magnitude in time. We further discuss analysis of such data sets, and with a special focus on the pause states described in the general literature on RNA polymerase pausing we compare and contrast the signatures of different reaction pathways.

Strand separation establishes a sustained lock at the Tus– Ter replication fork barrier

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.

High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers

Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20pN) for prolonged periods of time (>1000s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.

Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site

Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3′-end that allows for the de novo initiation of a second RdRp. This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.

What is all this fuss about Tus?: Comparison of recent findings from biophysical and biochemical experiments

Abstract Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The exact sequence of events by which Tus–Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein–protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus–Ter interactions at the nonpermissive face – interactions that result in a highly stable “locked” complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus–Ter mechanism – leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA–protein system further.

Real-time observation of replicative helicase assembly onto single-stranded DNA

Replicative helicases load onto DNA at the start of replication, and play a vital role by driving the replication fork forward. These helicases assemble into closed multimeric rings that need to encircle single-stranded (ss)DNA to be activated. Though helicase loading on substrates with accessible free ends has been well characterized for the T7 gp4 helicase, a model system for superfamily IV replicative helicases, the physiologically more relevant loading onto exposed ssDNA without free ends remains less well understood. Here, using a label-free assay that exploits changes in the DNA hairpin hopping dynamics to detect gp4 binding and activity, we characterize loading and activation of gp4 on exposed ssDNA without free ends, and find clear evidence of stepwise assembly of the helicase at the fork at physiologically relevant concentrations. The gradual loading onto ssDNA, rather than pre-forming in solution followed by spontaneous ring opening which appears favored at higher concentrations, suggests a new paradigm of stepwise assembly for the helicases in superfamily IV that do not require a separate loading enzyme.