Richard Janissen

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.

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.

Predicting Intra- and Intertypic Recombination in Enterovirus 71

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that: (1) EV-A71 strain-type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (2) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase (RdRp); (3) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination suggesting conservation in mechanism(s); and (4) sequencing of intertypic recombinant genomes indicates that template-switching is by a mechanism that requires some sequence homology at the recombination junction and that the triggers for template-switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.

dNTP-Dependent Conformational Transitions in the Fingers Subdomain of Klentaq1

DNA polymerases are responsible for the accurate replication of DNA. Kinetic studies indicate the requirement for multiple intermediate enzyme conformations in this process. Structural studies show a large conformational change in the fingers subdomain of DNA polymerase on binding of a correct dNTP. Using single molecule FRET we show that the conformational transition affecting the fingers subdomain also takes place in the apo and DNA-bound forms of the enzyme. In addition a third conformation is observed which is occupied in the presence of dNTP alone, and in the presence of a non base pairing dNTPs. The relative proportions of the identified states are altered dramatically depending on substrate. Binding of the correct nucleotide displaces this equilibrium dramatically towards the closed form, while binding of an incorrect nucleotide favors a more open conformation. The results suggest that the closed state is by design less energetically favored, providing a thermodynamic brake on incorrect nucleotide insertion.