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. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsBerghuis, B. A., Dulin, D., Xu, Z., van Laar, T., Cross, B., Janissen, R., Jergic, S., Dixon, N. E., Depken, M. & Dekker, N. H. (2015). Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier. Nature Chemical Biology, 11 (8), 579-585. Authors

The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor’s response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor’s performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level.

No abstract

Flap endonuclease 1 (FEN1) and XPG are essential 5 0 nuclease superfamily endonucleases in DNA replication and repair. FEN1 incises in the dsDNA region adjacent to 5 0 flaps, while XPG incises in the dsDNA region adjacent to DNA bubbles. We have used a hybrids method analysis combining crystallography, Small Angle X-ray Scattering (SAXS), Electron Microscopy (EM), and computation to characterize FEN1 specificity and activity on double flap substrates, and in the presence of sliding clamps 9-1-1 and PCNA and XPG specificity for DNA bubbles. Our crystallographic work shows a 5 0 nuclease superfamily conserved mechanism for resolving aberrant DNA structures that involves both structural motifs and flexibility. Our structures of product and substratebound complexes suggested FEN1 resolves 5 0 flaps using a dsDNA binding -ssDNA incision mechanism. The structure revealed FEN1 binding to a bent duplex DNA structure and an active site shielded by a helical gateway that would select for single-stranded DNA or RNA to reach the active site. Key structural elements mediating duplex DNA binding, substrate specificity and activity are superfamily conserved in our XPG catalytic domain crystal structure. Further computational and EM work with FEN1 complexes to sliding clamps 9-1-1 and PCNA showed distinct functionally-relevant differences in how FEN1 interacts with the sliding clamps and how the sliding clamps interact with upstream duplex DNA. We propose that the PCNA complex is more dynamic, consistent with PCNA's role in replication and that the 9-1-1 complex is more stable, consistent with 9-1-1 acting locally at the DNA damage.

Scientific Reports 7: Article number: 43285; published online: 07 March 2017; updated: 18 May 2018 The Acknowledgements section in this Article is incomplete. “We thank Seungkyu Ha, Yera Ye. Ussembayev, Richard Janissen, and Hubertus J. E. Beaumont for discussions, Richard M. Berry and Ren Lim for providing the strains, and Theo van Laar for the remaining help with the bacteria.