High-Speed Magnetic Tweezers Studies of the E-coli Replisome

The replisome is the nucleoprotein assembly responsible for replicating DNA prior to cell division. In E. coli, it comprises roughly a dozen proteins, including DNA polymerase III, DnaB helicase, the beta processivity clamp (b2), and theclamp loader complex. Through a complex hierarchy of dynamic interactions of varying strength, the E. coli replisome can incorporate nearly one thousand nucleotides per second with extremely high fidelity. As such, it represents an ideal model molecular motor, not to mention a potential and heretofore unexploited antibiotic target. In order to study the dynamic behavior of single active replisomes, we employ a magnetic tweezers (MT) assay incorporating a highspeed (several kHz) camera. DNA incorporating a hairpin is tethered between a microscope coverslip and a magnetic bead under applied force. Nucleotide
incorporation changes the bead height, allowing us to follow replication with higher spatiotemporal resolution than current single-molecule fluorescence approaches
allow. We have previously used this approach to measure rates and identify transient pausing in phage polymerases and present our recent results from the E. coli replisome here.