Abstract
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 (β2), and the clamp 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 high-speed (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.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call