Super-resolution Imaging of DNA Replisome Architecture and Dynamics in Live Bacillus Subtilis

Abstract

DNA replication happens in all living organisms and assures that the genome is accurately copied and maintained. The replisome is the molecular machine in cells that replicates DNA, and this protein assembly includes DNA polymerases which directly synthesize DNA by adding nucleotides. Although the bacterial replisome has been studied extensively in vitro, single-molecule microscopy is now providing a new perspective on the dynamics and architecture of replisome components in vivo. In this thesis, I studied the architecture and dynamics of several highly conserved replisome components in vivo in the model organism Bacillus subtilis. Photoactivated localization microscopy (PALM) and single-molecule tracking enable us to localize and track every single protein molecule with a resolution of 20 – 40 nm. I investigated the dynamics during normal DNA replication of a number of replisomal proteins, including the replicative DNA polymerases PolC and DnaE and the β-clamp loader DnaX. I then extended these investigations to replication arrest by two different pathways: PolC disruption by the damage-independent 6-hydroxy-phenylazo-uracil (HPUra) and cross-linking with mitomycin C (MMC), a damaging agent. I quantified the real-time behavior of different replisomal proteins during the DNA synthesis process. The results presented in this thesis show that all of these replisomal proteins are highly dynamic and exchange more rapidly than previously expected, and I characterized the molecular-scale distribution of each replisome component as well as responses to cellular mutations and external stimuli with a combination of single-molecule tracking, time-lapse imaging, and spatiotemporal image correlation spectroscopy. Finally, I developed a new approach to characterize the stoichiometry of replisomal proteins by counting the photobleaching steps with Bayesian statistics. Overall, these new insights into DNA replication indicate that the activities of bacterial replisomal proteins may be regulated in cells by coordinating and modulating the dynamics of protein recruitment, binding, and unbinding at the site of DNA synthesis.