Single-Molecule Tracking Reveals Altered Dynamics of a Transcription Regulator Expressed at Similar Levels from Different Gene Expression Systems

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 it is composed of several different proteins, including DNA polymerases, which directly synthesize DNA by adding nucleotides. Although the bacterial replisome has been studied extensively in vitro, little is known about the dynamics and architecture of replisome components in vivo. Here we use Bacillus subtilis, a Gram-positive bacterium commonly found in soil, as a model organism in which to study the architecture and dynamics of several replisome components in vivo. Photoactivated localization microscopy (PALM) and single-molecule tracking give a resolution of 20-40 nm, far below the diffraction limit of conventional microscopy, enabling us to localize and track every single protein molecule. In our study, we investigated the dynamics of a number of replisome components under different conditions, including the DNA polymerases PolC and DnaE, and the b-clamp loader DnaX. We can watch the real time behavior of different replisome components during the DNA synthesis process, and study them quantitatively. Surprisingly, our investigations have revealed that all these replisome components are highly dynamic and exchange more rapidly than previously expected, and we characterize the molecular scale distribution of each replisome component within the dynamic replication machinery.