The bacterial replisome: design principles for a dynamic molecular machine
Molecular Horizons and School of Chemistry & Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia.
The E. coli replisome is a complex and dynamic assembly of more than 20 protein subunits that include the multi-protein Pol III chromosomal replicase and the primosome (helicase/primase). The replisome works to achieve simultaneous copying of both strands at a replication fork at rates that approach 1000 bp/s, with near-perfect fidelity. In the textbook view, leading and lagging strand DNA replication are perfectly coordinated processes that are orchestrated to occur deterministically in discrete steps in space and time. However, there is no evolutionary pressure to achieve such elegance, nor do fundamental chemical principles allow it. I will integrate recent structural and single-molecule biophysical studies that are being used to develop a new picture of replisomal function that is messier than the textbook view. In particular, coupled leading and lagging strand replication assays with fully assembled replisomes containing fluorescent proteins enable protein exchange processes at a replication fork to be imaged in real time at the single-molecule level. We have shown, for example, that Pol III replicase complexes undergo frequent exchange at the fork, on a time scale that depends on their concentration in solution [Lewis et al., 2017, eLife, 6, e23932], while single-stranded DNA-binding protein (SSB) can be retained at the fork through multiple cycles of Okazaki fragment synthesis on the lagging strand (Lisanne Spenkelink, unpublished).