Oral Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Single-molecule visualisation of protein dynamics in the eukaryotic replisome. (#41)

Lisanne M. Spenkelink 1 , Jacob S. Lewis 1 , Grant D. Schauer 2 , Dan Zhang 2 , Michael E. O'Donnell 2 , Antoine M. van Oijen 1
  1. University of Wollongong, Wollongong, NSW, Australia
  2. Rockefeller University, New York, USA

DNA replication plays a crucial role in transmitting hereditary information from cell to cell. Replication of chromosomal DNA is a fundamentally complex and carefully orchestrated process, yet occurs at amazingly high rates with extraordinarily high fidelity. Although the enzymatic activities of the replicative DNA polymerases are well understood, its dynamics within the replisome are not. We tested the accepted view that polymerases remain stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labelled polymerases to visualise DNA replication by a reconstituted eukaryotic replisome at the single-molecule level for the first time. We demonstrate that polymerases are exchanged during processive DNA replication. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment. We show that the stoichiometry of polymerases at the replication fork is controlled by the concentration of the RFC clamploader. Furthermore our data suggests that the lagging-strand polymerase Pol δ is stably associated to the replisome. It can, therefore be, recycled for the synthesis of tens of Okazaki fragments. Our studies show highly dynamic behaviour of the replisome, contrary to the current textbook view.