Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia; Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands
Slobodan Jergic
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
Elizabeth A Wood
Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
Enrico Monachino
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia; Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands
Nicholas P Horan
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
Karl E Duderstadt
Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands; Max Planck Institute of Biochemistry, Martinsried, Germany; Physik Department, Technishche Universität München, Garching, Germany
Michael M Cox
Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
Andrew Robinson
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. 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.
