title: Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.
abstract: The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
authors.affiliation: Zernike Institute for Advanced Materials and Centre for Synthetic Biology, University of Groningen, 9700 AB Groningen, the Netherlands; Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; Physik Department, Technische Universität München, 85748 Garching, Germany. Electronic address: duderstadt@biochem.mpg.de.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Duderstadt
authors.affiliation: Zernike Institute for Advanced Materials and Centre for Synthetic Biology, University of Groningen, 9700 AB Groningen, the Netherlands.
authors.forename: Hylkje J
authors.initials: HJ
authors.lastname: Geertsema
authors.affiliation: Zernike Institute for Advanced Materials and Centre for Synthetic Biology, University of Groningen, 9700 AB Groningen, the Netherlands.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Stratmann
authors.affiliation: Zernike Institute for Advanced Materials and Centre for Synthetic Biology, University of Groningen, 9700 AB Groningen, the Netherlands.
authors.forename: Christiaan M
authors.initials: CM
authors.lastname: Punter
authors.affiliation: Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Arkadiusz W
authors.initials: AW
authors.lastname: Kulczyk
authors.affiliation: Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
authors.affiliation: Zernike Institute for Advanced Materials and Centre for Synthetic Biology, University of Groningen, 9700 AB Groningen, the Netherlands; Centre for Medical and Molecular Bioscience, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address: vanoijen@uow.edu.au.
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D019915:DNA Primase / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D000072760:Single Molecule Imaging / Q000379:methods; D059008:Time-Lapse Imaging / Q000379:methods
chemical_list: D004279:DNA, Viral; C022262:Okazaki fragments; D004247:DNA; D019915:DNA Primase
keywords: DNA replication; bacteriophage T7; flow stretching; single molecule