title: Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery.
abstract: Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.
authors.affiliation: Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Hylkje J
authors.initials: HJ
authors.lastname: Geertsema
authors.forename: Arkadiusz W
authors.initials: AW
authors.lastname: Kulczyk
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D017123:Bacteriophage T7 / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005453:Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D013997:Time Factors
chemical_list: D046912:Multiprotein Complexes; C022262:Okazaki fragments; D004247:DNA; D004259:DNA-Directed DNA Polymerase
keywords: fluorescence microscopy; polymerase exchange; single molecule