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Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

Duderstadt, K. E., Geertsema, H. J., Stratmann, S. A., Punter, C. M., Kulczyk, A. W., Richardson, C. C. & van Oijen, A. M., 15-Dec-2016, In : Molecular Cell. 64, 6, p. 1035-1047 13 p.

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  • Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals

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DOI

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 multi-dimensional 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.
Original languageEnglish
Pages (from-to)1035-1047
Number of pages13
JournalMolecular Cell
Volume64
Issue number6
Publication statusPublished - 15-Dec-2016

    Keywords

  • EUKARYOTIC REPLICATION FORK, COLI DNA-REPLICATION, ESCHERICHIA-COLI, BACTERIOPHAGE T7, PRIMER SYNTHESIS, POLYMERASE, MOLECULE, PRIMASE, PROTEINS, MECHANISM

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