Skip to main content
placeholder image

Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes

Journal Article


Download full-text (Open Access)

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


  •   Duderstadt, Karl E. (external author)
  •   Geertsema, Hylkje J. (external author)
  •   Stratmann, Sarah A. (external author)
  •   Punter, Christiaan M. (external author)
  •   Kulczyk, Arkadiusz W. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2016

Citation


  • Duderstadt, K. E., Geertsema, H., Stratmann, S. A., Punter, C. M., Kulczyk, A. W., Richardson, C. C. & van Oijen, A. M. (2016). Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes. Molecular Cell, 64 (6), 1035-1047.

Scopus Eid


  • 2-s2.0-85007189929

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2018&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/993

Number Of Pages


  • 12

Start Page


  • 1035

End Page


  • 1047

Volume


  • 64

Issue


  • 6

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


  •   Duderstadt, Karl E. (external author)
  •   Geertsema, Hylkje J. (external author)
  •   Stratmann, Sarah A. (external author)
  •   Punter, Christiaan M. (external author)
  •   Kulczyk, Arkadiusz W. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2016

Citation


  • Duderstadt, K. E., Geertsema, H., Stratmann, S. A., Punter, C. M., Kulczyk, A. W., Richardson, C. C. & van Oijen, A. M. (2016). Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes. Molecular Cell, 64 (6), 1035-1047.

Scopus Eid


  • 2-s2.0-85007189929

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2018&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/993

Number Of Pages


  • 12

Start Page


  • 1035

End Page


  • 1047

Volume


  • 64

Issue


  • 6