Article | Published:

The mechanism of DNA replication termination in vertebrates

Nature volume 525, pages 345350 (17 September 2015) | Download Citation

Abstract

Eukaryotic DNA replication terminates when replisomes from adjacent replication origins converge. Termination involves local completion of DNA synthesis, decatenation of daughter molecules and replisome disassembly. Termination has been difficult to study because termination events are generally asynchronous and sequence nonspecific. To overcome these challenges, we paused converging replisomes with a site-specific barrier in Xenopus egg extracts. Upon removal of the barrier, forks underwent synchronous and site-specific termination, allowing mechanistic dissection of this process. We show that DNA synthesis does not slow detectably as forks approach each other, and that leading strands pass each other unhindered before undergoing ligation to downstream lagging strands. Dissociation of the replicative CMG helicase (comprising CDC45, MCM2-7 and GINS) occurs only after the final ligation step, and is not required for completion of DNA synthesis, strongly suggesting that converging CMGs pass one another and dissociate from double-stranded DNA. This termination mechanism allows rapid completion of DNA synthesis while avoiding premature replisome disassembly.

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Acknowledgements

We thank C. Richardson and members of the Walter laboratory for feedback on the manuscript. We thank K. J. Marians and J. T. Yeeles for plasmids and the LacI purification protocol. J.C.W. was supported by NIH grants GM62267 and GM80676. J.C.W. is an investigator of the Howard Hughes Medical Institute.

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Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • James M. Dewar
    • , Magda Budzowska
    •  & Johannes C. Walter
  2. Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Johannes C. Walter

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Contributions

J.M.D. and J.C.W. designed the experiments. J.M.D. performed the experiments. M.B. developed methodologies for plasmid pull downs and HIS6-Ub immunoprecipitations. J.M.D. and J.C.W. interpreted the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Johannes C. Walter.

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https://doi.org/10.1038/nature14887

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