1. Chromosomes and Gene Expression
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Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

  1. Michael O Nguyen
  2. Manisha Jalan
  3. Carl A Morrow
  4. Fekret Osman
  5. Matthew C Whitby  Is a corresponding author
  1. University of Oxford, United Kingdom
Research Article
  • Cited 32
  • Views 2,178
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Cite this article as: eLife 2015;4:e04539 doi: 10.7554/eLife.04539

Abstract

The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ~10 minutes of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ~60 minutes, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change.

Article and author information

Author details

  1. Michael O Nguyen

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Manisha Jalan

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Carl A Morrow

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Fekret Osman

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Matthew C Whitby

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    For correspondence
    matthew.whitby@bioch.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Stephen C Kowalczykowski, University of California, Davis, United States

Publication history

  1. Received: August 28, 2014
  2. Accepted: March 24, 2015
  3. Accepted Manuscript published: March 25, 2015 (version 1)
  4. Version of Record published: April 23, 2015 (version 2)

Copyright

© 2015, Nguyen et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

    1. Chromosomes and Gene Expression
    Manisha Jalan et al.
    Research Advance Updated

    Homologous recombination helps ensure the timely completion of genome duplication by restarting collapsed replication forks. However, this beneficial function is not without risk as replication restarted by homologous recombination is prone to template switching (TS) that can generate deleterious genome rearrangements associated with diseases such as cancer. Previously we established an assay for studying TS in Schizosaccharomyces pombe (Nguyen et al., 2015). Here, we show that TS is detected up to 75 kb downstream of a collapsed replication fork and can be triggered by head-on collision between the restarted fork and RNA Polymerase III transcription. The Pif1 DNA helicase, Pfh1, promotes efficient restart and also suppresses TS. A further three conserved helicases (Fbh1, Rqh1 and Srs2) strongly suppress TS, but there is no change in TS frequency in cells lacking Fml1 or Mus81. We discuss how these factors likely influence TS.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Molly Brothers, Jasper Rine
    Research Article

    The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3-M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3 overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.