Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

  1. Allison McClure
  2. John Diffley  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom

Abstract

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Data availability

All source files for data have been uploaded to this site.

Article and author information

Author details

  1. Allison McClure

    The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. John Diffley

    The Francis Crick Institute, London, United Kingdom
    For correspondence
    john.diffley@crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5184-7680

Funding

Wellcome Trust (106252/Z/14/Z)

  • John Diffley

Wellcome Trust (219527/Z/19/Z)

  • John Diffley

European Commission (669424-CHROMOREP)

  • John Diffley

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Bruce Stillman, Cold Spring Harbor Laboratory, United States

Publication history

  1. Preprint posted: April 10, 2021 (view preprint)
  2. Received: April 24, 2021
  3. Accepted: August 12, 2021
  4. Accepted Manuscript published: August 13, 2021 (version 1)
  5. Version of Record published: August 25, 2021 (version 2)

Copyright

© 2021, McClure & Diffley

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.

Metrics

  • 1,528
    Page views
  • 321
    Downloads
  • 8
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Allison McClure
  2. John Diffley
(2021)
Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation
eLife 10:e69726.
https://doi.org/10.7554/eLife.69726

Further reading

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Hirotaka Araki, Shinjiro Hino ... Mitsuyoshi Nakao
    Research Article

    Skeletal muscle exhibits remarkable plasticity in response to environmental cues, with stress-dependent effects on the fast-twitch and slow-twitch fibers. Although stress-induced gene expression underlies environmental adaptation, it is unclear how transcriptional and epigenetic factors regulate fiber type-specific responses in the muscle. Here, we show that flavin-dependent lysine-specific demethylase-1 (LSD1) differentially controls responses to glucocorticoid and exercise in postnatal skeletal muscle. Using skeletal muscle-specific LSD1-knockout mice and in vitro approaches, we found that LSD1 loss exacerbated glucocorticoid-induced atrophy in the fast fiber-dominant muscles, with reduced nuclear retention of Foxk1, an anti-autophagic transcription factor. Furthermore, LSD1 depletion enhanced endurance exercise-induced hypertrophy in the slow fiber-dominant muscles, by induced expression of ERRγ, a transcription factor that promotes oxidative metabolism genes. Thus, LSD1 serves as an ‘epigenetic barrier’ that optimizes fiber type-specific responses and muscle mass under the stress conditions. Our results uncover that LSD1 modulators provide emerging therapeutic and preventive strategies against stress-induced myopathies such as sarcopenia, cachexia, and disuse atrophy.

    1. Chromosomes and Gene Expression
    2. Developmental Biology
    Nan Wang, Jing He ... Kehkooi Kee
    Research Article Updated

    Non-coding RNAs exert diverse functions in many cell types. In addition to transcription factors from coding genes, non-coding RNAs may also play essential roles in shaping and directing the fate of germ cells. The presence of many long non-coding RNAs (lncRNAs) which are specifically expressed in the germ cells during human gonadal development were reported and one divergent lncRNA, LNC1845, was functionally characterized. Comprehensive bioinformatic analysis of these lncRNAs indicates that divergent lncRNAs occupied the majority of female and male germ cells. Integrating lncRNA expression into the bioinformatic analysis also enhances the cell-type classification of female germ cells. Functional dissection using in vitro differentiation of human pluripotent stem cells to germ cells revealed the regulatory role of LNC1845 on a transcription factor essential for ovarian follicle development, LHX8, by modulating the levels of histone modifications, H3K4me3 and H3K27Ac. Hence, bioinformatical analysis and experimental verification provide a comprehensive analysis of lncRNAs in developing germ cells and elucidate how an lncRNA function as a cis regulator during human germ cell development.