CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

  1. Francisco de Asis Balaguer
  2. Clara Aicart-Ramos
  3. Gemma LM Fisher
  4. Sara de Bragança
  5. Eva M Martin-Cuevas
  6. Cesar L Pastrana
  7. Mark Simon Dillingham  Is a corresponding author
  8. Fernando Moreno-Herrero  Is a corresponding author
  1. Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Spain
  2. University of Bristol, United Kingdom

Abstract

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPgS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum-dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.

Data availability

All DNA sequences used are included in Supplementary Information.Data sets for Fig. 1E, 1F; Fig. 1-S1B, Fig.1-S1C; Fig.1-S4C, Fig1-S4D; Fig. 2C, 2G; Fig. 4C, 4D, 4E, 4F, 4G; Fig. 4-S1A, Fig. 4-S1B, Fig. 4-S1C; Fig. 5A, 5B; Fig. 6A, 6B, 6D, 6F, have been provided.

Article and author information

Author details

  1. Francisco de Asis Balaguer

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  2. Clara Aicart-Ramos

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  3. Gemma LM Fisher

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Sara de Bragança

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4039-1993
  5. Eva M Martin-Cuevas

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  6. Cesar L Pastrana

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  7. Mark Simon Dillingham

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    For correspondence
    mark.dillingham@bristol.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4612-7141
  8. Fernando Moreno-Herrero

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    For correspondence
    fernando.moreno@cnb.csic.es
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4083-1709

Funding

European Research Council (681299)

  • Fernando Moreno-Herrero

Wellcome Trust (100401/Z/12/Z)

  • Mark Simon Dillingham

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

Copyright

© 2021, Balaguer 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.

Metrics

  • 2,149
    views
  • 333
    downloads
  • 49
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Francisco de Asis Balaguer
  2. Clara Aicart-Ramos
  3. Gemma LM Fisher
  4. Sara de Bragança
  5. Eva M Martin-Cuevas
  6. Cesar L Pastrana
  7. Mark Simon Dillingham
  8. Fernando Moreno-Herrero
(2021)
CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS
eLife 10:e67554.
https://doi.org/10.7554/eLife.67554

Share this article

https://doi.org/10.7554/eLife.67554

Further reading

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Artem K Velichko, Nadezhda V Petrova ... Omar L Kantidze
    Research Article

    We investigated the role of the nucleolar protein Treacle in organizing and regulating the nucleolus in human cells. Our results support Treacle’s ability to form liquid-like phase condensates through electrostatic interactions among molecules. The formation of these biomolecular condensates is crucial for segregating nucleolar fibrillar centers from the dense fibrillar component and ensuring high levels of ribosomal RNA (rRNA) gene transcription and accurate rRNA processing. Both the central and C-terminal domains of Treacle are required to form liquid-like condensates. The initiation of phase separation is attributed to the C-terminal domain. The central domain is characterized by repeated stretches of alternatively charged amino acid residues and is vital for condensate stability. Overexpression of mutant forms of Treacle that cannot form liquid-like phase condensates compromises the assembly of fibrillar centers, suppressing rRNA gene transcription and disrupting rRNA processing. These mutant forms also fail to recruit DNA topoisomerase II binding protein 1 (TOPBP1), suppressing the DNA damage response in the nucleolus.

    1. Chromosomes and Gene Expression
    2. Evolutionary Biology
    Gülnihal Kavaklioglu, Alexandra Podhornik ... Christian Seiser
    Research Article

    Repression of retrotransposition is crucial for the successful fitness of a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, is an RNA-binding protein that is expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it is found to be essential for maintaining pluripotency. In cancer, L1TD1 expression is highly correlative with malignancy progression and as such considered a potential prognostic factor for tumors. However, its molecular role in cancer remains largely unknown. Our findings reveal that DNA hypomethylation induces the expression of L1TD1 in HAP1 human tumor cells. L1TD1 depletion significantly modulates both the proteome and transcriptome and thereby reduces cell viability. Notably, L1TD1 associates with L1 transcripts and interacts with L1 ORF1p protein, thereby facilitating L1 retrotransposition. Our data suggest that L1TD1 collaborates with its ancestral L1 ORF1p as an RNA chaperone, ensuring the efficient retrotransposition of L1 retrotransposons, rather than directly impacting the abundance of L1TD1 targets. In this way, L1TD1 might have an important role not only during early development but also in tumorigenesis.