1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

  1. Timothy J Wendorff
  2. James M Berger  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Johns Hopkins University School of Medicine, United States
https://doi.org/10.7554/eLife.31724
Share this article
Cite this article
  1. Timothy J Wendorff
  2. James M Berger
(2018)
Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage
eLife 7:e31724.
https://doi.org/10.7554/eLife.31724
  2. Download BibTeX
  3. Download .RIS

Abstract

Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to engage two DNA segments and link ATP turnover to DNA transport is poorly understood. Using multiple biochemical approaches, we show that Top6B, which harbors the ATPase activity of topo VI, recognizes and exploits the DNA crossings present in supercoiled DNA to stimulate subunit dimerization by ATP. Top6B self-association in turn induces extensive DNA bending, which is needed to support duplex cleavage by Top6A. Our observations explain how topo VI tightly coordinates DNA crossover recognition and ATP binding with strand scission, providing useful insights into the operation of type IIB topoisomerases and related meiotic recombination and GHKL ATPase machineries.

Data availability

The following previously published data sets were used

Article and author information

Author details

  1. Timothy J Wendorff

    Biophysics Graduate Program, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  2. James M Berger

    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
    For correspondence
    jberge29@jhmi.edu
    Competing interests
    James M Berger, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0666-1240

Funding

National Institutes of Health (RO1 CA077373)

  • James M Berger

National Science Foundation (DGE 1106400)

  • Timothy J Wendorff

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

Copyright

© 2018, Wendorff & Berger

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

  • 3,300
    views
  • 506
    downloads
  • 25
    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. Timothy J Wendorff
  2. James M Berger
(2018)
Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage
eLife 7:e31724.
https://doi.org/10.7554/eLife.31724

Share this article

https://doi.org/10.7554/eLife.31724

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome

    Kira A Cozzolino, Lynn Sanford ... Dylan J Taatjes
    Research Article

    Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by Trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 75 min to 24 hr timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), via rapid suppression of IFN-responsive transcription factor (TF) activity. We also discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways in cell type-specific ways, and broad upregulation of anti-inflammatory lipid mediators occurred specifically in kinase-inhibited cells during hyperactive IFNγ signaling. A subset of these lipids (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing mechanistic links between Mediator kinases, lipid metabolism, and nuclear receptor function. Collectively, our results establish CDK8/CDK19 as context-specific metabolic regulators, and reveal that these kinases control gene expression not only via TFs, but also through metabolic changes and splicing. Moreover, we establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

    1. Biochemistry and Chemical Biology
    2. Cancer Biology

    Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

    Flavie Coquel, Sing-Zong Ho ... Philippe Pasero
    Research Article

    Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or inhibit checkpoint kinases involved in the DNA damage response. Thus far, treatments that combine these two strategies have shown promise but also have severe adverse effects. To identify novel, better-tolerated anticancer combinations, we screened a collection of plant extracts and found two natural compounds from the plant, Psoralea corylifolia, that synergistically inhibit cancer cell proliferation. Bakuchiol inhibited DNA replication and activated the checkpoint kinase CHK1 by targeting DNA polymerases. Isobavachalcone interfered with DNA double-strand break repair by inhibiting the checkpoint kinase CHK2 and DNA end resection. The combination of bakuchiol and isobavachalcone synergistically inhibited cancer cell proliferation in vitro. Importantly, it also prevented tumor development in xenografted NOD/SCID mice. The synergistic effect of inhibiting DNA replication and CHK2 signaling identifies a vulnerability of cancer cells that might be exploited by using clinically approved inhibitors in novel combination therapies.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    eIF3 engages with 3’-UTR termini of highly translated mRNAs

    Santi Mestre-Fos, Lucas Ferguson ... Jamie HD Cate
    Research Article

    Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.