Poly(ADP-ribose) polymerase 1 searches DNA via a 'Monkey Bar' mechanism

  1. Johannes Rudolph  Is a corresponding author
  2. Jyothi Mahadevan
  3. Pamela Dyer
  4. Karolin Luger  Is a corresponding author
  1. University of Colorado, Boulder, United States

Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a 'monkey bar' mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA.

Data availability

We have provided Excel files for all the figures and tables.

Article and author information

Author details

  1. Johannes Rudolph

    Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, United States
    For correspondence
    Johannes.rudolph@colorado.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Jyothi Mahadevan

    Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Pamela Dyer

    Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0142-5073
  4. Karolin Luger

    Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, United States
    For correspondence
    karolin.luger@colorado.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5136-5331

Funding

National Cancer Institute (R01 CA218255)

  • Karolin Luger

Howard Hughes Medical Institute

  • Karolin Luger

University of Colorado Cancer Center Pilot Funding Grant (ST63501792)

  • Karolin Luger

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

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Publication history

  1. Received: April 24, 2018
  2. Accepted: August 3, 2018
  3. Accepted Manuscript published: August 8, 2018 (version 1)
  4. Version of Record published: September 12, 2018 (version 2)

Copyright

© 2018, Rudolph 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

  • 2,489
    Page views
  • 444
    Downloads
  • 21
    Citations

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

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. Johannes Rudolph
  2. Jyothi Mahadevan
  3. Pamela Dyer
  4. Karolin Luger
(2018)
Poly(ADP-ribose) polymerase 1 searches DNA via a 'Monkey Bar' mechanism
eLife 7:e37818.
https://doi.org/10.7554/eLife.37818

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Rajesh Sharma et al.
    Research Article

    Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here we present a crystal structure of PKG Iβ in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yitong Li et al.
    Research Article

    Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryo-EM structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multi-partite contacts at structured cores to activate the methylesterase. B56-interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56-interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.