1. Structural Biology and Molecular Biophysics
  2. Chromosomes and Gene Expression
Download icon

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

  1. Chun Zhou
  2. Sergei Pourmal
  3. Nikola P Pavletich  Is a corresponding author
  1. Memorial Sloan Kettering Cancer Center, United States
Research Article
  • Cited 45
  • Views 5,131
  • Annotations
Cite this article as: eLife 2015;4:e09832 doi: 10.7554/eLife.09832

Abstract

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

Article and author information

Author details

  1. Chun Zhou

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Sergei Pourmal

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Nikola P Pavletich

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    For correspondence
    pavletin@mskcc.org
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. John Kuriyan, Howard Hughes Medical Institute, University of California, Berkeley, United States

Publication history

  1. Received: July 2, 2015
  2. Accepted: October 20, 2015
  3. Accepted Manuscript published: October 22, 2015 (version 1)
  4. Accepted Manuscript updated: November 2, 2015 (version 2)
  5. Version of Record published: December 30, 2015 (version 3)

Copyright

© 2015, Zhou 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

  • 5,131
    Page views
  • 1,103
    Downloads
  • 45
    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)

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

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

Further reading

    1. Structural Biology and Molecular Biophysics
    Achinta Sannigrahi et al.
    Research Article

    Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, ALS. Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association mediated toxic aggregation and survival time scale after ALS diagnosis.

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics
    Robert J Nichols et al.
    Research Article

    Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartments in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.