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

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

  1. Fahad Rashid
  2. Paul D Harris
  3. Manal S Zaher
  4. Mohamed A Sobhy
  5. Luay I Joudeh
  6. Chunli Yan
  7. Hubert Piwonski
  8. Susan E Tsutakawa
  9. Ivaylo Ivanov
  10. John A Tainer
  11. Satoshi Habuchi
  12. Samir M Hamdan  Is a corresponding author
  1. King Abdullah University of Science and Technology, Saudi Arabia
  2. Georgia State University, United States
  3. Lawrence Berkeley National Laboratory, United States
Research Article
  • Cited 23
  • Views 2,801
  • Annotations
Cite this article as: eLife 2017;6:e21884 doi: 10.7554/eLife.21884

Abstract

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Article and author information

Author details

  1. Fahad Rashid

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  2. Paul D Harris

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  3. Manal S Zaher

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  4. Mohamed A Sobhy

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  5. Luay I Joudeh

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  6. Chunli Yan

    Department of Chemistry, Georgia State University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Hubert Piwonski

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8666-3945

  8. Susan E Tsutakawa

    Lawrence Berkeley National Laboratory, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Ivaylo Ivanov

    Department of Chemistry, Georgia State University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. John A Tainer

    Lawrence Berkeley National Laboratory, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. Satoshi Habuchi

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    Competing interests
    The authors declare that no competing interests exist.

  12. Samir M Hamdan

    Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
    For correspondence
    samir.hamdan@kaust.edu.sa
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5192-1852

Funding

King Abdullah University of Science and Technology (2201 CRG3)

  • Fahad Rashid
  • Paul D Harris
  • Manal S Zaher
  • Mohamed A Sobhy
  • Luay I Joudeh
  • Hubert Piwonski
  • John A Tainer
  • Satoshi Habuchi
  • Samir M Hamdan

National Science Foundation (MCB-1149521)

  • Chunli Yan
  • Ivaylo Ivanov

NIH Clinical Center (R01GM110387)

  • Chunli Yan
  • Ivaylo Ivanov

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

Reviewing Editor

  1. James M Berger, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: September 27, 2016
  2. Accepted: February 20, 2017
  3. Accepted Manuscript published: February 23, 2017 (version 1)
  4. Version of Record published: March 20, 2017 (version 2)

Copyright

© 2017, Rashid 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

  • 2,801
    Page views
  • 678
    Downloads
  • 23
    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)

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology
    2. Cancer Biology

    Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia

    Margaret A M Nelson et al.
    Research Article

    Typified by oxidative phosphorylation (OXPHOS), mitochondria catalyze a wide variety of cellular processes seemingly critical for malignant growth. As such, there is considerable interest in targeting mitochondrial metabolism in cancer. However, notwithstanding the few drugs targeting mutant dehydrogenase activity, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. The present project was based on the premise that the development of efficacious mitochondrial-targeted anti-cancer compounds requires answering two fundamental questions: 1) is mitochondrial bioenergetics in fact different between cancer and non-cancer cells? and 2) If so, what are the underlying mechanisms? Such information is particularly critical for the subset of human cancers, including acute myeloid leukemia (AML), in which alterations in mitochondrial metabolism are implicated in various aspects of cancer biology (e.g., clonal expansion and chemoresistance). Herein, we leveraged an in-house diagnostic biochemical workflow to comprehensively evaluate mitochondrial bioenergetic efficiency and capacity in various hematological cell types, with a specific focus on OXPHOS dynamics in AML. Consistent with prior reports, clonal cell expansion, characteristic of leukemia, was universally associated with a hyper-metabolic phenotype which included increases in basal and maximal glycolytic and respiratory flux. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic limitations in oxidative ATP synthesis (i.e., OXPHOS). Remarkably, by performing experiments across a physiological span of ATP free energy (i.e, ΔGATP), we provide direct evidence that, rather than contributing to cellular ΔGATP, leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of OXPHOS kinetics proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Taken together, these findings argue against ATP being the primary output of mitochondria in leukemia and provide proof-of-principle that restoring, rather than disrupting, OXPHOS and/or cellular ΔGATP in cancer may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

    1. Biochemistry and Chemical Biology

    Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

    M'Lynn E Fisher et al.
    Research Article Updated

    The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    A subcellular map of the human kinome

    Haitao Zhang et al.
    Tools and Resources Updated

    The human kinome comprises 538 kinases playing essential functions by catalyzing protein phosphorylation. Annotation of subcellular distribution of the kinome greatly facilitates investigation of normal and disease mechanisms. Here, we present Kinome Atlas (KA), an image-based map of the kinome annotated to 10 cellular compartments. 456 epitope-tagged kinases, representing 85% of the human kinome, were expressed in HeLa cells and imaged by immunofluorescent microscopy under a similar condition. KA revealed kinase family-enriched subcellular localizations and discovered a collection of new kinase localizations at mitochondria, plasma membrane, extracellular space, and other structures. Furthermore, KA demonstrated the role of liquid-liquid phase separation in formation of kinase condensates. Identification of MOK as a mitochondrial kinase revealed its function in cristae dynamics, respiration, and oxidative stress response. Although limited by possible mislocalization due to overexpression or epitope tagging, this subcellular map of the kinome can be used to refine regulatory mechanisms involving protein phosphorylation.