The Mu transpososome structure sheds light on DDE recombinase evolution

Journal name:
Nature
Volume:
491,
Pages:
413–417
Date published:
DOI:
doi:10.1038/nature11602
Received
Accepted
Published online

Abstract

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of the Mu transpososome—Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA—determined from data that extend anisotropically to 5.2Å, 5.2Å and 3.7Å resolution, in conjunction with previously determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains have different roles when bound to different sites. The structure also provides explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved ‘DDE’ catalytic domain, comparisons among the limited set of available complex structures indicate that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

At a glance

Figures

  1. Transposition pathway and structure determination.
    Figure 1: Transposition pathway and structure determination.

    a, Cartoon of transposition. The transposase (MuA) pairs the bacteriophage genome ends (blue and red). At each end, the same active site catalyses the attack of H2O at the phage–host junction and then the direct attack of the phage 3′-OH on target DNA (‘strand transfer’). Target binding is nonspecific, and there is a 5-bp stagger between the sites of attack. Host and target DNAs may be entire circular replicons. After the ATP-dependent unfoldase ClpX disassembles the final strand transfer complex, the 3′ hydroxyls are used as replication primers, resulting in duplication of the bacteriophage genome. Our crystals contain the strand transfer product (third panel). b, Domain structure of MuA. c, Experimental electron density map after phase improvement with Parrot superimposed on the model (contours are 1.2 and 2σ).

  2. Transpososome structure.
    Figure 2: Transpososome structure.

    The complex sits on a crystallographic two-fold symmetry axis (vertical) that relates the blue and red halves. The pale- and dark-coloured subunits adopt different conformations within the homotetramer. DNA colours match Fig. 1. a, Cartoon. Catalytic sites are marked as yellow and tan stars (facing the viewer or the background, respectively) and domains of the blue subunits are labelled. b, Ribbon drawing, with the scissile phosphate groups shown as yellow spheres. c, Same drawing as in b, rotated ~90° about a vertical axis.

  3. Stereo close-up view of interactions near the Mu DNA-target junction.
    Figure 3: Stereo close-up view of interactions near the Mu DNA–target junction.

    Colours are the same as in Fig. 2. A segment of DNA from a symmetry-related complex (yellow) binds the positively charged domain IIIα of the R2-bound subunit (cyan). If the red Mu end DNA were extended to include flanking host DNA, it could lie where the yellow DNA does. The yellow sphere marks the phosphate group at the Mu–target DNA junction, and the main chains of the two active site D residues are also yellow (a third active site residue lies on a helix that could not be modelled). The loop that extends from domain IIα (~amino acids 410–430) to interact with the black target DNA is circled on the red subunit.

  4. Model for a transpososome assembled on full left (reddish) and right (blue) bacteriophage ends.
    Figure 4: Model for a transpososome assembled on full left (reddish) and right (blue) bacteriophage ends.

    The N terminus of each domain Iβ is marked with a red sphere to show the approximate position of domain Iα, which transiently binds the enhancer. Domains discussed in the text are labelled. Inset: cartoon of the bacteriophage Mu genome ends and internal enhancer element.

  5. Comparison of DDE recombinase-DNA complexes.
    Figure 5: Comparison of DDE recombinase–DNA complexes.

    The mobile element ends are red and blue, and target DNA (where included) is black. Subunits that carry out the chemical reactions are red and blue; additional subunits are pink and cyan. Active site residues, scissile phosphate groups, and the two β-strands of the conserved catalytic domain that carry the catalytic D residues are in yellow. Mos1 is a Tc1/mariner family eukaryotic DNA transposon; Tn5 is a bacterial DNA transposon; and PFV is a mammalian retrovirus5, 6, 7. Mos1 and Tn5 require only a dimer for activity, whereas Mu transposase and PFV integrase require tetramers. In the PFV structure, only the catalytic domains of the additional subunits were visible (pink and cyan).

Videos

  1. Ribbon drawing of the transpososome structure rotating 360°
    Video 1: Ribbon drawing of the transpososome structure rotating 360°
    The complex is rotating about the crystallographic twofold axis that relates the red and blue halves. Colours are as in the main text: bacteriophage Mu end DNAs are red and blue, target DNA black, and the scissile phosphate and active site residues are yellow. The darker-colored subunits catalyze DNA cleavage and strand transfer and the lighter-colored subunits aid in complex assembly and stability.
  2. Closeup view of the experimental electron density, after improvement with Parrot, and contoured at 1.3 and 2.3 Sigma, rotating 360°
    Video 2: Closeup view of the experimental electron density, after improvement with Parrot, and contoured at 1.3 and 2.3 Sigma, rotating 360°
    The rotation axis and colors are as in the main text and Supplementary Video 1.

Accession codes

Primary accessions

Protein Data Bank

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Author information

Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA

    • Sherwin P. Montaño,
    • Ying Z. Pigli &
    • Phoebe A. Rice

Contributions

S.P.M. carried out most of the crystallographic work, Y.Z.P. grew the first diffracting transpososome crystals and assisted with all other aspects of the project, and P.A.R. designed the project and assisted in computational work and interpretation of the results.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Coordinates and structure factors were deposited at the Protein Data Bank under accession 4fcy.

Author details

Supplementary information

Video

  1. Video 1: Ribbon drawing of the transpososome structure rotating 360° (4,785 KB, Download)
    The complex is rotating about the crystallographic twofold axis that relates the red and blue halves. Colours are as in the main text: bacteriophage Mu end DNAs are red and blue, target DNA black, and the scissile phosphate and active site residues are yellow. The darker-colored subunits catalyze DNA cleavage and strand transfer and the lighter-colored subunits aid in complex assembly and stability.
  2. Video 2: Closeup view of the experimental electron density, after improvement with Parrot, and contoured at 1.3 and 2.3 Sigma, rotating 360° (9,536 KB, Download)
    The rotation axis and colors are as in the main text and Supplementary Video 1.

PDF files

  1. Supplementary Information (1.1 MB)

    This file contains Supplementary Figures 1-4, Supplementary References and Supplementary Table 1.

Additional data