Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages

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Science  04 Sep 2015:
Vol. 349, Issue 6252, pp. 1120-1124
DOI: 10.1126/science.aaa9046
  • Fig. 1 Identification of pNOB8 centromere and AspA-centromere structure.

    (A) AspA (0 to 1000 nM) was incubated with three DNA fragments upstream of its gene or the pNOB8 traG gene and subjected to electrophoretic mobility shift assays. White triangles denote location of the palindromic centromere. Arrows, unbound DNA; brackets, high-molecular-weight AspA-DNA complexes. The percentage of bound DNA was plotted against the concentration of AspA. (B) DNase I footprint identifying the DNA site bound by AspA (left, 0 to 750 nM) and its ability to spread at higher concentrations (right, 0 to 3000 nM). First lane, AG ladder; last lane, bovine serum albumin control. The inverted centromere repeat is in green and the aspA gene in blue. Below footprints, the sequence shaded in gray indicates the extent of the AspA footprint on top and bottom strands at low-to-medium protein concentrations. (C) (Top) AspA–32-oligomer (AspA-32mer) structure. Three interacting AspA dimers coat the DNA, which generates a continuous superstructure. (Bottom) Close-up showing contacts between recognition helices occupying the same major groove (31). (D) AspA N-terminal arm-DNA contacts. (E) Schematic diagram showing AspA-centromere interactions (31).

  • Fig. 2 AspA-DNA superhelix formation.

    (A) AspA-DNA superstructure. The centrally bound dimer is magenta, and dimers spreading in the 5′ and 3′ directions are green and blue. (B) (Top) ITC of wtAspA binding to the 32-oligomer (32mer) DNA, revealing a binding stoichiometry of three AspA dimers to one DNA duplex. (Bottom) ITC analysis of AspA(A53K)-32mer interaction showing a stoichiometry of one AspA dimer to one DNA duplex. (C) DNase I footprints performed with wtAspA and AspA(A53K) binding to the pNOB8 par upstream region (as in Fig. 1B). Unlike wtAspA, AspA(A53K) does not spread.

  • Fig. 3 Structure of ParB-N and assembly onto the AspA-DNA superhelix.

    (A) Increasing concentrations of wtParB-N (blue) or ParB-N α11-α12-α13 truncation mutant (red) were added to a preformed AspA-DNA complex to analyze binding. mP, millipolarization. (B) ITC analyses revealing that ParB-N does not bind DNA (left) but binds AspA-DNA (right). (C) (Left) pNOB8 ParB-N structure. (Right) Overlay of ParB-N onto T. thermophilus Spo0J. Notable structural differences are denoted by asterisks. (D) AspA–ParB-N model obtained by docking the AspA and ParB-N crystal structures into the SAXS envelope. (E) AspA–DNA–ParB-N model obtained by fitting the ParB-N–AspA model onto the AspA-DNA superhelix. The AspA-DNA superhelix is blue, and ParB-N molecules are shown as coarse cartoon surfaces, with individual molecules colored red and magenta.

  • Fig. 4 pNOB8 ParB-C contains a eukaryotic histone–CenpA-like fold, and ParA harbors a bacterial Walker-box fold.

    (A) Superimposition of the ParB-C and Kluyveromyces lactis Cse4-CenpA structures (rmsd = 2.5 Å for Cα atoms). (B) ParB-C modeled onto DNA to show its electrostatic surface. (C) pNOB8 ParA–AMP-PNP subunit structure. (D) Superimposition of apo and AMP-PNP–bound ParA subunits showing relation of other subunit.

Supplementary Materials

  • Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages

    Maria A. Schumacher, Nam K. Tonthat, Jeehyun Lee, Fernando A. Rodriguez-Castañeda, Naga Babu Chinnam, Anne K. Kalliomaa-Sanford, Irene W. Ng, Madhuri T. Barge, Porsha L. R. Shaw, Daniela Barillà

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Tables S1 and S2
    • References

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