ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA

Abstract

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Fig. 1.

Detection of higher molecular weight ParA2 structures with a pelleting assay. In each case, ParA2 was 10 μM and nucleotides were used at 1 mM. For the DNA titrations, 1.5 fmol–1.5 pmol of pSM829 (B–D) or pSM833 (E), corresponding to ParA2∶DNA molar ratios varying from 200,000∶1 to 200∶1 were used. (S) and (P) denote the supernatant and pellet fractions, respectively.

Fig. 2.

ParA2 binding and hydrolysis of ATP. (A) ParA2 nucleotide binding as assessed by photoaffinity cross-linking. Reactions contained 5 μM ParA2 and 167 μM [α ³²P]-ATP; excess cold nucleotide competitors were added to a final concentration of 2 mM. (B–D) ParA2 ATP hydrolysis detected by TLC. (C) ParA2 incubated with 7.5 pM–7.5 nM pSM829, corresponding to a ParA2∶DNA molar ratio range from 200,000∶1 to 200∶1. (D) ParA2 incubated with 0.38–3.0 μM ParB2, corresponding to a ParA2∶ParB2 molar ratio range from 4∶1 to 1∶2. Values in (C) and (D) were averaged from two independent experiments.

Fig. 3.

ParA2 interacts with DNA in a nonsequence specific manner. EMSA of ParA2 binding to radio-labeled parS2-A DNA (A) or random DNA (B). ParA2 ranged from 0–1280 ng (0–1.4 μM). (C) ParA2 protection of pSM829 from restriction by Sau3AI. ParA2 ranged from 0.14–17 μg, corresponding to a molar ratio range of ParA2∶DNA of 82∶1–8237∶1.

Fig. 4.

Electron micrographs of negatively stained ParA2-dsDNA filaments formed in the presence of ATP (A), ParA2-dsDNA filaments formed in the presence of ADP (B), and ParA2-dsDNA filaments (C). The scale bar (B) is 500 Å and applies to (A–C). The inset in (A) depicts a fast freeze/deep etch micrograph of a ParA2-DNA filament formed in the presence of ATP. Three-dimensional reconstructions of ParA2-dsDNA filaments formed with ATP (D) and ParA2-dsDNA filaments formed in the presence of ADP (E).

Fig. 5.

The three-dimensional reconstruction of ParA2-dsDNA filaments formed with ATP (transparent gray surface) can be fit by the unmodified crystal structure (5) of a P1 ParA-ADP dimer (PDB 3EZ2), shown as cyan ribbons. The side view (A) and the top view (B) show the good match between the density envelope and the high resolution protein structure. Atoms in P1 ParA residues 318 and 349 are represented as red spheres, and these correspond to residues 189 and 218 in Soj which have been shown to be crucial for sequence-independent DNA-binding (7). Atoms in P1 ParA residue 351 are shown as blue spheres, and this corresponds to residue 340 of SopA which has also been shown to be essential for sequence-independent DNA-binding (8). The HTH-domain is involved in both lateral (A, blue arrowhead) and longitudinal (B, red arrows) contacts within the ParA2-dsDNA-ATP filament. (C) The geometry of ParA2 binding to B-form DNA. One strand of the DNA is shown in magenta, and the other in yellow. The DNA has ∼10.4 bp per turn, with a pitch of ∼35 Å. Every eighth nucleotide along the magenta strand is indicated with the space-filling atoms. The helical path connecting these residues is shown by the gray transparent tube, which makes one left-handed turn every 120 Å, contacting 4.4 of the space-filling atoms per turn. These are the helical parameters of the ParA2 filament in (A).

Fig. 6.

Nicked circular ϕX174 molecules were covered with ParA2 in the presence of ATP (A) or ADP (C). The histograms represent the contour lengths measured for ParA2 polymerized with ATP (B) or with ADP (D).