The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere

  1. Gemma LM Fisher
  2. César L Pastrana
  3. Victoria A Higman
  4. Alan Koh
  5. James A Taylor
  6. Annika Butterer
  7. Timothy Craggs
  8. Frank Sobott
  9. Heath Murray
  10. Matthew P Crump
  11. Fernando Moreno-Herrero
  12. Mark S Dillingham  Is a corresponding author
  1. University of Bristol, United Kingdom
  2. Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Spain
  3. Newcastle University, United Kingdom
  4. University of Antwerp, Belgium
  5. University of Sheffield, United Kingdom
  6. University of Leeds, United Kingdom
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
A hypothetical model for ParB-mediated condensation of the origin of replication region.

(A) Domains and regions as identified in (Bartosik et al., 2004; Kusiak et al., 2011). (B) ParB is thought to be anchored at parS (grey) via the HtH motif found in the CDBD (red). ParB protomers …

https://doi.org/10.7554/eLife.28086.002
Figure 1—figure supplement 1
Structural models for genomic ParB.

(A–C) Schematics of different domain arrangements for dimeric B. subtilis ParB. Whilst it is known that ParB is dimeric in solution, the primary dimerisation interface is unclear and possibly …

https://doi.org/10.7554/eLife.28086.003
Figure 2 with 1 supplement
The R149 residue within the HtH motif is essential for specific binding to parS, but not required for non-specific binding and condensation.

(A) Representative TBM-EMSAs for wild type ParB and ParBR149G monitoring binding of parS-containing or non-specific 147 bp dsDNA. (B) Schematic of the magnetic tweezer assay used to monitor …

https://doi.org/10.7554/eLife.28086.004
Figure 2—figure supplement 1
The R149 residue within the HtH motif is not required for non-specific binding and condensation.

(A) SDS-PAGE of wild type ParB and mutant protein preparations. (B) Non-specific DNA binding isotherms for wild type ParB and ParBR149G measured using a PIFE assay. The data were fitted to the Hill …

https://doi.org/10.7554/eLife.28086.005
Figure 3 with 1 supplement
Solution NMR structure of the dimeric ParB C-terminal domain.

(A) Ensemble overlay of the 14 lowest-energy CTD structures. Red and blue depict separate monomers within the dimer. (B) Secondary structure elements are identified. α1 indicates the N-terminus of …

https://doi.org/10.7554/eLife.28086.006
Figure 3—source data 1

NMR assignment, structure calculation and validation statistics.

acommonly assigned groups (i.e. excluding OH, Asp/Glu side-chain carbonyl, Lys amide and Arg guanidinium groups as well as tertiary aromatic carbons); bresidues 229–282; cvalues reported by ARIA 2.3 (Rieping et al., 2007); dordered residues (230-254, 259-278) as calculated by PSVS 1.5 (Bhattacharya et al., 2007); evalues reported by Procheck (Laskowski et al., 1993); fvalue reported by PDB validation software; g residues in secondary structure (231-245, 249-254, 257-264, 267-277). The structural validation programs used were as follows: CING (Doreleijers et al., 2012), Verify3D (Lüthy et al., 1992), Prosa II (Sippl, 1993), Procheck (Laskowski et al., 1993), MolProbity (Lovell et al., 2003).

https://doi.org/10.7554/eLife.28086.008
Figure 3—figure supplement 1
Solution NMR structure of the dimeric ParB CTD domain.

(A) Assigned 1H-15N HSQC spectra of CTD. Arginine ε and η peaks are aliased once and twice, respectively. Starred peaks indicate additional (usually minor) species. (B) The C-terminal domain of B. …

https://doi.org/10.7554/eLife.28086.007
Figure 4 with 1 supplement
The CTD binds DNA via a lysine-rich surface.

(A–C) TBE-EMSAs for the titration of full length ParB and CTD against 147 bp DNA. Wild type and mutant proteins, K255A + K257A and K252A + K255A + K259A, are indicated. (D) Native mass spectrometry. …

https://doi.org/10.7554/eLife.28086.009
Figure 4—figure supplement 1
The CTD binds DNA via a lysine-rich surface.

(A) TBE-EMSA showing binding of the CTD to the 10 bp hairpin DNA substrate used for NMR experiments. (B) Assigned 1H-15N HSQC spectra of the CTD prior (blue) and after (red) titration with a 10 bp …

https://doi.org/10.7554/eLife.28086.010
Figure 5 with 1 supplement
DNA binding by the CTD is required for efficient DNA condensation in vitro.

(A) Mean force-extension curves of DNA molecules co-incubated with ParB variants at the indicated concentrations. Non-condensed (protein-free) DNA data is fitted to the worm-like chain model. Solid …

https://doi.org/10.7554/eLife.28086.011
Figure 5—figure supplement 1
DNA binding by the CTD is required for efficient DNA condensation in vitro.

(A) Representative traces of condensation assays monitored by magnetic tweezers. The expected DNA extension in the absence of ParB at each force is indicated by the dashed lines. (i), (ii) and (ii) …

https://doi.org/10.7554/eLife.28086.012
Figure 6 with 2 supplements
The CTD of ParB both inhibits and disrupts ParB-dependent DNA condensation.

(A) Mean force-extension curves for DNA molecules co-incubated with 1 µM ParB2 in the presence or absence of 5 µM CTD2. Non-condensed (protein-free) DNA data is fitted to the worm-like chain model. …

https://doi.org/10.7554/eLife.28086.013
Figure 6—figure supplement 1
The CTD of ParB both inhibits and disrupts ParB-dependent DNA condensation.

(A) Representative traces of condensation assays for ParB in the presence and absence of excess free CTD. The expected DNA extension in the absence of ParB at each force is indicated by the dashed …

https://doi.org/10.7554/eLife.28086.014
Figure 6—figure supplement 2
Possible mechanisms to explain the dominant negative effect of the CTD on full length ParB.

(A) A working model for ParB network formation and concomitant DNA condensation incorporating ideas from this work and other published studies (see main text for details). At least two DNA:protein …

https://doi.org/10.7554/eLife.28086.015
Figure 7 with 1 supplement
DNA-binding and dimerisation by the CTD is critical for ParB function in vivo.

(A) Variant ParB-GFP mutants form abnormal foci in B. subtilis. Cells were grown overnight in slow growth conditions before dilution (1:100) into fast growth media, and were allowed to achieve at …

https://doi.org/10.7554/eLife.28086.016
Figure 7—figure supplement 1
DNA-binding and dimerisation by the CTD is critical for ParB function in vivo.

(A–B) Western blot analysis. Expression levels of ParBL270D+L274D and ParBK252A+K255A+K259A with a C-terminal gfp fusion were similar to wild type ParB. DivIVA is an abundant cell division protein …

https://doi.org/10.7554/eLife.28086.017

Tables

Table 1
Primer sequences used in ChIP-qPCR
https://doi.org/10.7554/eLife.28086.018
PlasmidGenotypeGenome location
oqAKPCR35’-AGCCGGATTGATCAAACATC-3’359.32°
oqAKPCR45’-AGAGCCGATCAGACGAAAAC-3’359.32°
oqAKPCR55’-GAGGCAAGCAAAGCTCACTC-3’359.45°
oqAKPCR65’-TGCCATGACAGAGCTGAAAC-3’359.45°
oqAKPCR75’-CTTTTCCAAGGCCTTTAGCC-3’359.22°
oqAKPCR85’-TCACGGAAAACCCATCATTT-3’359.22°
oqAKPCR95’-TATTGGCCTGCTTCATACCC-3’359.65°
oqAKPCR105’-TGGAGATTCTGTCCACGAAA-3’359.65°
oqPCR95’-AAAAAGTGATTGCGGAGCAG-3’359.16°
oqPCR105’-AGAACCGCATCTTTCACAGG-3’359.16°
oqPCR255’-TCCATAATCGCCTCTTGGAC-3’359.37°
oqPCR265’-AAGCGCATGCTTATGCTAGG-3’359.37°
oqPCR315’-GATCCGAAGGTCTGTCTACG-3’359.76°
oqPCR325’-CGATTGCGATTGTACGGTTG-3’359.76°
oqPCR575’-TTTGCATGAACTGGGCAATA-3’146.52°
oqPCR585’-TCCGAACATGTCCAATGAGA-3’146.52°

Additional files

Download links