Crystallization and X-ray diffraction analysis of the DNA-remodelling protein DnaD fromBacillus subtilis

Abstract

The DnaD protein is an essential component of the chromosome-replication machinery of the Gram-positive bacterium Bacillus subtilis and is part of the primosomal cascade that ultimately loads the replicative ring helicase DnaC onto DNA. Moreover, DnaD is a global regulator of DNA architecture, as it forms higher order nucleoprotein structures in order to open supercoiled DNA. Here, the crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis are reported. Crystals of the N-terminal domain are trigonal, with either P3 1 21 or P3 2 21 space-group symmetry, and diffracted X-rays to 2.0 Å resolution; crystals of the C-terminal domain are hexagonal, with space group P6 1 or P6 5 , and diffracted X-rays to 2.9 Å resolution in-house. Determination of the structure of the DnaD domains will provide insight into how remodelling of the nucleoid is associated with priming of replication in the model Gram-positive organism B. subtilis.

Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation

“…Crystallization of the native DnaD Nd was carried out as described before 44. For obtaining phase information, samples of the DnaD Nd were prepared in the presence of selenomethionine (SeMet) by transforming the expression plasmid in methionine auxotroph E. coli B834 (DE3) cells.…”

“…Protein expression was then induced by adding isopropyl-β- d -thioga-lactopyranoside to a final concentration of 1 mM, and cultures were further grown overnight. Purification and crystallization of SeMet Nd were carried out as described for the native protein but at the lower protein concentration of 10 mg mL −1 , in contrast to 20 mg mL −1 for the unlabeled protein 44…”

“…The SeMet derivative crystals diffracted to 2.2 Å (peak) and 2.1 Å (remote) spacing. Data on the native DnaD Nd crystals were collected in-house as previously described 44. All data sets were processed with the programs MOSFLM46 and SCALA47 of the CCP4 suite 48…”

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD

“…The structure of the N-terminal domain of B. subtilis DnaD has been determined by X-ray crystallography ( 16 , 28 , 29 ), revealing a winged helix-turn-helix fold. Whilst proteins with such a motif commonly, but not exclusively, have a DNA-binding function, this is not the case for the DnaD N-terminal domain, which instead facilitates formation of extensive protein scaffolds ( 14–16 ).…”

Single-Molecule Atomic Force Spectroscopy Reveals that DnaD Forms Scaffolds and Enhances Duplex Melting

“…Nd is responsible for scaffold formation in the absence of DNA, while Cd binds to DNA and oligomerizes 18. Both domains have been crystallized and structural determination is underway 28. Topoisomerase I-based assays revealed that full-length DnaD or Cd binding to DNA induces untwisting of the duplex, thus converting it from plectonemic to paranemic 19.…”