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

Crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis is reported

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase

Helicase loading in Gram-positive Bacillus

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mapping protein dynamics conformational changes

Understanding how proteins interact and behave in large dynamic multi-protein complexes is an important area of significance in many human diseases that are associated with faults in their interactions. Increasing understanding of the well characterized bacterial DNA replication machinery (the replisome) will continue to enrich our understanding of other dynamic complexes, including those that carry out human DNA replication. Although replication proteins are not themselves highly conserved between eubacteria and higher organisms, the replication mechanisms generally are. We focussed on pairwise interactions that involve one of the key organizational centres of the bacterial replisome, the DnaI helicase loader and the DnaB helicase. As a first step, we measured the dynamics of the Bacillus sp. helicase loader DnaI and the DnaB helicase separately, using elastic, quasielastic and inelastic neutron scattering at four instrumental resolutions. This provides a basis for future experiments on the DnaI-DnaB complex. The measurement established that internal molecular flexibility and diffusive motions are significantly higher in DnaB helicase compared to those evaluated in DnaI. The structural rigidity was also studied. The analysis revealed that DnaI structural rigidity is larger than that of DnaB. Furthermore, the method allowed the extraction of vibrational density of states, showing that DnaI has more vibrational modes than DnaB

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis Dnal helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase

Mapping protein dynamics conformational changes in replisomal macromolecular assemblies

Understanding how proteins interact and behave in large dynamic multi-protein complexes is an important area of significance in many human diseases that are associated with faults in their interactions. Increasing understanding of the well characterized bacterial DNA replication machinery (the replisome) will continue to enrich our understanding of other dynamic complexes, including those that carry out human DNA replication. Although replication proteins are not themselves highly conserved between eubacteria and higher organisms, the replication mechanisms generally are. We focussed on pairwise interactions that involve one of the key organizational centres of the bacterial replisome, the DnaI helicase loader and the DnaB helicase. As a first step, we measured the dynamics of the Bacillus sp. helicase loader DnaI and the DnaB helicase separately, using elastic, quasielastic and inelastic neutron scattering at four instrumental resolutions. This provides a basis for future experiments on the DnaI-DnaB complex. The measurement established that internal molecular flexibility and diffusive motions are significantly higher in DnaB helicase compared to those evaluated in DnaI. The structural rigidity was also studied. The analysis revealed that DnaI structural rigidity is larger than that of DnaB. Furthermore, the method allowed the extraction of vibrational density of states, showing that DnaI has more vibrational modes than DnaB

A Novel Zinc-Binding Fold in the Helicase Intreraction Domain of Bacillus Subtilis Dnai Helicase Loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase

domain of the

novel zinc-binding fold in the helicase interactio