Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.
Bottom Line: Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo.Localization of GFP-SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA.The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.
Affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK.Show MeSH
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Mentions: The stoichiometry of the SirA–DnaADI complex was determined using size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). In these experiments, samples are fractionated on a gel-filtration column and the absorbance at 280 nm and the refractive index of the eluate are monitored together with the multi-angle laser light scattering of the sample. This enables the weight average molecular weight (Mw) of species in the eluate to be calculated continuously. Samples of DnaADI and SirA–DnaADI were analysed at a series of protein concentrations. As shown in Fig. 1A, DnaADI elutes from the size exclusion column as a single A280 peak at ∼ 27.5 min and has an experimentally determined molecular mass of ∼ 11 kDa. This suggests that DnaADI from B. subtilis is a monomer (calculated molecular mass = 9.7 kDa) in contrast to E. coli DnaADI which is reported to form dimers under similar conditions (Abe et al., 2007).
Affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK.