Figure 5.

Monomeric Soj specifically prevents DnaA oligomerization in vitro. (A) In vitro oligomer formation assay with DnaA^Sup proteins. DnaA proteins (3 μM) and Soj^G12V (12, 24, and 36 μM) were incubated in oligomer formation buffer in the presence of ATP (2 mM) and DNA (pBsoriC4; 3 nM) for 15 min prior to the addition of BMOE. DnaA proteins were separated by SDS–PAGE and visualized by western blotting. (B) Monomeric Soj is unable to disassemble pre-formed DnaA oligmers. Reaction conditions are the same as in (A). DnaA was crosslinked at T=15 min. Lane 1, DnaA was added to the reaction buffer at T=13. Lane 2, Soj^G12V was added at T=0 and DnaA added at T=13. Lane 3, DnaA was added at T=0 and Soj^G12V added at T=13. (C) Monomeric Soj does not affect DnaA ATP binding. DnaA^R313A (3 μM) and/or Soj^K16A (36 μM) were incubated with α-P³² ATP before being isolated from the reaction using magnetic nickel beads and denatured with methanol. The released nucleotides were separated on a PEI cellulose TLC plate and visualized by autoradiography. Density values were measured using ImageJ (n=3). (D) Monomeric Soj disrupts DnaA oligomers independent of DnaA ATPase activity. In vitro oligomer formation assay with the ATP hydrolysis deficient DnaA protein. DnaA^R313A (3 μM) and Soj^G12V (12, 24, and 36 μM) were incubated in oligomer formation buffer in the presence of ATP (2 mM) and DNA (pBsoriC4; 3 nM) for 15 min prior to the addition of BMOE. DnaA proteins were separated by SDS–PAGE and visualized by western blotting.