Abstract
Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.
Highlights
Ministry of Education, Culture, Sports, Technology and Science of Japan. □S The on-line version of this article contains supplemental Fig. S1 and additional references. 1 To whom correspondence should be addressed: Dept. of Molecular Biology, formation of the prepriming complex [6, 7]
Together with the data from the oriC pulldown exper- tagged DnaA and DiaA did not directly bind to streptavidin iments (Fig. 2, C and D, and supplemental Fig. S1B), these beads (Fig. 3D, lanes 1, 4, 7, and 12). These results indicate that results demonstrate a crucial role for DnaA Phe-46 in the DiaA- DnaA F46A as well as wild-type DnaA can form a mixed muldependent efficient formation of initiation complexes
We used NMR analysis and a pulldown assay to demonstrate that the presence of DnaA Phe-46, which occurs within DnaA domain I, is a prerequisite for DiaA binding (Figs. 1 and 2)
Summary
E. coli oriC carries a dozen DnaA-binding sites, including the high affinity 9-mer DnaA boxes (R1 and R4 sites) and ATPDnaA-preferential low affinity sites (ADLAS), which include the I and sites [20, 27]. The interaction of ATP-DnaA with ADLAS is important for the activation of DnaAoriC complexes. DiaA stimulates the cooperative binding of ATP-DnaA on oriC, especially on ADLAS, resulting in the formation of open complexes [15]. We have previously determined the tertiary structure of the DnaA domain I and found that DnaA Glu-21, within this domain, is a DnaB interaction site, required for DnaB loading onto open complexes [23]. We found that this site is required for DiaA-dependent stimulation of initiation complex formation and that only a subgroup of DnaA molecules, assembled on oriC, is sufficient for DiaA stimulation. Like the DiaA stimulation, the stimulation of DnaB loading requires only a subgroup of DnaA molecules assembled on oriC. In addition we propose a novel model for the structure of initiation complexes that includes DiaA and suggest possible modes of interactions for DiaA and DnaB on the initial complexes
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