Abstract
DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein) leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.
Highlights
All cells must be able to integrate environmental and internal physiological cues into the decision when to commence the duplication of the genome in order to initiate the proliferation cycle
DnaA binds to several high affinity binding sites at the origin region of replication on the bacterial chromosome, triggers the unwinding of the DNA duplex nearby, and supports loading of the DNA helicase, which in turn leads to the establishment of the DNA replication machinery
Using two different fluorescence microscopy techniques, we show that DnaA binding and unbinding to oriC is very rapid in two bacterial species and occurs in the range of few seconds
Summary
All cells must be able to integrate environmental and internal physiological cues into the decision when to commence the duplication of the genome in order to initiate the proliferation cycle. Nature appears to have invented the process of replication initiation only once, because the key players, called ORC in eukaryotic and in archaeal cells, and DnaA in bacteria, are conserved AAA (ATPases Associated with diverse cellular Activities) proteins, whose ATPase activity leads to conformational changes that are transduced into mechanical force or into switch-like processes. For DnaA, ATP hydrolysis confers a crucial role in initiation control [1, 2]. A failure to limit initiation of replication to once per cell cycle leads to growth defects and in severe cases to lethality
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