Abstract
DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.
Highlights
DnaA is the replication initiator and transcription factor and a AAA+ ATPase found in virtually all bacteria
DnaA is the highly conserved replication initiation protein found in virtually all bacteria [reviewed in 1, 2–6]
The overall goal of our experiments was to identify all regions in the B. subtilis genome capable of binding DnaA, and to compare the binding properties of ATP-DnaA and ADP-DnaA to these regions to gain a better understanding of how DnaA binding is regulated
Summary
DnaA is the highly conserved replication initiation protein found in virtually all bacteria [reviewed in 1, 2–6]. DnaA is a AAA+ ATPase that is present in cells as both ATP-DnaA and ADP-DnaA. Both ATP-DnaA and ADP-DnaA bind DNA, where analyzed, ATP-DnaA is required for replication initiation [3, 14,15,16,17,18,19]. For E. coli DnaA, there are some sites that appear to bind ATP-DnaA and ADP-DnaA well, and others that have a preference for ATP-DnaA [reviewed in 3]. The activity of Bacillus subtilis DnaA appears to be largely regulated by several proteins, all of which affect its ability to bind cooperatively to DNA [22,23,24]
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