Mapping the Escherichia coli DnaA-binding landscape reveals a preference for binding pairs of closely spaced DNA sites.

ABSTRACT

DnaA is a widely conserved DNA-binding protein that is essential for the initiation of DNA replication in many bacterial species, including Escherichia coli. Cooperative binding of ATP-bound DnaA to multiple 9mer sites ('DnaA boxes') at the origin of replication results in local unwinding of the DNA and recruitment of the replication machinery. DnaA also functions as a transcription regulator by binding to DNA sites upstream of target genes. Previous studies have identified many sites of direct positive and negative regulation by E. coli DnaA. Here, we use a ChIP-seq to map the E. coli DnaA-binding landscape. Our data reveal a compact regulon for DnaA that coordinates the initiation of DNA replication with expression of genes associated with nucleotide synthesis, replication, DNA repair and RNA metabolism. We also show that DnaA binds preferentially to pairs of DnaA boxes spaced 2 or 3 bp apart. Mutation of either the upstream or downstream site in a pair disrupts DnaA binding, as does altering the spacing between sites. We conclude that binding of DnaA at almost all target sites requires a dimer of DnaA, with each subunit making critical contacts with a DnaA box.