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.

Keywords: ChIP-seq; DnaA; DnaA box.

Fig. 1.. DnaA preferentially binds pairs of DNA sites separated by 2 or 3 bp. (a) Sequence logo illustrating the enriched DNA sequence motif associated with DnaA-bound regions. (b) Schematic of the double DnaA box upstream of purH, with the sequence of each individual DnaA box shown in uppercase. Underlined bases indicate matches to the DnaA box consensus. (c) ChIP-qPCR measurements of DnaA association with the purH upstream region containing a wild-type or mutated double DnaA box. The nature of the changes is indicated by the schematic to the left of the graph. Black boxes indicate consensus DnaA boxes. Grey boxes indicate DnaA boxes with two base changes from the consensus. Boxes with pairs of diagonal lines indicate fully mutated DnaA boxes. Mutants (g, h) have altered spacing between the DnaA boxes. Full sequences for the wild-type and mutated boxes are shown in Fig. S2. Note that mutant (c) has an additional mutation (C to A substitution) 28 bp downstream of the double DnaA box. (d) Genomic DNA sequences were scored by how well they match a single DnaA box motif or a double DnaA box motif (both 2 bp and 3 bp spacing were considered). In the cumulative frequency plot, the x-axis indicates the rank of a genomic region with respect to how well it matches the single or double DnaA box motif. The y-axis indicates the number of DnaA-bound regions associated with a genomic region of a given rank or better. For example, the top 50 ranked genomic regions, according to their match to a double DnaA box motif, are associated with 81 % of all DnaA-bound regions.