DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis1. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities2. DnaA monomers recognise the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand3–5. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over thirty years, the sequence specificity for single-strand DNA binding remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating 3-mer motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilise DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element comprises part of the core oriC structure. The discovery and characterisation of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms.

Genome duplication is essential for cell proliferation, and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. In bacteria, the master initiator DnaA binds the chromosome origin ( oriC ) and unwinds the DNA duplex to permit helicase loading. However, despite decades of research it remained unclear how the information encoded within oriC guides DnaA‐dependent strand separation. To address this fundamental question, we took a systematic genetic approach in vivo and identified the core set of essential sequence elements within the Bacillus subtilis chromosome origin unwinding region. Using this information, we then show in vitro that the minimal replication origin sequence elements are necessary and sufficient to promote the mechanical functions of DNA duplex unwinding by DnaA. Because the basal DNA unwinding system characterized here appears to be conserved throughout the bacterial domain, this discovery provides a framework for understanding oriC architecture, activity, regulation and diversity.

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