The bacterial DnaA-trio replication origin element specifies ssDNA initiator binding

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.