The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding

The bacterial chromosome replication origin contains an indispensable element composed of a repeating trinucleotide motif, termed the DnaA-trio, that stabilizes DnaA binding on single-stranded DNA. Recognition motifs at DNA replication origins DnaA is a bacterial DNA replication initiation protein which, in common with replication initiators in higher organisms, contains an AAA+ (ATPases associated with various cellular activities) domain. At the origin, DnaA binds to double-stranded DNA in a recognition reaction, then unwinds the DNA and extends along the single-strand DNA (ssDNA). Heath Murray and colleagues show that the replication origin contains a repeating trinucleotide motif, which they term the DnaA-trio, that is recognized by DnaA and then stabilizes DnaA binding on the ssDNA. The authors suggest that this recognition element is a conserved component of the core bacterial replication origin, and that similar specific recognition motifs may be present in replication origins of higher organisms. 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 synthesis 1 . In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities 2 . DnaA monomers recognize 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 strand 3 , 4 , 5 . While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize 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 is part of the core oriC structure. The discovery and characterization 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 findi