The Escherichia coli replication origin oriC contains the initiator ATP-DnaA-Oligomerization Region (DOR) and its flanking duplex unwinding element (DUE). In the Left-DOR subregion, ATP-DnaA forms a pentamer by binding to R1, R5M and three other DnaA boxes. The DNA-bending protein IHF binds sequence-specifically to the interspace between R1 and R5M boxes, promoting DUE unwinding, which is sustained predominantly by binding of R1/R5M-bound DnaAs to the single-stranded DUE (ssDUE). The present study describes DUE unwinding mechanisms promoted by DnaA and IHF-structural homolog HU, a ubiquitous protein in eubacterial species that binds DNA sequence-non-specifically, preferring bent DNA. Similar to IHF, HU promoted DUE unwinding dependent on ssDUE binding of R1/R5M-bound DnaAs. Unlike IHF, HU strictly required R1/R5M-bound DnaAs and interactions between the two DnaAs. Notably, HU site-specifically bound the R1-R5M interspace in a manner stimulated by ATP-DnaA and ssDUE. These findings suggest a model that interactions between the two DnaAs trigger DNA bending within the R1/R5M-interspace and initial DUE unwinding, which promotes site-specific HU binding that stabilizes the overall complex and DUE unwinding. Moreover, HU site-specifically bound the replication origin of the ancestral bacterium Thermotoga maritima depending on the cognate ATP-DnaA. The ssDUE recruitment mechanism could be evolutionarily conserved in eubacteria.

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