Single-stranded DNA recruitment mechanism in replication origin unwinding by DnaA initiator protein and HU, an evolutionary ubiquitous nucleoid protein

• Published in: Nucleic acids research 2023-07, Vol.51 (12), p.6286-6306
• Main Authors: Yoshida, Ryusei, Ozaki, Shogo, Kawakami, Hironori, Katayama, Tsutomu
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; DNA Replication; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Genome Integrity, Repair and; Origin Recognition Complex - genetics; Origin Recognition Complex - metabolism; Protein Binding; Replication Origin
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad389

Abstract 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. Graphical Abstract Graphical Abstract The left-oriC subregion responsible for DUE unwinding is shown. ATP-DnaA constructs a specific pentamer, causing initial unstable DNA bending and DUE unwinding, which are stabilized by bent DNA-preferential binding of HU protein and specific ssDUE-DnaA binding.