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

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.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle

“…DnaA-ATP assembles into a pentamer via its binding to DnaA boxes in one half of the DOR and, progressively, via its binding to the single-stranded motifs in the DUE, which stabilizes the unwound DUE. This unwinding is promoted by the nucleoid-associated protein (NAP), IHF, or indeed by HU [ 14 ]. Katayama’s group analysed, also by P1 nuclease sensitivity, the opening of M13 oriC DNA by DnaA in vitro at the DUE using various types of mutated oriC and IHF; the results obtained were consistent with those obtained in vivo [ 64 ].…”

“…The consequence of this interaction is the opening of oriC , which allows the insertion of DNA helicase into oriC in order to start DNA synthesis after the loading of DNA polymerase III. This interaction between DnaA and DnaA boxes seems to be important in most of the processes in which DnaA is involved [ 13 , 14 ]. DnaA has four domains [ 15 , 16 ] ( Figure 1 ).…”

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA

“…The oriC region comprises a duplex-unwinding element (DUE) and a flanking DnaA oligomerization region (DOR), which contains a cluster of DnaA-binding sites (DnaA boxes). ATP-bound DnaA, but not ADP-bound DnaA, forms specific oligomers with the DOR that promote DUE unwinding in concert with IHF or its homologue HU ( Cassler et al, 1995 ; Shimizu et al, 2016 ; Sakiyama et al, 2017 ; Kasho et al, 2021 ; Yoshida et al, 2023 ). IHF specifically binds to a consensus sequence (TAAnnnnTTGATW, where W is A or T, and n is any nucleotides) at a DUE-proximal site within the DOR and induces sharp DNA bending (120°–180°) ( Figure 1 ; Aeling et al, 2006 ).…”

“…Domain II is a flexible linker ( Nozaki and Ogawa, 2008 ). Domain III contains AAA+ motifs involved in ATP/ADP binding ( Kawakami et al, 2006 ), ATP hydrolysis ( Nishida et al, 2002 ), and domain III–domain III interactions ( Kawakami et al, 2005 ; Erzberger et al, 2006 ; Noguchi et al, 2015 ), in addition to a specific motif for ssDUE binding ( Ozaki et al, 2008 ; Duderstadt et al, 2011 ; Yoshida et al, 2023 ). Domain IV binds specifically to the 9-mer DnaA box consensus sequence, TTAWnCACA (where W is A and T) ( Fujikawa et al, 2003 ).…”

Dynamics of chromosome organization in a minimal bacterial cell

“…Given these considerations, we hypothesize that HU’s lingering essentiality in Syn3A is a reflection of it only acting through an interaction specific to the high-affinity binding mode. In E. coli , HU is known to interact with replication initiator protein DnaA ( Chodavarapu et al, 2007 ), HU α /DNA stoichiometry has been shown to increase for faster-growing E. coli cells ( Abebe et al, 2017 ), and experimental evidence suggests a mechanism of HU promoting duplex unwinding at the oriC replication origin ( Yoshida et al, 2023 ). Additionally, HU is essential for replication initiation in Gram-positive B. subtilis ( Karaboja and Wang, 2022 ; Schramm and Murray, 2022 ), whose replication origin is similarly a DnaA-based oriC .…”