Regulatory dynamics in the ternary DnaA complex for initiation of chromosomal replication in Escherichia coli

Abstract

In Escherichia coli, the level of the ATP–DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP–DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE–DnaA-box-R1–IBS–DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP–DnaA predominantly promotes ATP–DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP–DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.

Identification of a basal system for unwinding a bacterial chromosome origin

“…In E. coli , the origin appears to have undergone further modification by varying the affinity of upstream DnaA‐boxes towards DnaA and with the addition of a specific site for the nucleoid‐associated protein IHF (grey) (Leonard & Grimwade, ). It has been proposed that DNA bending by DnaA and IHF induces topological strain that promotes strand separation, after which a DNA loop delivers ssDNA at the unwinding site to an upstream DnaA oligomer (Sakiyama et al , ; Grimwade et al , ).…”

“…Such studies using the well‐characterized E. coli oriC plasmids indicated that a significant number of the DnaA‐boxes and binding sites for architectural proteins were essential for origin activity (Asai et al , ; Woelker & Messer, ; Roth et al , ; Langer et al , ; McGarry et al , ). However, several laboratories subsequently showed that this plasmid system does not faithfully reflect oriC within its native environment and mutagenesis of the endogenous E. coli oriC has revealed that none of the protein binding sites is individually essential for origin function (Weigel et al , ; Stepankiw et al , ; Kaur et al , ; Noguchi et al , ; Sakiyama et al , ). Thus, despite isolation of a bacterial replication origin almost forty years ago, the minimal essential sequences that are necessary and sufficient to support a specific DnaA‐dependent unwinding mechanism remain unknown.…”

“…Second, the ability of the DnaA ATP to specifically bind and stretch ssDNA (Speck & Messer, 2001;Duderstadt et al, 2011;Cheng et al, 2015;Richardson et al, 2016) suggests that a DnaA filament could promote origin unwinding by assembling onto one strand of dsDNA and destabilizing the double helix. Third, a combination of biochemical, genetic and modelling data using the Escherichia coli replication system suggests that DnaA binds dsDNA as a monomer proximal to the unwinding region and as an oligomer distal to the unwinding region (Ozaki & Katayama, 2012a;Kaur et al, 2014;Noguchi et al, 2015;Shimizu et al, 2016;Sakiyama et al, 2017). Subsequent binding of an architectural protein between the proximal and distal sites introduces a sharp bend in the dsDNA that connects DnaA proteins from the two regions to promote unwinding and recruit ssDNA to the dsDNA-bound DnaA oligomer.…”

Specific basic patch‐dependent multimerization of Saccharomyces cerevisiae ORC on single‐stranded DNA promotes ATP hydrolysis

“…The cross‐linking assays were carried out using a method developed for bacterial DnaA (Sakiyama, Kasho, Noguchi, Kawakami, & Katayama, ) with minor modifications. ORC was incubated with DL15 for 10 min at room temperature in the same buffer (5 μl) used for the EMSA, but without competitor and BSA.…”

When size matters – coordination of growth and cell cycle in bacteria

“…Accumulation of a critical amount of DnaA-ATP bound to the oriC eventually results in the unwinding of the AT-rich DUE region (Kurokawa et al, 1999;Ozaki & Katayama, 2012;Sakiyama et al, 2017). Then further replication proteins are recruited to the so-called "open complex", namely DnaB helicase is loaded with the aid of DnaC, followed by DnaG primase and multi-subunit DNA polymerase III (Katayama, 2017).…”