## 5 The Specific Case of DnaA in the _E. coli_ Cell Cycle
### DnaA Boxes and Dat4  
The binding of DnaA to its sites plays a central role in its activity. There are around 350 DnaA proteins per origin (but up to a total of 2500 DnaA in a fast growing cell where there are also many origins) [109] and these proteins can bind with differing affinities to around 300 DnaA boxes in the chromosome [110]. These boxes are found in regions that include _oriC_ and _datA_, which are able to bind 45 and 370 DnaA proteins with apparent dissociation constants of \(8.6\times 10^{-9}\) M and \(1.7\times 10^{-8}\) M respectively [111]. DnaA binding to _datA_ is consistent with a titration role in initiation because such binding appears to delay initiation or prevent hyper-initiation [81; 112; 113].  
In this titration role, _datA_ does not fit readily into the class of equilibrium structures that are subject to quantity sensing (if it were to perform quantity sensing, we might expect, for example, the number of copies of _datA_ to accumulate--which they do not--and, in accumulating, progressively bind only ADP-DnaA to give an increased ratio of free ATP-DnaA:ADP-DnaA); unless, of course, _datA_ is physically part of an _equilibrium_ origin hyperstructure--certainly, it does seem close to _oriC_[71; 81]. In this case, the equilibrium material that accumulates (and that is sensed by the origin hyperstructure in its equilibrium state) might include some of the Nucleoid-Associated Proteins or _NAPs_, which are central to the organization, replication, segregation, repair and expression of bacterial chromosomes, and which are essential if a new cell cycle is to be completed successfully. The NAPs include HU, H-NS, Fis, Integration Host Factor (IHF), StpA and Dps, each of which binds to several hundred specific sites throughout the chromosomes and which together bind non-specifically to the chromosome at an average of one NAP per hundred base pairs [114; 115; 116; 117; 118]. Several NAPs, includingIHF, HU and Dps, associate with Dna [91, 92]. IHF, for example, binds to many sites on the chromosome [16], and, in bending DNA, often acts together with other NAPs and transcription factors; for example, IHF interacts with DnaA and Fis to initiate replication via IHF binding to a site in _oriC_ to stimulate unwinding [119, 120]. This makes IHF a good candidate for playing a role in quantity sensing. It is therefore significant that IHF binds to _datA_ and that this binding increases the number of DnaA proteins that bind _datA_[93]. It will be apparent to the reader at this stage that the DNA-binding protein best-placed for the lead role in a quantity sensing hyperstructure--perhaps as the first amongst equals--is DnaA itself.  
Instead of _datA_ belonging to the class of quantity sensing, equilibrium hyperstructures, could it belong to the class of intensity sensing, non-equilibrium hyperstructures? Here, one is looking for evidence, for example, that the superhelicity of the _datA_ region increases during the build-up to initiation and that this releases DnaA. We know of no direct evidence for this (although DnaA binding to DNA is affected by superhelicity, see below). It is, of course, conceivable that a _datA_-IHF-DnaA hyperstructure is involved in _intensity sensing_. Such a role could be related to the proximity to _oriC_ of the genes encoding subunits of ATP synthase which themselves are likely to create an ATP synthase transertion hyperstructure with a size and topology that should change with continued growth (and also to the distribution of gyrase sites as discussed in [27]); in the case in which spiral lipid domains result from transertion, it is significant that disruption of the domains of MreB plus fluid phospholipids affects the activity of LacY and the F\({}_{1}\)F\({}_{0}\) ATP synthase [154].