# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 5 \"The Initiation Mess\" Revisited: Conundrums
### The DnaA-ATP:DnaA-ADP Conundrum  
Very recently, a fluorescence-based study found that the intracellular distribution of DnaA is homogeneous in contrast to the distribution of HU (and, by extension, DNA), which is heterogeneous with respect to the halves of the cell corresponding to the future daughters. These variations in the DnaA/DNA ratio in the future daughter cells were proposed to cause variations in the time of initiation [115]. It has been repeatedly suggested that fluctuations in the ATP/ADP ratio are crucial for timing replication initiation. However, only 15 to 30% of the more than 1000 DnaA proteins present in the cell are in the ATP form [116]. ATP pools must double during growth (\(\times\)2.4) to maintain a constant concentration over the cell cycle. ATP pool sizes peak at the time of cell division and cell birth, and then they decline to a steady state level [117]. The initiation factor DnaA is active only when bound to ATP. The ATP/ADP ratios, in the form of DnaA-ATP/DnaA-ADP ratios (for example), might, therefore, determine the observed growth rate invariant cell mass per origin at which initiation occurs, the so-called \"initiation mass\" (_Mi_) [118]. However, this proposal faces several caveats. Put simply, it is difficult to envisage how cell mass, per se, could determine when in the cell cycle to initiate DNA replication [119]. While ATP concentrations are also independent of the growth rate and vary only 12% between slow and fast growth (compared to a variation in \(Mi\) of about 20%) [117; 120], ATP/ADP oscillations seem unlikely to explain \(Mi\) and replication initiation timing, at least in a global regulatory role as a \"timer\" or \"sizer\" mechanism since ATP pools, for example, are at millimolar (from 1.2 to 3.6 mmol [117]) or supersaturating concentrations throughout the cell cycle [121]. The cellular need for excess ATP has been a longstanding mystery (see below). In line with previous studies [122], the concentration of DnaA has recently been shown to be constant over the cell cycle [115]. DnaA has a high affinity for both ATP and ADP in the micromolar range with KD of 0.03 and 0.1 [49], suggesting that DnaA is bound to ATP throughout most of the cell cycle: [DnaA] = constant; [DnaA-ATP] = constant; and [DnaA-ADP] = constant at all growth rates and at all phases of the cell cycle [123]. Additionally, the DnaA-ATP-to-DnaA-ADP transition is not essential for wild-type initiation control except at high growth rates [65]. DnaA-ATP/DnaA-ADP ratios are, therefore, dispensable in determining \(Mi\) and initiation timing. The idea that the concentration of DnaA-ATP regulates initiation and determines the initiation mass is incompatible with the fact that the concentration of DnaA (saturated by the high concentration of ATP) is higher than needed for replication initiation (200-500/_oriC_ versus 5-20/_oriC_) [59; 124; 125]. Since DnaA, like ATP, is continuously present at supersaturating levels, its level can be doubled without affecting initiation [126]. Another complication arises from the fact that DnaA is competent for initiation in the absence of ATP binding, although cell cycle regulation of its initiation competence is compromised. Mutations that result in hyper-initiation or asynchronous initiation, such as _dnaAcos_, _dnaA46_, and _hda_, all cause defects in either binding or hydrolyzing ATP, though the mutants remain allosterically competent for initiation [127]. The _dnaAcos_ hyper-initiation phenotype is highly toxic to the cell and results in fork collisions that produce lethal DNA double-strand breaks [128], a problem not encountered in in vitro systems. The Hda-directed post-initiation hydrolysis of ATP would, therefore, serve primarily to inactivate DnaA that is already bound to _oriC_. Yet another complication arises from the need to sequester _oriC_ following initiation. DnaA-ADP detaches from _oriC_, allowing the sequestration protein SeqA to bind the hemimethylated DNA and block DnaA-ATP from rebinding _oriC_ post-initiation.