# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 3 The DnaA Protein
### Detailed Analysis of oriC Opening by DnaA In Vitro
#### 3.3.2 _oriC_ Opening Without ATP  
The importance of ATP in the opening of _oriC_ by DnaA, as described above, raises the question of how the _dnaA5_ and _dnaA46_ mutants, which are deficient in ATP binding, can open _oriC_. [PERSON] and [PERSON] found that the DnaA protein can perform its role without ATP using conditions that differed considerably from those used previously [53]. Firstly, the _oriC_ substrate was in the form of minicircles, which were negatively supercoiled (and, at 641 bp, one-tenth the size of those used previously), and which were prepared by over-night incubation at 15 \({}^{\circ}\)C in a glass capillary with ligase and with BET. BET introduces negative supercoiling. Secondly, the substrate and DnaA were incubated at 30 \({}^{\circ}\)C for 30 min, conditions in which most of the minicircles are opened. This reaction is less efficient than P1 nuclease sensitization performed during 3 min of incubation at 38 \({}^{\circ}\)C with ATP and may be unable to open multiple copies of _oriC_ simultaneously. This would fit with the asynchronous initiation phenotype of the _dnaA46_ mutant [34], which is presumably due to the slow opening of _oriC_ at the permissive temperature reducing the probability of having all the copies opening at the same time.  
This result is consistent with the presence of an active site on the DnaA protein other than the AAA\({}^{+}\)ATP-binding site; two domains, IIIa with its single-strand DNA attaching  
Figure 1: Loop-back model (adapted from [52]): _oriC_ containing DUE (duplex unwinding element), DnaA boxes (R1, R5M, R2, and R4), IHF (and the IHF-binding site), and Fis (and the Fis-binding site) is folded by IHF facilitating interactions between DnaA and DUE.  
site and IV with its possible DnaA-recognition capacity, are candidates for allowing _oriC_ opening without ATP. These sites may be thermolabile in the DnaA5 and DnaA46 proteins. We speculate that _oriC_ opening by DnaA without ATP depends, firstly, on the spontaneous breathing of DnaA boxes or partial opening of DUE by structural constraints as occurring in negatively supercoiled _oriC_ minicircles, and, secondly, on a single-stranded region being enlarged by the insertion of DnaA. This speculation is supported by the observation of [PERSON]'s group that an increase in the culture temperature by 10 \({}^{\circ}\)C induces an extra cycle of DNA replication in _E. coli_ that depends on both DnaA and the DUE of _oriC_[54]. This could be explained by the denaturation of the DUE by heat, followed by the insertion of DnaA.  
Another explanation for _oriC_ opening by DnaA without ATP was provided by [PERSON]'s group. They found that DnaA protein prepared from a _dnaA5_ or _dnaA46_ mutant with a defective ATP-binding site failed to replace the wild-type protein to allow _oriC_ plasmid replication in [PERSON]'s in vitro replication system with purified proteins. Seeking factors that could reactivate these thermosensitive proteins in cell-free extracts, they isolated two heat shock proteins, DnaK and GrpE; pretreatment of DnaA5 in the presence of ATP (which is necessary for heat shock proteins to function) restores the initiation activity at 30 \({}^{\circ}\)C but not at 40 \({}^{\circ}\)C. This result led them to speculate that these heat-shock proteins reactivated DnaA5 by inducing conformational changes that promote _oriC_ opening, performed by some domains of DnaA [55] (see below).