## 8 What Is the Relationship between Strand Opening and DnaA Binding? [PERSON]'s group first showed that DnaA opens _oriC_ in vitro depending on the presence of ATP [61]. The opening of _oriC_ was detected by P1 nuclease sensitivity in this case, and repeatedly shown by other techniques such as KMnO\({}_{4}\) modification [62] and DMS footprinting [63]. _oriC_ contains (1) the DnaA-ATP-Oligomerization Region (DOR), with twelve DnaA boxes, and (2) the neighbouring Duplex Unwinding Element (DUE), which contains three AT-rich 13-mer repeats along with DnaA binding motifs. 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]. [PERSON]'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]. That said, it is difficult to follow the kinetics of _oriC_ opening with these techniques. [PERSON]'s group performed a single molecule analysis on DnaA-_oriC_(2 kb) interaction using an optical magnetic tweezer to follow the rapid kinetics of double-stranded DNA opening. They observed formation of stable complexes between supposedly DnaA-ATP oligomers and _oriC_ with different degrees of positive supercoiling. The formation of these complexes occurred using an _oriC_ that lacked the DUE, raising the question of whether they were studying a non-canonical reaction. Other questions include why the kinetics of the complex formation was not studied, why the formation of the complex did not occur constantly [65], and whether DnaA was actually present in the complex. It should be pointed out that the use of optical magnetic tweezers is technically demanding: it requires the attachment of _oriC_ DNA to a magnetic bead followed by the selection of intact _oriC_-containing beads (which are easily damaged and consequently in a minority). Techniques based on minicircles of DNA facilitate the detection of fine-scale modifications to the DNA structure. Using an _oriC_ minicircle of 641 bp with three negative supercoils, [PERSON] and [PERSON] found that around 80% of this substrate was positively twisted three times during incubation with DnaA and that the efficiency of unwinding was affected by the degree of negative superhelicity of the minicircle (three negative turns proved more effective in causing unwinding than two or four negative turns). Unwinding of this _oriC_ minicircle by DnaA was verified by Bal31 sensitivity (rather than by P1 nuclease sensitivity), whilst the presence of DnaA on the unwound minicircle was confirmed by an anti-DnaA antiserum. The problem raised by this work is that the unwinding did not require ATP [66]. It should also be noted that the above work on _oriC_ minicircles depends on a sophisticated technique that requires the formation of circles from a linear 641 bp _oriC_ fragment that can only be achieved in a glass capillary after overnight incubation in the presence of DNA ligase and ethidium bromide, which introduces superhelicity; modification of the superhelicity of minicircles resulting from DnaA action is scored after Topo I treatment and is not directly measured. This work raised the question of whether or not the ATP-dependent opening of _oriC_ by DnaA (as demonstrated by P1 nuclease sensitivity) is the unique pathway for the initiation of replication. The fact that the mutant isolated first, _dnaA46_, which has lost the ATP binding site, can grow normally at a low temperature indicates the existence of an alternative pathway whereby _oriC_ can fire without ATP. Consistent with this, the growth of _dnaA46_ is more sensitive than the wild type to gyrase inhibitors [67], whilst the opening of _oriC_ minicircles by DnaA is sensitive to negative supercoiling densities [66](see above).