## 17 Miscellaneous Questions  
[PERSON] and collaborators performed a series of experiments that largely entailed shifting a growing culture of a _dnaA(ts)_ strain (and sometimes a _dnaC(ts)_ strain) to the non-permissive temperature for an hour and then returning that culture to the permissive temperature in the presence or absence of chloramphenicol; in the former case, this resulted in four to five initiation events, as opposed to just one in the latter case [152]. Only ten minutes of inhibition of protein synthesis were needed to produce these extra initiations [153]. Could seeing initiation in terms of hyperstructure dynamics help explain these results? For example, is it possible that the inhibition of protein synthesis, which would disrupt an MMS hyperstructure (or perturb a hyperstructure to which the MMS operon would normally contribute), would, therefore, inhibit an initiation hyperstructure? A complementary possibility is that the drop in temperature resulted in the decondensation of ions from _oriC_ and associated proteins within a hyperstructure, leading to the opening of the strands and initiation [97]. As mentioned above (see Section 15**Does an Initiation Hyperstructure Sense DNA Supercoiling? **), _E. coli_ can grow despite the inactivation of _oriC_ and _dnaA_, provided cells lack enzymes such as RNase H, which removes RNA-DNA hybrids in the form of R-loops [154]. This is because replication can be initiated at multiple ectopic _oriK_ sites (for which a consensus sequence has yet to be defined) elsewhere on the chromosome [155; 156]. It has been proposed that this \"constitutive stable replication\" may be a relic of the replication used by early cells [157]. Assuming that increasing transcription at an _oriK_ increases the probability of replication, it is tempting to speculate that such coupling could provide an intensity-sensing mechanism to allow replication of DNA before it becomes limiting for growth [158]. That said, it is difficult to square this simple mechanism with the apparently normal timing of minichromosome replication in conditions in which chromosome replication itself is random (see below). [PERSON] and [PERSON] used an integratively suppressed strain to investigate minichromosome replication [159]. In this strain, the chromosomal _ori_C is inactive and replication occurs at random from a plasmid origin (P1); despite this, the rounds of replication of minichromosomes as seen using density shifts occurred at cell cycle intervals, consistent with a signal for initiation still being generated at the normal time [159]. The authors argued against an artefact due to a lengthening of the eclipse period--a period during which a newly replicated origin is refractory to a second initiation event [160]--but, rather, proposed that the minichromosome replication they observed was not being triggered by the process of chromosome replication; in other words, the system that normally triggers chromosome replication continued working even when chromosome replication was random [159]. This result, therefore, appears to call into question models based on the chromosome being an integral part of the cell cycle clock. Is it possible to explain the result by invoking the operation of a \"primitive\" physico-chemical clock based, for example, on hyperstructure dynamics? In particular, could the result be explained by the cyclically changing states of metabolic, non-equilibrium hyperstructures [97]? Could it even be based on some sort of long-term cellular memory based on hyperstructures, analogous to the memory of exposure to inducer conferred by the existence of a Lac hyperstructure that, once created by a level of inducer, maintains the capacity to metabolise lactose in the subsequent absence of this high level [161; 162; 163]? If such a memory depended on the segregation of hyperstructures with the DNA strands over the generations, it could give a distribution of growth rates and corresponding cell cycle periods in the population [164].