## 16 Is Ribonucleotide Reductase an Essential Constituent of the Initiation Hyperstructure?
coli_ that usually map to the _dnaA_ gene in both _E. coli_ and _B. subtilis_[135; 140], while suppressors of the mutant _ldaa_ gene, which overinitiates DNA replication, map to the _nrdAB_ gene [141; 142]. Suppressors of the _dnaAcos_ mutant, which also overinitiates DNA replication, likewise map to the _nrdAB_ locus [143; 144]. As noted above, initiation in _E. coli_ is a complex process involving the formation of a multi-component replication hyperstructure (composed of RNR, DnaA, and possibly SeqA and other factors) that activates initiation at a specific cell mass called the \"initiation mass\" [129]. The initiation mass is independent, it appears, of cell growth rate. How the cell \"senses\" the initiation mass, and therefore \"knows\" when to initiate chromosome duplication, has remained a mystery since the initiation mass concept was first introduced over fifty years ago. It is known, however, that neither DnaA, which controls the frequency of initiation, nor RNR, which controls the rate of DNA synthesis, appears to be the primary determinant of the timing of initiation or of the setting of the initiation mass [78; 139] (see above Section 11: **Is DnaA a Controller of Chromosomal Copy Numbers Rather than a Timer? **)  
Cells with reduced dNTP levels, however, initiate DNA replication earlier in the cell cycle (immediately after cell division) compared to wild type cells, but at a relatively larger cell size and hence at the same initiation mass [127]. This observation might suggest a link between the rate of elongation and the initiation mass itself, since DNA replication is coupled to cell growth, albeit by an unknown mechanism [134]. This raises an interesting question: does the rate of elongation, which is coupled to cell growth in the mother cell, set the initiation mass in the daughter cells, instead of the initiation mass setting the time of initiation in the daughter cell cycles? If so, then the rate of elongation, rather than the initiation mass itself, might be the decisive parameter that determines the major eventsdriving the bacterial cell cycle. Rephrasing the question more precisely: is the initiation mass a passive consequence of the coupling between replication and growth or is it, as is commonly believed, an active cause of initiation and its timing in the cell cycle? The independence of the initiation mass from the cell growth rate strongly suggests a metabolic link to the signalling of replication initiation, a link that remains poorly elucidated to date. In eukaryotes, DNA synthesis takes place during the reductive (biosynthetic) phase of the cell cycle and coincides with an abrupt rise in reactive oxygen species (ROS) at the G1 oxidative phase/ S phase transition, which suggests that the cellular metabolic state plays a role in signalling the timing of initiation at a critical cell physiology/mass, at least in eukaryotes [145; 146; 147]. As mentioned above (see above Section 6: **Does the Initiation Hyperstructure Contain Glycolytic Enzymes? **), in _B. subtilis_, a number of enzymes involved in metabolism have been shown to be associated with both replication initiation and elongation (specifically, the DnaC helicase, DnaG primase, and DnaE lagging strand polymerase), whilst in _E. coli_, carbon metabolism plays an important role in DNA replication fidelity and correlates with DNA synthesis [148; 149; 150]. It is tempting to speculate that levels and balances in metabolite pool sizes play a significant regulatory role in signalling and controlling important cell cycle processes, such as the accumulation of the initiation mass, the timing of the initiation of DNA replication, the rate of chromosome elongation, and cell division. Clearly, these events are tightly coordinated and co-regulated. One way is via post-translational modifications such as the acetylation of DnaA (see above Section 10: **What Modifications Does DnaA Undergo and What Are Their roles?