# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 6 The Nucleotypic Effect, \(Mi\), and Growth Rate \"Invariance\"  
RNR, as the regulator of elongation, and DnaA, as the regulator of initiation, may form what has been termed a \"homeostatic pair\" (Figure 4): the two main replication factors cooperate in setting and coordinating initiation at a growth rate-dependent \(Mi\) (either variant or invariant; see: [118; 174] and references therein); that is, a cell mass that is necessary and sufficient to guarantee a growth-rate invariant DNA/mass ratio for each division cycle. If the nucleotypic effect applies to bacteria, then it would not be surprising that the amount of DNA and the amount of cell mass accumulate in tandem under different (physiological) growth conditions in order to supply equivalent DNA/mass ratios to daughter cells--whether or not \(Mi\) is growth-rate invariant. Figure 4: Model of DnaA:RNR homeostatic pair (adapted from [112]). (**a**) Expression of the \(dnaA\) gene (black) and the expression of the \(nrdA\) gene (red) oscillate out of phase in the first half of the cell cycle (see Extended Data Figure 6 in [112]): \(dnaA\) expression increases after cell division (div) when \(nrdA\) expression is low. Low levels of newly formed DnaA-ATP inversely correlate with high levels of \(dnaA\) expression (due to autoregulation) and stimulate \(nrdA\) expression. As DnaA levels increase, DnaA inhibits \(nrdA\) expression [110]. Consequently, DnaA protein will repress its own expression at or after initiation and will concomitantly limit RNR production by repressing \(nrdA\) expression during the S phase/C period, presumably to a level of expression that is in equilibrium with the number of active replication forks. DnaA and RNR thus act as a homeostatic pair to regulate the number of forks according to growth rate. The cytoplasm in \(C\). _crescentus_ is reduced in G1. At the G1/S transition (initiation of DNA replication), the cytoplasm switches to a highly oxidized state that  
becomes increasingly reduced during the S phase until G2 when the cytoplasm switches back to a reduced state [149; 153; 156]. This gated redox cycle also occurs in yeast and metazoan cells. It has been proposed to be a universal feature of cell cycle control [149; 156] and, therefore, might explain the coordination between cell growth/mass and the major cell cycle events, including the initiation of DNA replication, which coincides with a relatively constant \(Mi\). (**b**) Steps in the hypothetical relationship between licensing in the mother cell and initiation of replication in daughter cells. (1) DnaA binds _oriC_ immediately after sequestration leading to its full licensing in late C- or D-periods; (2) licensing in the mother cell titrates DnaA prior to cell division; (3) low levels of DnaA at mother cell division and daughter cell birth induce _dnaA_ expression in the B-period of the daughter cell cycle; (4) low levels of DnaA in the B-period of the daughter cell stimulate expression of _nrdAB_ prior to initiation (late B- and early C-period); (5) high levels of DnaA in the early C-period repress _nrdAB_ and limit RNR levels post-initiation (_nrdAB_ expression is not necessary for elongation, but its expression is absolutely necessary for initiation, underlining the important role dNTP synthesis plays in the cell cycle regulation of replication initiation) [159; 169]. An equilibrium (homeostasis) is established between rates of dNTP synthesis and the number of active forks, an equilibrium that serves to protect the cell against the mutagenic effects of abnormally high levels of RNR and/or rates of dNTP production (in conjunction with dATP allosteric regulation of RNR and NrdR repression of _nrdAB_[175]). It should be noted that during fast growth, there is no B-period. DnaA, therefore, licenses _oriC_ in the mother cell (immediately after sequestration), and DnaA:RNR activates initiation prior to cell division. Black curve: _dnaA_ gene expression level; red curve: _nrdAB_ expression level.