# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 7 Discussion  
The rate of fork movement relative to the growth rate of the cell (tau\({}_{\text{CYC}}\)/tau) determines the amount of DNA in the cell. This means that a cell with a slower rate of fork movement (causing a longer C-period) relative to its growth rate has higher concentrations of _oriC_ and/or chromosomal DNA and, in consequence, is proportionally larger in size (the nucleotypic effect). This is consistent with a hypothesis in which the rate of fork movement in the mother cell influences the cell mass and the time at which initiation occurs in the daughter cell. In other words, the amount of DNA (complete, fully replicated, chromosomal equivalents) in the individual cell is a determinant of its mass and, hence, the amount of DNA is a determinant of the initiation mass, _Mi_. In this hypothesis, the _Mi_ could, therefore, be considered more of a consequence or a correlate of initiation rather than its actual cause. How might a nucleotypic effect operate in bacteria? Ribosome synthesis, which is limited by the metabolic rate [177], controls the rate and amount of cell mass accumulation (protein synthesis). RNR, likewise, controls the rate and amount of DNA synthesis. It seems plausible then that the production of these two factors (among others) is coordinated via RNA polymerase, such that an initiation mass emerges from their parallel expression: cell mass accumulating concomitantly with DNA synthesis. As the chromosome is being duplicated, the cell volume expands to accommodate the increasing amount of DNA until a critical DNA/mass ratio is reached before cell division. It remains to be shown if and how RNApolymerase activity might coordinate DNA synthesis with protein synthesis, but the in vivo requirement of RNA polymerase might reflect the need to couple DNA synthesis to cell mass accumulation in a manner establishing a growth rate invariant DNA/mass ratio. One way to test this hypothesis would be by performing the following experiment: simultaneous, long-term (>12 h) overproduction of DnaA and RNR should result either in cell lysis (programmed cell death) due to perturbed DNA/mass homeostasis, or in a DNA/cell mass homeostasis in which cell sizes are directly proportional to complete fully replicated chromosomal copy numbers, perhaps resembling filamentous cells [178]. An _E. coli_ mutant deficient for SAM synthesis, for example, is defective in cell division but maintains an otherwise normal cell cycle. The extremely elongated cells (_cei_ phenotype) result in filaments that are 700 times longer than the usual cell length/size [162]. A second way to test the hypothesis would be to examine the level of RNR activity and dNTP pool sizes in the _rpoC_ mutant, the expectation being that dNTP pool sizes will be elevated two-fold in order to account for the two-fold increase in chromosome copy number in these cells [104]. It might also be possible to isolate mutants other than _rpoC_ that have a higher-than-normal chromosomal copy number. [PERSON] et al., for example, have shown that the delta-_lda_ strain overexpressing _nrdAB_ has an elevated number of genomes compared to the wild type [179]. A number of questions remain to be addressed concerning the _hda_, _dnaA_, and _dnaN_ initiation hyperstructure and its regulatory inter-relationship with _nrdAB_ expression and the coordination of replication initiation with elongation, presumably in the form of a hyperstructure involving _oriC_ and _nrdAB_. These questions include:  
1. Is the _nrdAB_ gene co-located with _oriC_ at initiation when the hyperstructure forms? 2. Although some evidence exists that _nrdAB_ levels are elevated in the _hda_ mutant in certain genetic backgrounds (but not wild type), the _hda_ gene itself is found in only a limited number of bacterial species [109]. How, then, is dNTP synthesis coordinated with replication initiation in Hda-deficient species? 3.