# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 5 \"The Initiation Mess\" Revisited: Conundrums
### The Conundrum of the Initiation-Elongation Correlation  
It has long been noted that there is a negative correlation between the cell cycle frequency of initiation and the rate of elongation (replication fork speed): hyper-initiation (more than one initiation event per chromosome per cell cycle) results in slower replication forks and, conversely, slower replication forks result in hyper-initiation (excess replication forks) [163]. This observation appears to be universal: a negative correlation between origin density (initiation frequency per kilobase) and fork rate has repeatedly been reported since 1975 in yeast and metazoan cells [164]. Experimentally perturbing fork progression (slowing forks) results in the activation of \"dormant\" origins and a reduction in average replicon size (for a proposed mechanism, see [165]). Hence, there is a clear homeostatic relationship between the frequency of initiation during a single cell cycle and the rate of replication fork elongation. DnaA might license initiation of DNA synthesis from _oriC_[166; 167], but it cannot actually cause this initiation until dNTP pool sizes are permissive for DNA synthesis. This proposal is supported by the observation that dNTP pool sizes are sub-saturating for replication fork rates at all growth rates--in contrast to DnaA and ATP, which are maintained at supersaturating concentrations throughout the cell cycle [168]. Deoxynucleotide pool sizes may, therefore, be limiting not only for replication fork progression but also for the initiation of DNA replication at _oriC_. However, this hypothesis remains to be verified. It has been shown that over-expressing the cell cycle-regulated enzyme RNR not only expands dNTP pool sizes but also accelerates fork rates and shortens the C-period of _E. coli_ independently of the growth rate. Conversely, either inhibiting or titrating RNR reduces pool sizes, slows fork rates, and lengthens the C-period [163; 168; 169]. The reduced fork rate when RNR is titrated in vivo results in a three-fold increase in chromosomal DNA and, concomitantly, a threefold increase in cell size (Figure 3)--a phenomenon well known in eukaryotes and referred to as the \"nucleotide effect\" in which the C-value (defined as the total amount of DNA contained within a cell's haploid chromosome set) determines cell size [17]. The nucleotypic effect inverses the assumed relationship between cell mass and cellular DNA content: DNA synthesis and, therefore, DNA content, drive cell mass accumulation during the cell cycle. Thymine auxotrophic strains of _E. coli_ can grow in a steady state despite the concentration of thymine in the medium being limiting [170]. In these conditions, the time taken to replicate the chromosome is lengthened [171], which is accompanied by morphological changes occurring to the chromosome interpreted as corresponding to changes in the number of \"replication positions\". This number, termed \"nucleoid complexity\", has been defined by the ratio of time taken to replicate the chromosome to the generation time (c/\(\tau\)) [19], in contrast to \"chromosome complexity\" (defined as the _oriC/ter_ ratio). It could be argued that the increase in nucleoid complexity during thymine deprivation reflects not only the slowing of replication but also an increase in the number of initiations. Indeed, inhibiting elongation under a variety of experimental conditions induces _nrdAB_ expression and elevates levels of RNR [172]. Cell size depends on the growth rate and the rate of elongation during DNA synthesis (duration of the C-period). Experiments have shown in all organisms that fork rate depends on dNTP supply or RNR activity. High levels of RNR shorten the C-period, and low levels lengthen it [159; 168]. However, the growth rate is largely unaffected and, hence, cells continue to grow and increase their masses when fork rate is experimentally impeded [163; 171; 173]. At the same time, initiation will continue, increasing nucleoid complexity or the number of replication origins and forks per chromosome.