# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 5 \"The Initiation Mess\" Revisited: Conundrums
### The SeqA, Dam, SAM Conundrum: The Bacterial Cell Cycle as a Redox Oscillator
Elongation occurs at a constant rate depending on RNR activity and dNTP pool sizes at the replication forks. Topo IV is activated concomitantly with DnaA-ATP licensing of _oriC_ after SeqA disengages from the fully methylated DNA (Elongation/Licensing). Segregation and decatanation then occur (Segregation/Decatanation). The mother cell divides, and fully licensed sister cells are born (Division/Birth). Protein synthesis, including DnaA synthesis and RNR synthesis, prime the licensed _oriC_ for initiation when the right metabolic conditions are met (Initiation/Sequestraction). The cycle is then repeated in the daughter cells. It is proposed here that licensing in the mother cell confers an adaptive advantage to the daughter cells by allowing them to respond rapidly to both external (temperature shift, etc.) and internal (stalled forks) challenges. in the respiratory chain or in the environment [154; 155]. Significantly, oscillation of the intracellular redox state during the cell cycle of _C. crescentus_ controls the activity of NstA, a protein that inhibits the decatenation activity of TopoIV [156]. SeqA has long been considered an inhibitor of premature reinitiation. However, like NtsA in _C. crescentus_, SeqA specifically inhibits TopoIV, which is required for segregation and, consequently, for cell division [80; 157]. One possibility is that SeqA participates in the timing of Dna-ATP licensing of _oriC_ (post-sequestration or during the late C-period) for the initiations that will occur in the subsequent cell cycles of the daughter cells, or in fast-growing cells before cell division. The asynchrony phenotype in _seqA_ mutants might, therefore, reflect aberrant licensing and the anomalous premature, stochastic activation of initiation before chromosome segregation and cell division have been completed. In the case of RNR, it was found that inhibiting DNA replication increases the synthesis of _nrd_ mRNA and that this increase depends on protein synthesis [158]. Moreover, the quantities of both _nrd_ mRNA and RNR protein increase during the _E. coli_ cell cycle to reach a maximum at the time of the initiation of DNA replication [159], as does GSH, indicating that an expansion in dNTP pool sizes occurs at or before the onset of DNA synthesis; dNTP pools then decline fivefold during the cell cycle from the peak at or before initiation [121], consistent with cell cycle pool size oscillations of GSH and dNTPs [144]. In _C. crescentus_, which relies essentially on Trx as a reducing agent, the need to precisely control Trx abundance is crucial to cell growth and underlies the importance of redox regulation of cell cycle progression, presumably via DNA synthesis [153]. In _E. coli_, mutation of the gene encoding the thioredoxinine-like protein, YbbN, results in over-initiation but does not affect replication in vitro, which has been attributed to its acting as a chaperone for three enzymes in the DNA polymerase 3 holoenzyme, namely DnaN, HolE, and a subunit of HolB [160]. Alternatively, the mutation might affect RNR and perturb replication elongation in vivo, thus resulting in the observed over-initiation. GSH is essential not only for initiation, but it also plays a role in coordinating cell division with cell growth [144], which might explain the positive correlation between cell age at replication initiation and cell age at constriction. As previously suggested: \"Some event before or at chromosome initiation triggers the onset of cell division\" [161]. A critical level of GSH might be one of these factors. Interestingly, SAM is also required for cell division [162], suggesting that these metabolites might serve as signals activated at the initiation of DNA replication that eventually trigger cell division or, indeed, that simultaneously trigger the process leading to cell division [16].