# 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  
The above points support the argument that, while DnaA-ATP is active and DnaA-ADP is inactive, the ratio between them does not determine the _Mi_, and, therefore, the principal role of the transition between the two forms--in conjunction with SeqA--is to prevent free DnaA-ATP from re-binding _oriC_ until DNA becomes fully methylated [78; 79; 139]. SeqA binding to hemimethylated _oriC_, in the latter scenario, would serve primarily to coordinate DNA segregation, DNA fork movement, and nucleoid restructuring by inhibiting TopoIV [78; 140; 141; 142]. Inhibition of initiation, therefore, might be a simple consequence of sequestration, while benefiting the cell with the adaptive advantage of maintaining a critical distance between multiple replication forks on a single chromosome until sister chromosomes are fully segregated. The above proposal is supported by the observation that overproduction of SeqA increases the period of sequestration at _oriC_ and delays nucleoid segregation and cell division [78], apparently without impeding fork movement. Because both segregation and cell division are inhibited under these conditions, the persistence of SeqA at _oriC_ argues in favour of SeqA playing a regulatory role in licensing _oriC_ upstream from nucleoid segregation and cell division (Figure 2). DnaA production might, therefore, be needed for the post-sequestration licensing of _oriC_, in addition to (or instead of) the actual firing of initiation. Dam methylation of the _dnaA_ gene, for example, is required for its post-sequestraction expression, which is necessary to replenish DnaA, and, in _dam_ mutants, DnaA levels decline to one-third the level in wild-type cells [139]. Whether or not the disassembly of SeqA foci serves as a signal to trigger segregation and division remains unclear. Since sequestration lasts during only one-third of the cell cycle, an important question arises: What factor or event is responsible for disengaging SeqA from hemimethylated _oriC_, which depends on protein synthesis? The _dam_ methyltransferase gradually replaces SeqA as it dissociates from hemimethylated DNA; but under physiological conditions, the methyltransferase does not actively displace SeqA [143], which indicates that some factor other than the _dam_ methylase itself is involved. S-adenosylmethionine (SAM) oscillates during the cell cycle and peaks at the S-phase in _C. crescentus_[144]. This might suggest that, in _E. coli_, SAM could be involved in mediating the detachment of SeqA from _oriC_. SAM, for example, allosterically enhances the affinity and binding of _dam_ methyltransferase to DNA [145; 146]. Once SeqA disengages from _oriC_, DnaA-ATP, would immediately re-bind to _oriC_ post-sequestraction. What, then, might prevent DNA synthesis from firing prior to cell division at a fully licensed _oriC_? In addition to SAM, a number of energy and redox metabolites involved in replication initiation such as ATP, NAD(P)H, GSH, and dNTPs fluctuate in phase with the cell cycle [144; 147; 148], suggesting that the bacterial cell cycle is a redox oscillator that couples, or gears, major cell cycle events to changing rates of cell growth (Figure 2) [149; 150]. NAD(P)H drives DNA synthesis in the S-phase of the eukaryotic cell cycle, providing the electrons needed to reduce RNR and activate dNTP synthesis [151], which occurs via the GSH and thioredoxin pathways [152]. The Trx pathway is induced in _Caulobacter crescentus_, for example, immediately before replication initiation and oscillates in phase with the bacterial cell cycle [153], presumably in response to reactive oxygen species generated  
Figure 2: Mother cell licensing model. Licensing of _oriC_ by DnaA-ATP occurs immediately after SeqA disengages from hemimethylated _oriC_, which is then rapidly methylated to form fully methylated DNA along the chromosome. During the C-period, _dnaA_ expression is low due to elevated amounts of DnaA and auto-regulation of the _dnaA_ gene.